Full Journal - Journal of Cell and Molecular Biology - Haliç Üniversitesi

STANBUL 

1998 

VOLUME 1 • NO. 2 • 2002 • ISSN 1303-3646 

GOLDEN HORN UNIVERSITY 

FACULTY OF ARTS AND SCIENCES 

Journal of Cell and 

Molecular Biology

Golden Horn University 

Faculty of Arts and Sciences 

Journal of Cell and Molecular Biology 

Founder 

Prof. Dr. Gündüz GED‹KO⁄LU 

President of Board of Trustee 

Rights held by 

Prof. Dr.Ahmet YÜKSEL 

Rector 

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Summaries of all articles in this journal are 

available free of charge from www.halic.edu.tr 

ISSN 1303-3646 

Igor ALEXANDROV, Dubna, Russia 

Çetin ALGÜNEfi, Edirne, Turkey 

Aglaia ATHANASSIADOU, Patros, Greece 

fiehnaz BOLKENT, ‹stanbul, Turkey 

Nihat BOZCUK, Ankara, Turkey 

‹smail ÇAKMAK, ‹stanbul, Turkey 

Adile ÇEV‹KBAfi, ‹stanbul, Turkey 

Beyaz›t ÇIRAKO⁄LU, ‹stanbul, Turkey 

Ayfl›n ÇOTUK, ‹stanbul, Turkey 

Zihni DEM‹RBA⁄, Trabzon, Turkey 

Mustafa DJAMGOZ, London, UK 

Advisory Board 

Journal of Cell and 

Molecular Biology 

Published by Golden Horn University 

Faculty of Arts and Sciences 

Editor 

Atilla ÖZALPAN 

Associate Editor 

Narç›n PALAVAN ÜNSAL 

Editorial Board 

Çimen ATAK 

Atok OLGUN 

P›nar ÖZKAN 

Damla BÜYÜKTUNÇER 

Özge EM‹RO⁄LU 

Mehmet Ali TÜFEKÇ‹ 

Kürflat ÖZD‹LL‹ 

Asl› BAfiAR 

Ünal EGEL‹, Bursa, Turkey 

Candan JOHANSEN, ‹stanbul, Turkey 

As›m KADIO⁄LU, Trabzon, Turkey 

Valentine KEFEL‹, Pennsylvania, USA 

Göksel OLGUN, Edirne, Turkey 

Zekiye SULUDERE, Ankara, Turkey 

‹smail TÜRKAN, ‹zmir, Turkey 

Mehmet TOPAKTAfi, Adana, Turkey 

Meral ÜNAL, ‹stanbul, Turkey 

Mustafa YAT‹N, Boston, USA 

Ziya Z‹YLAN, ‹stanbul, Turkey


CONTENTS Volume 1, No.2, 2002 

Review articles 

Phytochelatin biosynthesis and cadmium detoxification 

Fitokelatin biyosentezi ve kadmiyum detoksifikasyonu 

G. Bayçu 

Polyamines in living organisms 

Canl› organizmalarda poliaminler 

M. Yatin 

Research Papers 

The histopathological changes in the mouse thyroid depending on the aluminium 

Fare tiroid bezinde alüminyuma ba¤l› histopatolojik de¤ifliklikler 

T. Aktaç, E. Bakar 

Inhibitory effect of 57 % hepatectomized mice serum on the growth of L-cells 

% 57 hepatektomi uygulanm›fl fare serumunun L-hücrelerinin ço¤almas›n› bask›lay›c› etkisi 

S. Altun, M. Topçul, G. Özcan Ar›can 

Effect of epirubicin and tamoxifen on labelling index in FM3A cells 

FM3A hücrelerinin iflaretlenme indeksi üzerine epirubisin ve tamoksifenin etkisi 

M. Topçul, G. Özcan Ar›can, N. Erensoy, A. Özalpan 

The effect of adriamycin on Ehrlich ascites tumor cells iinn vviittrroo and iinn vviivvoo 

Adriamisinin in vitro ve in vivo koflullarda Ehrlich ascites tümör hücrelerine etkisi 

G. Ulako¤lu 

Camalexin is not required for the function of RRPPPP11 and RRPPPP1133 resistance genes 

in AArraabbiiddooppssiiss tthhaalliiaannaa inoculated with PPeerroonnoossppoorraa ppaarraassiittiiccaa 

Kamaleksin Peronospora parasitica ile inokulasyonundan sonra Arabidopsis 

thaliana’n›n RPP1 ve RPP13 dayan›kl›l›k genlerinin ifllevleri için gerekli de¤ildir. 

F. Mert-Türk, E. B. Holub 

Retardation of senescence by mmeettaa-topolin in wheat leaves 

Meta-topolinin bu¤day yapraklar›nda senesensi geciktirmesi 

N. Palavan-Ünsal, S. Ça¤, E. Çetin, D. Büyüktunçer 

Book Reviews 

Instructions for authors 

Volume content 

Author index 

45-55 

57-67 

69-72 

73-79 

81-85 

87-91 

93-99 

101-108 

109-110 

111-112 

113-114 

115

Journal of Cell and Molecular Biology 1: 45-55, 2002. 

Golden Horn University, Printed in Turkey. 


Gülriz Bayçu 

University of ‹stanbul, Faculty of Science, Department of Biology, 34460, Süleymaniye, ‹stanbul, Turkey 

Received 10 May 2002; Accepted 27 June 2002 

Abstract 

Heavy metal contamination in the environment is increasing due to human industrial activity and heavy metal 

causes major environmental problems. Ions like Cd, Hg, Cr, or Pb are non essential heavy metals which are 

potentially highly toxic even at very low concentrations. Heavy metal detoxification and tolerance in plants can be 

achieved by a number of different mechanisms on the molecular basis. Such as the production of metal-binding 

compounds, metal deposition in vacuoles, alterations of membrane structures, synthesis of stress metabolites; but 

the mechanism which has been studied most closely in recent years is chelation. One reaction of plants to excess 

Cd concentration is the formation of Cd-binding polypeptides, or the chelation by a family of peptide ligands, the 

phytochelatins (PCs). PCs are produced by higher plants, algae and some fungi in order to detoxify Cd by 

sequestration to form PC-Cd complexes which play a pivotal role in heavy metal, primarily Cd tolerance by 

decreasing their free concentrations. PCs are derived from glutathione (GSH) and related thiols by the action of PC 

synthase. Understanding the genetic and molecular basis of PC biosynthesis mechanism is an important goal in 

developing plants for the phytoremediation of contaminated environments. This review summarizes present 

knowledge in the field of PC biosynthesis and Cd detoxification . 

KKeeyy wwoorrddss:: Phytochelatins, plants, cadmium, detoxification, tolerance 


Özet 

‹nsan›n endüstriyel aktivitesi sonucunda çevredeki a¤›r metal kirlenmesi artmakta ve a¤›r metal toksisitesi önemli 

çevresel problemlere neden olmaktad›r. Cd, Hg, Cr, Pb gibi gerekli olmayan a¤›r metaller çok düflük 

konsantrasyonlarda bile oldukça toksiktir. Bitkilerdeki a¤›r metal detoksifikasyonu ve tolerans moleküler anlamda, 

metal-ba¤layan bilefliklerin üretimi, vakuollerde metal birikimi, membran yap›s›nda de¤ifliklik, stres 

metabolitlerinin sentezi gibi birçok farkl› mekanizmalar taraf›ndan yürütülür. Ancak son y›llarda araflt›rma konusu 

olarak en fazla ilgi duyulan mekanizma kelat oluflumudur. Bitkilerin afl›r› Cd konsantrasyonuna gösterdi¤i 

tepkilerden biri Cd-ba¤layan polipeptidler, ya da bir peptid ligand grubu ile kelat meydana getiren fitokelatinlerdir 

(PC). PC'ler yüksek bitkiler, algler ve baz› mantarlar taraf›ndan üretilir, PC-Cd bileflikleri oluflturulur ve serbest 

metal konsantrasyonu azalt›larak özellikle Cd detoksifikasyonu ile Cd tolerans›nda önemli rol oynarlar. Bu 

bileflikler glutationdan (GSH) ve iliflkili tiollerden PC sentaz aktivitesi ile meydana gelirler. PC biyosentezinin 

genetik ve moleküler temelinin anlafl›labilmesi, kirlenmifl çevrelerin fitoremediasyonunda kullan›lacak bitkilerin 

gelifltirilebilmesi aç›s›ndan büyük önem tafl›maktad›r. Bu derleme, PC biyosentezi ve Cd detoksifikasyonu 

alan›ndaki bilgileri özetlemektedir. 

AAnnaahhttaarr ssöözzccüükklleerr:: Fitokelatinler, bitkiler, kadmiyum, detoksifikasyon, tolerans 

45

46 Gülriz Bayçu 

Cd Contamination in the environment and toxic 

effects on plants 

Heavy metals such as cadmium (Cd), chromium (Cr), 

copper (Cu), mercury (Hg), lead (Pb), aluminium 

(Al), nickel (Ni), etc., are important environmental 

pollutants particularly in areas where there is high 

antropogenic pressure (Abrahamson et al.,1992; 

Sanità di Toppi and Gabbrielli, 1999). This group of 

elements is toxic to all organisms at varying 

concentrations (Speiser et al., 1992). Cd is a toxic 

element that normally occurs in very low 

concentrations in soils (Wagner, 1993). However, it is 

released into the environment by urban traffic, metalworking 

industries, heating systems, power stations, 

cement factories, waste incinerators and phosphate 

fertilizers (Sanità di Toppi and Gabbrielli, 1999). In 

the areas that have been subjected to mining, the 

concentration can be high, varying from 100-600 mg 

kg -1 dry weight. In addition, following the application 

of sewage sludge to agricultural land, Cd can 

accumulate in the top soil (Lombi et al., 2000). In 

humans, Cd is suspected carcinogen it displace Ca or 

Zn in proteins and can cause oxidative stress (Steffens 

and Bagchi, 1995). 

Photosynthetic organisms are the principal entry 

point of metals into the food chain leading to animals 

and man (Rauser, 1990). Heavy metals such as Cu 

and Zn are essential for normal plant growth, but 

elevated concentrations can result in growth 

inhibition and toxicity symptoms. Non-essential 

metals like Cd and Pb are potentially highly toxic and 

show toxic effects even at very low concentrations 

(Hall, 2002). However, a number of plants (termed 

hyperaccumulators) that grow on metalliferous soils, 

are able to translocate Cd from the roots and 

accumulate it in high concentrations in the shoots 

(Chardonnens et al., 1998). It has been suggested that 

such plants would be of considerable value in the 

remediation of soils that are heavily contaminated 

with heavy metals (Zhu et al., 1999). 

Several mechanisms are known to allow plants 

and other organisms to tolerate the presence of toxic 

non-essential metal ions inside the cell. Physiological 

studies indicate that heavy metal tolerance is one of 

the prerequisites of heavy metal hyperaccumulation 

in plants (Raskin et al.,1997). Wide information is 

available on ecological impact of Cd, especially as 

concerns its motility in soil and uptake by plants 

(Leita et al., 1991). Although Cd is not an essential 

nutrient for plants, the metal ion is taken up rapidly by 

the roots and on most occasions causes inhibition of 

growth (Leita et al., 1991). It has been demonstrated 

that Cd is not only easily available to plants from soil 

or other substrates, but also it is toxic to them at much 

lower concentrations than other heavy metals like Zn, 

Pb or Cu. The degree to which higher plants are able 

to take up Cd depends on its bioavailability, 

modulated by the presence of organic matter, pH, 

redox potential, temperature and concentrations of 

other elements (Sanità di Toppi and Gabbrielli, 

1999). Cd is believed to penetrate the root through the 

cortical tissue and it is readily taken up and 

transported within the plant through an apoplastic 

and/or a symplastic pathway (Page et al., 1981; Salt et 

al., 1997). Phytotoxicity has been observed to be 

dependent upon plant species as well as Cd 

concentration in substrate (Leita et al., 1991). Cd 

interferes with plant metabolism and in a very general 

way, causes leaf roll, chlorosis, growth and yield 

reduction (Leita et al., 1991). Inhibition of 

ribonuclease and nitrate reductase activity, interaction 

with the water balance, decrease in chlorophyll 

content, inhibition of stomatal opening, reduction of 

normal H + /K + exchange, production of oxidative 

stress and enhanced lipid peroxidation are the most 

important phytotoxic effects of Cd (Sanità di Toppi 

and Gabbrielli, 1999). 

Heavy metal stress and detoxification mechanisms 

All living cells are confronted with the dilemma that 

on one side they need certain amounts of free heavy 

metal ions (such as Zn, Cu , Ni , etc.) for their normal 

metabolic function, and on the other side they have to 

protect themselves from an intracellular excess of 

these metal ions which would lead to cell death. This 

dilemma can only be overcome by a stringent 

regulation of free metal ion concentrations within the 

cells (Tomsett and Thurman, 1988; Gekeler et al., 

1989). 

In general, resistance to excessively available 

chemical elements can be based on avoidance, i.e. 

exclusion of the element from the body, or on 

tolerance, i.e. the ability to survive, grow and 

reproduce with the element present at elevated 

concentrations in the body (Levitt, 1980; Ernst et al.,

1990). Heavy metal toxicity can elicit adaptive and 

constitutive responses in plants (Sanità di Toppi and 

Gabbrielli, 1999) and plants possess a range of 

potential cellular mechanisms that may be involved in 

the detoxification of heavy metal ions and thus 

tolerance to metal stress. A regulated network of 

metal transport including metal-binding to cell walls 

and reduced transport across cell membrane, 

trafficking and sequestration activities such as active 

efflux, vacuolar compartmentalization and chelation 

functions to provide the uptake, distribution and 

detoxification of metal ions (Steffens, 1990; 

Neumann et al., 1994). Plants appear to accumulate 

metal-chelating compounds upon exposure to 

excessive metal availability levels, such as amino 

acids and amino acid derivatives, citric acid, malic 

acid, peptides, polypeptides and phytochelatins 

(Cobbett, 2000). 

Heavy metals can induce the synthesis of 

thiol-rich metal-chelating peptides (Grill et al., 1985; 

Gekeler et al., 1989; Rauser, 1990; Steffens, 1990). 

The physiologically relevant aspect of metal binding 

is only evident with complexes to liberate the 

constituent metal-free polypeptides. In 1957 a 

cysteine-rich, Cd-binding protein devoid of aromatic 

amino acids was isolated from equine kidney 

(Margoshes and Vallee, 1957; Nussbaum et al., 1988) 

and subsequently called "metallothionein". Induction 

of these cysteine-rich, 4 to 8 kDa small metal-binding 

peptides confer tolerance to a broad range of metals in 

mammals, Drosophila, Neurospora crassa, 

Saccharomyces cerevisiae and they also appear to be 

involved in both metal detoxification and homeostasis 

(Speiser et al., 1992; Vatamaniuk et al., 2000). 

Cd-detoxification and Cd-binding polypeptides: 

Phytochelatins 

Higher plants, algae and certain organisms of the 

kingdom fungi produce class III metallothioneins, 

which are atypical, nontranslationally synthesized 

metal thiolate polypeptides (Grill et al., 1985; Rauser, 

1990; Steffens, 1990; Vatamaniuk et al., 2000). A 

variety of metals including Cd, Cu, Zn, Pb, Hg, Ni, 

Bi, Ag and Au; in addition, multiatomic anions 

including SeO4 -2 , SeO4 -3 and AsO4 -3 also induce their 

synthesis (Grill et al., 1991; Rauser, 1991). Among 

Phytochelatin and cadmium 

47 

the common metals, Cd is a strong inducer, whereas 

Zn appears to be a weak inducer, requiring high 

external levels for induction (Steffens, 1990). These 

cysteine-rich peptides capable of binding heavy metal 

ions via thiolate coordination (Grill et al., 1989; 

Leopold et al., 1999) have been shown to bind Cd and 

Cu directly and are believed to bind Pb and Hg by 

competition with Cd (Speiser et al., 1992) which 

mostly contribute to Cd detoxification and to Cu 

homeostasis (Grill et al., 1988, 1989; Tukendorf and 

Rauser, 1990). With regard to Zn it is difficult to 

demonstrate binding to these polypeptides because of 

the low affinity of the ligand for Zn ions (Grill et al., 

1989). 

This inducible Cd-binding compounds show 

properties distinctly different from metallothioneins 

(Grill et al., 1985). Similarities were high cysteine 

content and comparable circular dichroism spectra, 

indicating the lack of aromatic residues and the 

possible binding of heavy metals by mercaptide 

complexes (Nussbaum et al., 1988). This type of 

biological response to heavy metal stress involving 

the synthesis of small heavy metal complexing 

peptides in higher plants, algae and in some fungi 

termed as "phytochelatins" (PCs) (Grill et al., 1985; 

Rauser, 1990; Steffens, 1990; Cobbett, 2001). They 

are also called as cadystins, Cd-binding peptides, 

γ-glutamyl metal-binding peptides or (γ-EC)nG 1 ( 1 E, 

L-glutamic acid; C, L-cysteine; G, glycine). The 

primary structure of these polypeptides show a series 

of molecules of poly (γ-glutamyl-cysteinyl)-glycine 

(Figure 1) and consists of the repeated dipeptide 

γ-Glu-Cys attached to a carboxy-terminal glycine, 

with a structure of (γ-Glu-Cys)n-Gly, where n=2 

Figure 1: Structure of phytochelatins (from Manunza et al., 

1997).


Figure 2: Optimized structure of the Cd(PC2)2 complex. 

Only the S atoms enter the coordination sphere of Cd 

(from Manunza et al., 1997). 

through 11 depending on the source (Rauser, 1995). 

These complexes generally contain predominantly 

Cd, a range of cysteine-rich-polypeptides, and 

acid-labile sulfide (Figure 2). Glutamic acid found in 

the composition is linked to each sulfur containing 

cysteine by a γ-peptide linkage and because of the 

repetitive γ-glutamic acid bonds, PCs can not be 

regarded as primary gene products (Speiser et al., 

1992). In a few members of the order Fabales, such 

as in Vicia faba, PCs are substituted by a peptide 

family containing a ß-alanine carboxy terminus 

instead of the glycine. These peptides are termed 

homo-phytochelatins, h-PCn or (γ-Glu-Cys)n-ß-Ala 

(n=2-7) (Gekeler et al., 1989). 

PCs are secondary metabolites synthesized 

enzymatically from glutathione (GSH) by 

γ-glutamyl-cysteine dipeptidyl transpeptidase (PC 

synthase, GCS), a 25 kDa protein that removes a 

γ-glutamyl-cysteine moiety from one molecule of 

GSH and couples it to another GSH. PC biosynthesis 

can be induced very rapidly in roots and tissue culture 

cells and is accompanied by a fall in the concentration 

of GSH during early PC accumulation, following the 

addition of Cd or other heavy metals (Grill et al., 

1989). Studies indicate GSH as a substrate for PC 

synthesis. Kinetic data for plant cells show that 

synthesis of PC2 (i.e., n=2) is faster than that of PC3, 

which is faster than that of PC4, as if the shorter PC is 

the precursor to the longer PC (Tukendorf and Rauser, 

1990). The number of repeating units varies with the 

conditions of Cd exposure (Speiser et al., 1992). Cd 

activates the PC synthase to form PCs and synthesis 

stops when free Cd is no longer present (Cobbett, 

2000) which suggests that the structure of PC 

consisting thiol and carboxyl groups are essential for 

the formation of tight PC-Cd complexes (Satofuka et 

al., 2001). 

In laboratory conditions, PC complexes elute as 

broad peaks from gel permeation columns. The 

molecular weight (Mr) PC-Cd complexes were found 

about 3 to 10 kDa depending upon ionic strength . The 

lower Mr observed at high ionic strength suggests that 

complexes possess a trimeric or tetrameric peptide 

stoichiometry. The high Mr observed at low ionic 

strength has been suggested to result both from 

electrostatic repulsion of the negatively charged free 

Glu-carboxylates of the polypeptides and from 

complex aggregation (Steffens, 1990). 

Owing to the high content of cysteine, PCs are 

able to create complex compounds with toxic ions of 

metals. These complexes transport heavy metal into 

the vacuole by the ABC transporter which is localized 

in the tonoplast (Ortiz et al., 1995), thus separating 

them from cell metabolism. PCs bind Cd with high 

affinity and localize together with Cd to the vacuole 

of intact cells (Vögeli-Lange and Wagner, 1990; 

Vatamaniuk et al., 1999). A vacuolar HMT1 

transporter catalyses MgATP-dependent uptake of 

both PC-Cd complexes and h-PCs. Activation of the 

detoxicative-PC system in the cytosol (Rauser, 1995; 

Zenk, 1996; Sanità di Toppi and Gabbrielli, 1999; 

Cobbett, 2000) may show that phytochelatins play an 

important part in detoxification and homeostatis 

(Cobbett, 2001; Piechalak et al., 2002). 

Some possible molecular bases of mechanisms of 

metal detoxification and tolerance involving PCs are 

as follows: 

a. Increased activity of PC biosynthesis, such as PC 

synthase in metal-tolerant plants, 

b. Increased activity of enzymes responsible to 

S 2- saturation of metal-PC complexes, 

c. Modified chloroplastic/extrachloroplastic 

compartmentation of one of the components; PC, 

S 2- , or metal, 

d. Modified transport of metal-PC complexes into 

the vacuole, 

e. Modified rates of PC turnover. 

Modification of some process not directly related 

to metal accumulation by PC, but which allows cell

survival and therefore continued complexation of 

metal by PC (Robinson, 1990; Rauser, 1995), 

(Figure 3). 

PCs were first discovered in the fission yeast 

Schizosaccharomyces pombe as components of 

metal-containing complexes from Cd-induced cells and 

the structure of PCs produced by S. pombe have been 

found identical to those of plants with the number of 

repeating units ranging from 2 to 11 (Speiser et al., 

1992). According to Gekeler et al. (1989), PCs were 

produced by all higher plants tested and they have also 

been shown to exist in the algae Chlorella fusca and the 

yeast Candida glabrata, which produces both PCs and a 

metallothionein. 

Several 10 kDa metal-binding peptides from 

differentiated tissues and suspension cell cultures of 

higher plants, such as Rauwolfia serpentina have been 

reported by Grill et al. (1985) and the induction of 

peptides by Cd was also observed in cell cultures of 

Berberis stolonifera, Malva sylvestris, Solanum 

marginatum. In all cases more than 90 percent of the Cd 

taken up by the cells was complexed to the PC peptides. 

Phytochelatin and cadmium 49 

Figure 3: A model of intracellular location of Cd-binding complexes and associated transport mechanisms 

(from Rauser, 1995). 

Some of the plant species have received special 

attention due to damages reported which are supposed 

to be antropogenic origin and heavy metal pollution 

who considered to be one possibility (Gekeler et 

al.,1989). It was therefore of interest to investigate the 

potential of some Gymnospermae and Angiospermae 

to PCs. Both suspension cultures and 1 year old 

rooted plants were used and they were found to have 

the ability to form PCs in varying chain length after 

exposure to sublethal concentrations of Cd. For 

example, Gingko biloba have produced PCs of the 

type (γ-Glu-Cys)n-Gly where n=2-5, Abies alba, n=2- 

3, Picea abies, n=2-3, Pinus pinea n=2-4, Pinus 

sylvestris n=2-4, Laurus nobilis n=2-6, Viola 

calaminaria n=2-5, Triticum aestivum n=2-4, 

Phoenix dactylifera n=2-5, Cyperus esculentum, n=2- 

5, Ananas comosus n=2-4, Asparagus officinales n=2- 

4. Some Fabaceae species such as Astragalus 

gummifer, Lotus ornithopodioides, Ononis natrix, 

and Lathyrus ochrus were also found active to 

produce homo-phytochelatins (h-PC2) (Gekeler et al., 

1989).


PC production was found to be the main response 

mechanism to Cd stress in the roots of higher plants 

such as: Avena sativa, Brassica juncea, Cucumis 

sativus, Glycine max, Hordeum vulgare, Lactuca 

sativa, Lupinus luteus, Lycopersicon esculentum, 

Oryza sativa, Phasealous vulgaris, Pisum sativum, 

Raphanus sativus, Sesamum indicum, Silene vulgaris, 

Zea mays (Klapheck, 1988; Leita et al.,1991; De 

Knecht et al., 1994; Inouhe et al., 1994; Klapheck et 

al., 1995; Salt et al., 1995; Chen et al., 1997; Sanità 

di Toppi and Gabbrielli, 1999). 

The fact that root tissue contains a much higher 

concentration of heavy metals as well as of PCs than 

the leaf tissue points to the fact that metals are 

obviously immobilized to a far greater extent at the 

site of metal uptake. Binding of Cd to PCs in Betula 

pendula roots has been suggested as an explanation 

for tolerance to Cd toxicity in this tree. It is assumed 

that PCs participate in protecting the root against Cd 

interferences with growth, possibly by restricting 

Cd-induced changes in the nutrient composition of 

the plant (Gussarsson et al., 1996). 

PC is demonstrated as the major intracellular Cd 

chelator in the microalga, Chlamydomonas 

reinhardtii. From Cd challenged algal cells high 

molecular weight (HMW) and low molecular weight 

(LMW) complexes were purified and characterized 

and these complexes differed in accumulation 

kinetics, PC profile, acid labile sulfide content, and in 

vivo turnover rate. According to this investigation, the 

accumulation of LMW complex appeared to be an 

early sign of metal stress and HMW complex 

contributed to stable Cd sequestration. (Hu et al., 

2001). In another investigation with B. juncea, it is 

suggested that the high levels of sulfur uptake by this 

plant may play an important role, because Cd 

exposure and the resulting burst of PC synthesis have 

been reported to deplete GSH faster than biosynthesis 

can replenish it (Tukendorf and Rauser, 1990). 

Therefore, the production of PCs with their 

advantages in stability over the LMW complex, could 

contribute to higher metal tolerance by more effective 

sequestration (Speiser et al., 1992). 

In Ailanthus altissima roots, 200 mM Cd supply 

for 1 week induced three strongly bound slow 

migrating (HMW) PCs and five fast migrating 

(LMW) PCs which include a very fast migrating and 

strongly bound PC-Cd complex. Binding of purified 

PCs from the root extract to 109 Cd 2+ were observed 

through non-denaturing gel electrophoresis and direct 

autoradiography. The Mr of the Cd-binding proteins 

were found with the following pattern: 10.5, 18, 22, 

23.5, 26, 50, 65, 75 kDa. The heterogeneity of PC- 

Cds was probably due to the difference in the number 

of PC-Cd chains, chain lengths, molecular weights, 

S 2- amounts and number of Cd 2+ ion bounds. 

Tentatively, they were assumed to represent PCs of 

the type (γ-Glu-Cys)n-Gly where n=2-6 and may play 

a role in Cd-detoxification and in Cd-tolerance 

mechanism in this pollution resistant tree (Bayçu, 

1998). 

A number of observations indicate that synthesis 

of PCs in response to Cd exposure is essential for 

expression of tolerance to this metal. Mutants of 

S. pombe that are unable to synthesize PCs have 

increased sensitivity to Cd. Therefore by sequestering 

the metal in a less toxic form, PCs appear to play a 

critical role in the mechanism that protects cellular 

metabolism from damage caused by Cd. Increased Cd 

tolerance is associated with higher concentrations of 

PCs and accumulation of HMW PCs (Gupta and 

Goldsbrough, 1991). 

Regulation of PC biosynthesis 

1. PC Synthase 

PC synthesis is regulated at a number of levels, most 

importantly through the activation of PC synthase by 

various heavy metal ions. PC synthase is a 

cytoplasmic constitutive enzyme and its activity is the 

expected major determinant of the rate of PC 

synthesis (Grill et al., 1989; Chen et al., 1997; 

Cobbett, 2000). Kinetic studies using plant cell 

cultures exposed to Cd demonstrated that PC 

biosynthesis occurs within minutes of exposure to the 

heavy metal and is independent of de novo protein 

synthesis (Rauser, 1999). Likewise, in in vitro studies 

of PC synthase expressed in E. coli or in S. cerevisiae, 

the enzyme was activated to varying extents by Cd, 

Cu, Ag, Hg, Zn and Pb ions (Clemens et al., 1999; Ha 

et al., 1999; Vatamaniuk et al., 2000). The mechanism 

by which PC synthase is activated appears to be 

relatively non-specific with respect to the activating 

metal ion, although some metals are more effective 

than the others. It is suggested that the conserved 

amino-terminal domain confers the catalytic activity

of this enzyme and the carboxy-terminal domain acts 

as a local sensor by binding heavy-metal ions 

(presumably via the multiple Cys residues) and 

bringing them into contact with the activation site in 

the amino-terminal catalytic domain. Activation of 

the enzyme probably arises from an interaction 

between residues and free metal ions or metal-GSH 

complexes (Cobbett, 2000). 

In a short-term treatment of potato tuber (Solanum 

tuberosum L.) discs with CdCl2 the induction of PC 

synthase biosynthesis was detected. The intensity of 

this process was found depended on the concentration 

of Cd ions (0.01 - 1 mmol·dm -3 ), exposure time and 

Cd resistance of tissues. In more resistant tissues, PC 

synthase activity was found much higher and PC 

synthase was more resistant to oxidative stress. It is 

suggested that these tissues possessed more efficient 

Cd detoxification system (Stroinski and Zielezinski, 

2001). 

Suspension-cultured cells of azuki bean (Vigna 

angularis) as well as the original root tissues were found 

hypersensitive to Cd (


plants and fungi (Cobbett et al., 1998; Vatamaniuk et 

al., 1999). Experiments with CAD1 mutants of 

A. thaliana conducted by Howden et al. (1995) 

proved the function of PCs in inactivation of heavy 

metals. It was found that these CAD1 mutants, which 

are unable to synthesize PCs, exhibited extreme 

sensitivity to Cd (Piechalak et al., 2002). As indicated 

by the hypersensitivity of PC-deficient Arabidopsis 

CAD1 mutants to Cd (Howden et al., 1995), PC 

synthase genes conribute most markedly to Cd 

detoxification in planta (Cobbett et al., 1998; 

Vatamaniuk et al., 1999). 

In another investigation with A. thaliana including 

a PC-deficient mutant, CAD1-3, and the wild type 

plants, the potential effect of prior exposure to 

different Cd concentrations on Cd uptake and 

accumulation is tested. Plants were grown for 1 week 

in nutrient solution containing different Cd 

concentrations (0.05-1.0 µM Cd(NO3)2), and 

thereafter they were subjected to 0.5 µM Cd labelled 

with 109 Cd for 2 h. According to these results, it has 

been shown that the PC-deficient mutant CAD1-3, 

accumulates less Cd than the wild type (Larsson et al., 

2002). The possibility that the differences in Cd 

accumulation in mutant and wild-type lines may be 

due to the cytosolic Cd regulation, which is inhibited 

by the complexation of Cd by PCs (Speiser et al., 

1992). 

Can PCs have a role in Cd detoxification at levels 

of Cd exposure relevant to plants in a natural 

environment? It has been estimated that solutions of 

non-polluted soils contain Cd concentrations of less 

than 0.3 µM (Wagner, 1993). Nonetheless, the 

sensitivity of the Arabidopsis CAD1-3 mutant to 

concentrations of Cd as low as 0.6 µM (Howden et 

al., 1995) suggests that PCs may have a role in 

heavy-metal detoxification in an unpolluted 

environment (Cobbett, 2000). 

PC synthase genes and Cd-detoxification in 

animals 

Cd, which is an environmental pollutant with 

well-known mutagenic, carcinogenic, and teratogenic 

effects is known to accumulate in the human kidney 

for a relatively long time and at high doses and it is 

also known to have harmful effects on the respiratory 

system and has been associated with bone disease. At 

the molecular and cellular levels, a lot of studies on 

apoptosis and stress kinase activation of Cd have been 

performed (Takagi et al., 2002). 

Although PCs have not yet been detected in 

animal species, unexpectedly genes with similar 

sequences to those encoding PC synthase, for 

example a gene similar to AtPCS1 found in 

A. thaliana have been identified in the nematode, 

Caenorhabditis elegans (Cobbett, 2000; Takagi et al., 

2002). The amino-terminal region of the predicted 

gene product is equally similar to the plant and yeast 

proteins. In contrast, the carboxy-terminal domain has 

little obvious similarity to the plant or yeast 

gene-products, except that it contains multiple pairs 

of Cys residues. The existence of PC-synthase-like 

genes in animals suggests that PCs play a wider role 

in heavy-metal detoxification than previously 

expected (Cobbett, 2000). 

Mammalian cells can not synthesize PC because 

of their lack of the key enzymes for PC biosynthesis, 

PC synthase. To determine whether or not PC can be 

synthesized in mammalian cells with the introduction 

of the PC synthase gene (AtPCS1) from A. thaliana 

the gene AtPCS1 gene was amplified by PCR. The 

apoptotic cell death caused by Cd were examined and 

the effects of PC on the detoxification of Cd were 

studied by attempting to express these plant-specific 

peptides (PCs) in mammalian cells by transfecting 

Jurkat cells with AtPCS1 (Takagi et al., 2002). With 

the addition of the chemically synthesized PC7 to the 

culture medium containing 10-100 µM CdCl2 the 

survival ratio of Jurkat CAGGS cells clearly 

recovered. In the case of Jurkat PCS cells treated with 

20, PC synthesis corresponding to PC2-PC6 could be 

clearly observed indicating that the PC synthase gene 

from A. thaliana could catalyze PC synthesis even in 

Jurkat cells. Indeed, the activity of the PC synthase in 

these cells was also stimulated by Cd. Therefore, not 

only the catalytic activity but also the activation 

mechanism of the plant PC synthase was functional in 

the mammalian cells. It has been found interesting 

that the plant AtPCS1 gene can be utilized to enhance 

the resistance to Cd toxicity of mammalian cells 

(Takagi et al., 2002). 

Conclusions 

Higher plants like other organisms are believed to 

possess intracellular metal buffer systems, i.e. metal 

chelating subtances, which serve to keep the

intracellular availability of essential metals within 

certain limits. They may as well serve to reduce the 

availability of nonessential metals. The capacity of 

these systems can be varied by de novo synthesis of 

the chelating compounds. The heavy metal-PC 

complexes may also be relevant for animal and 

human nutritional studies since these complexes 

reflect the state of heavy metal chelation within these 

plants (Gekeler et al.,1989). The "phytochelatin 

response" or synthesis of Cd-binding polypeptides, is 

one of the few examples in plant stress biology in 

which it can be readily demonstrated that the stress 

response (PC synthesis) is truly an adaptive stress 

response (Steffens, 1990). 

It has been shown that the energy necessary for PC 

production is considerable: since PCs derive from 

GSH, Cd-stressed cells have to restore the GSH used 

to form them, by activating the enzymes catalyzing 

GSH biosynthesis (Sanità di Toppi and Gabbrielli, 

1999). However as discussed by Ow (1996), other 

factors can be more important than the PC level for 

efficient Cd detoxification, such as a PC reductase 

enzyme which seems to be essential to guarantee 

sufficient reducing power to prevent the oxidation of 

Cd-induced PCs and ineffective Cd-binding. Also in 

higher plants high levels of PCs may not be sufficient 

for complete Cd detoxification. The rapid formation 

of HMW complex, highly stabilized by S 2- groups, 

seems to particularly decisive in Cd detoxification 

(Speiser et al., 1992; Zenk, 1996). 

Because of their ability to bind heavy metal ions, 

PCs are considered to play a role in cellular metal 

homeostasis and metal detoxification (Grill et al., 

1988; Rauser, 1990; Steffens, 1990). Moreover, it is 

suggested that PCs are involved in differential heavy 

metal tolerance, i.e. naturally or artificially selected 

intraspesific heritable differences in the ability to 

tolerate high levels of metal exposure. Increased 

metal tolerance could involve increased or 

accelerated production of PCs or the formation of 

more stable metal-PC complexes due to either an 

increase in the PC chain length or an increase in the 

incorporation of labile sulfide into the complex 

(Rauser, 1995). 

It is possible that the plants can cope effectively 

with Cd stress by means of the mechanisms of 

avoidance, detoxification and repair which are 

provided by PCs and vacuolar compartmentalizationi.e. 

amount of proteins, rapidity in HMW formation, 

number of γ-Glu-Cys units, high incorporation of 


S 2- , level of reduction of PCs. If Cd levels are low in 

the soil or culture medium but the exposure time is 

long, a general cellular homeostatic process may be 

the plant management to chronic Cd stress (Sanità di 

Toppi and Gabbrielli, 1999). If Cd levels are high or 

very high and the exposure time is short, plant can 

manage this acute Cd stress by detoxification 

(constitutive response) and repair. Cd tolerance 

(adaptive response), which is mostly observed in high 

concentrations of Cd with a long exposure time, in 

higher plants should be defined as the natural or 

artificially given capacity regulated by interacting 

genetic and environmental factors, thus the 

development of tolerance should be a long-term 

process (Sanità di Toppi and Gabbrielli, 1999). The 

mechanisms required to adapt to highly contaminated 

environments may involve just one of these processes 

(Meharg, 1994). 

Molecular genetic approaches have brought 

important advances in our understanding of PC 

biosynthesis (Clemens, 2001). The identification of 

PC-synthase genes from plants and other organisms 

and also the possibility to utilize the plant genes in 

mammalian cells to enhance the resistance to Cd 

toxicity by PC activity, is a significant breakthrough 

that will lead to a better understanding of the 

regulation of a critical step in PC biosynthesis. 

Nonetheless, we must keep in mind the numerous 

other aspects of PC biosynthesis and function, and the 

ways in which they, too, are regulated at a cellular and 

physiological level in response to heavy-metal 

exposure. These include aspects of sulfur 

assimilation, GSH and sulphide biosynthesis, PC 

compartmentalization and the signal pathways 

through which metal toxicity leads to gene regulation 

(Cobbett, 2000, 2001). 

Increasing pollution of the environment caused by 

heavy metals is becoming a significant problem in the 

modern world. The use of plants and the concept of 

phytoremediation of contaminated soils has been 

increasingly supported by research in recent years 

(Salt et al., 1998; Cobbett, 2000; Rennenberg and 

Will, 2000). To investigate the heavy-metal 

detoxification processes will allow us to explore the 

mechanisms by which some species are capable of 

hyper-accumulation of metals such as Cd and how 

they may be best used for phytoremediation. 

Understanding the genetic and molecular basis of 

such mechanisms is an important goal in developing 

plants for the phytoremediation of contaminated 

environments.


References 

Abrahamson SL, Speiser DM and Ow DW. A gel 

electrophoresis assay for phytochelatins. Anal 

Biochem. 200: 239-243, 1992. 

Bayçu G. Cadmium tolerance and cadmium-binding 

polypeptides in Ailanthus altissima. In: Progress in 

Botanical Research. Tsekos I and Moustakas M (Eds). 

Kluwer Academic Publishers, London. 1998. 

Chardonnens AN, Tenbookum WM, Kuijper LDJ, Verkleij 

JAC and Ernst WHO. Distribution of cadmium in 

leaves of cadmium tolerant and sensitive ecotypes of 

Silene vulgaris. Physiol Plant. 104: 75-80, 1998. 

Chen J, Zhou J and Goldsbrough PB. Characterisation of 

phytochelatin synthase from tomato. Physiol Plantarum. 

101 : 165-172, 1997. 

Clemens S. Molecular mechanisms of plant metal tolerance 

and homeostasis. Planta. 212: 475-486, 2001. 

Clemens S, Kim EJ, Neumann D and Schroeder JI. 

Tolerance to toxic metals by a gene family of 

phytochelatin synthases from plants and yeast. 

EMBO J. 18: 3325-3333, 1999. 

Cobbett CS, May MJ, Howden R and Rolls B. The 

glutathione-deficient, cadmium-sensitive mutant, 

cad2-1, of Arabidopsis thaliana is deficient in 

g-glutamylcysteine synthetase. Plant J. 16: 73-78, 1998. 

Cobbett CS. Phytochelatin biosynthesis and function in 

heavy-metal detoxification. Curr Opin Plant Biol. 

3: 211-216, 2000. 

Cobbett CS. Heavy metal detoxification in plants: 

phytochelatin biosynthesis and function. IUBMB Life. 

51: 183-188, 2001. 

De Knecht J, van Dillen M, Koevoets PLM, Schat H, 

Verkleij JAC and Ernst WHO. Phytochelatins in 

cadmium-sensitive and cadmium-tolerant Silene 

vulgaris. Plant Physiol. 104: 255-261, 1994. 

Ernst WHO, Schat H and Verkleij JAC. Evolutionary 

biology of metal resistance in Silene vulgaris. Evol 

Trends in Plants. 4:45-51, 1990. 

Gekeler W, Grill E, Winnacker EL and Zenk MH. Survey of 

the plant kingdom for the ability to bind heavy metals 

through phytochelatins. Z Naturforsch. 44: 361-369, 

1989. 

Grill E, Winnacker EL and Zenk MH. Phytochelatins: The 

principal heavy-metal complexing peptides of higher 

plants. Science. 230: 674-676, 1985. 

Grill E, Winnacker EL and Zenk MH. Occurence of heavy 

metal binding phytochelatins in plants growing in a 

mining refuse area. Experientia. 44: 539-540, 1988. 

Grill E, Loffler S, Winnacker EL and Zenk MH. 

Phytochelatins, the heavy-metal-binding peptides of 

plants, are synthesized from glutathione by a specific 

γ-glutamylcysteine dipeptidyl transpeptidase 

(phytochelatin synthase). Proc Natl Acad Sci USA. 86: 

6838-6842, 1989. 

Grill E, Loffler S, Winnacker EL and Zenk MH. 

Phytochelatins. In: Methods in Enzymology. 

Metallobiochemistry, Part B Metallothionein and 

Related Molecules. Riordan JF and Vallee BL (Eds). 

Academic Press, San Diego. 205: 333-341, 1991. 

Gupta SC and Goldsbrough PB. Phytochelatin 

accumulation and cadmium tolerance in selected tomato 

cell lines. Plant Physiol. 97:306-312, 1991. 

Gussarsson M, Asp H, Adalsteinsson S and Jensen P. 

Enhancement of cadmium effects on growth and 

nutrient composition of birch (Betula pendula) by 

buthionine sulphoximine (BSO). J Exp Botany. 

47: 211-215, 1996. 

Ha SB, Smith AP, Howden R, Dietrich WM, Bugg S, 

O'Connell MJ, Goldsbrough PB and Cobbett CS. 

Phytochelatin synthase genes from Arabidopsis and the 

yeast, Schizosaccharomyces pombe. Plant Cell. 

11: 1153-1164, 1999. 

Hall JL. Cellular mechanisms for heavy metal 

detoxification and tolerance. J Exp Botany. 

53: 1-11, 2002. 

Howden R, Goldsbrough PB, Andersen CR and Cobbett 

CS. Cadmium-sensitive, cad1, mutants of Arabidopsis 

thaliana are phytochelatin deficient. Plant Physiol. 

107: 1059-1066, 1995. 

Hu S, Lau KWK and Wu M. Cadmium sequestration in 

Chlamydomonas reinhardtii. Plant Sci. 161: 987-996, 

2001. 

Inouhe M, Ninomiya S, Tohoyama H, Joho M and 

Murayama T. Different characteristics of roots in the 

cadmium-tolerance Cd-binding complex formation 

between mono - and dicotyledonous plants. J Plant Res. 

107:201-207. Plant Physiol. 123: 1029-1036, 1994. 

Inouhe M, Ito R, Ito S, Sasada N, Tohoyama H and 

Joho M. Azuki bean cells are hypersensitive to 

cadmium and do not synthesize phytochelatins. 

Plant Physiol. 123: 1029-1036, 2000. 

Klapheck S. Homoglutathione: isolation, quantification and 

occurrence in legumes. Physiol Plantarum. 74: 727- 

732, 1988. 

Klapheck S, Schlunz S and Bergmann L. Synthesis of 

phytochelatins and homo-phytochelatins in Pisum 

sativum L. Plant Physiol. 107: 515-521, 1995. 

Larsson EH, Asp H and Bornman JF. Influence of prior 

Cd 2+ exposure on the uptake of Cd 2+ and other 

elements in the phytochelatin-deficient mutant, cad1-3, 

of Arabidopsis thaliana. J Exp Botany. 53: 447-453, 

2002. 

Leita L, Contin M and Maggioni A. Distribution of 

cadmium and induced Cd-bindind proteins in roots, 

stems and leaves of Phaseolus vulgaris. Plant Science. 

77:139-147, 1991. 

Leopold I, Gunther D, Schmit J and Neumann D. 

Phytochelatins and heavy metal tolerance. 

Phytochemistry. 50:1323-1328, 1999. 

Levitt J. Responses of Plants to Environmental Stresses. 

Vol. 1, Academic Press, New York. 1980. 

Lombi FJ, Zhao, SJ, Dunham S and McGrath SP. 

Cadmium accumulation in populations of Thlaspi

caerulescens and Thlaspi goesingense. New Phytol. 

145: 11-20, 2000. 

Manunza B, Deiana S, Pintore M, Solinas V and 

Gessa C. The complexes of cadmium with 

phytochelatins. A quantum mechanics study. 

http://antas.agraria.uniss.it/electronic_papers/eccc4 

/phytoc/Cdp4a.pdb. 1997. 

Margoshes M and Vallee BL. A cadmium protein from 

equine kidney cortex. J Am Chem Soc. 79: 4813-4814, 

1957. 

Meharg AA. Integrated tolerance mechanisms: constitutive 

and adaptive plant responses to elevated metal 

concentrations in the environment. Plant Cell Environ. 

17:989-993, 1994. 

Neumann G, Lichtenberger O, Günther D, Tschierch K 

and Nover L. Heat-shock proteins induce heavy-metal 

tolerance in higher plants. Planta. 194: 360-367, 1994. 

Nussbaum S, Schmutz D and Brunold C. Regulation of 

assimilatory sulfate reduction by cadmium in Zea mays 

L. Plant Physiol. 88:1407-1410, 1988. 

Ortiz DF, Ruscitti T, McCue KF and Ow DW. Transport of 

metal-binding peptides by HMT1, a fission yeast 

ABC-type vacuolar membrane protein. J Biol Chem. 

270: 4721-4728, 1995. 

Ow DW. Heavy metal tolerance genes:prospective tools for 

bioremediations. Res Concerv Recycl. 18:135-149,1996. 

Page AL, Bingham FT and Chang AC. Cadmium. In: Effect 

of Heavy Metal Pollution on Plants. Lepp NW (Ed). 

Applied Science Publishers, London. 1981. 

Piechalak A, Tomaszewska B, Baralkiewicz D and Malecka 

A. Accumulation and detoxification of lead ions in 

legumes. Phytochemistry. in press. 

Raskin I, Smith RD and Salt DE. Phytoremediation of 

metals: using plants to remove pollutants from 

environment. Curr Opin Biotechnol. 8:221-226, 1997. 

Rauser WE. Phytochelatins. Ann Rev Biochem. 

59: 61-86, 1990. 

Rauser WE. Cadmium-binding peptides from plants. In: 

Methods in Enzymology. Metallobiochemistry, Part B 

Metallothionein and Related Molecules. Riordan JF and 

Vallee BL (Eds). Academic Press, San Diego. 

205: 319-333, 1991. 

Rauser WE. Phytochelatins and related peptides. Plant 

Physiol. 109: 1141-1149, 1995. 

Rauser WE. Structure and function of metal chelators 

produced by plants; the case for organic acids, amino 

acids, phytin and metallothioneins. Cell Biochem 

Biophys. 31: 19-48, 1999. 

Rennenberg H and Will B. Phytochelatin production and 

cadmium accumulation in transgenic poplar (Populus 

tremula x Populus alba). In: Sulfur Nutrition and Sulfur 

Assimilation in Higher Plants. Brunold C et al. (Eds). 

Paul Haupt, Bern. 393-398, 2000. 

Robinson NJ. Metal-binding polypeptides in plants. 

In: Heavy Metal Tolerance in Plants, Evolutionary 

Aspects. Shaw AJ (Ed). CRC Press, Boca Raton. 

195-214, 1990. 


Salt DE, Prince RC, Pickering IJ and Raskin I. Mechanisms 

of cadmium mobility and accumulation in Indian 

mustard. Plant Physiol. 109: 1427-1433, 1995. 

Salt DE, Pickering IJ, Prince RC, Gleba D, Dushenkov S, 

Smith RD and Raskin I. Metal accumulation in 

aquacultured seedlings of Indian mustard. Environ Sci 

Technol. 31: 1631-1644, 1997. 

Salt DE, Smith RD and Raskin I. Phytoremediation. Ann 

Rev Plant Physiol Plant Mol Biol. 49: 643-668, 1998. 

Sanità di Toppi L and Gabbrielli R. Response to cadmium 

in higher plants. Environ Exp Bot. 41: 105-130, 1999. 

Satofuka H, Fukui T, Takagi M, Atomi H and Imanaka 

T. Metal-binding properties of phytochelatin-related 

peptides. J Inorg Biochem. 86: 595-602, 2001. 

Speiser DM, Abrahamson SL, Banuelos G and Ow DW. 

Brassica juncea produces a phytochelatin-cadmiumsulfide 

complex. Plant Physiol. 99: 817-821, 1992. 

Steffens JC. The heavy- metal binding peptides of plants. 

Ann. Rev Plant Physiol Plant Mol Biol. 41: 553-575, 

1990. 

Steffens SJ and Bagchi D. Oxidative mechanisms in the 

toxicity of metal ions. Free Radic Biol Med. 

18: 321-336, 1995. 

Stroinski A and Zielezinska M. Cadmium and oxidative 

stress influence on phytochelatin synthase activity in 

potato tuber. Acta Physiol Plant. 23:157-160, 2001. 

Takagi M, Satofuka H, Amano S, Mizuno H, Eguchi Y, 

Kazumasa H, Kazuhisa M, Fukui K and Imanaka T. 

Cellular toxicity of cadmium ions and their 

detoxification by heavy metal spesific plant peptides, 

phytochelatins, expressed in mammalian cells. 

J Biochem. 131: 233-239, 2002. 

Tomsett AB and Thurman DA. Molecular biology of metal 

tolerances in plants. Plant Cell Environ. 11: 313-394, 

1988. 

Tukendorf A and Rauser WE. Changes in glutathione and 

phytochelatins in roots of maize seedlings exposed to 

cadmium. Plant Science. 70: 155-166, 1990. 

Vatamaniuk OK, Mari S, Lu YP and Rea PA. AtPCS1, 

a phytochelatin synthase from Arabidopsis: isolation 

and in vitro reconstitution. Proc Natl Acad Sci USA. 

96: 7110-7115, 1999. 

Vatamaniuk OK, Mari S, Lu YP and Rea PA. Mechanism 

of heavy metal ion activation of phytochelatin synthase. 

J Biol Chem. 275: 31451-31459, 2000. 

Vögeli-Lange R and Wagner GJ. Subcellular localization of 

cadmium and cadmium-binding peptides in tobacco 

leaves. Plant Physiol. 92: 1086-1093, 1990. 

Wagner GJ. Accumulation of cadmium in crop plants and its 

consequences to human health. Adv Agron. 

51: 173-212, 1993. 

Zenk MH. Heavy metal detoxification in higher plants-a 

review. Gene. 179:21-30, 1996. 

Zhu YL, Pilon-Smits EAH, Jouanin L and Terry N. 

Overexpression of glutathione synthetase in Indian 

mustard enhances cadmium accumulation and 

tolerance. Plant Physiol. 119: 73-79, 1999.




Mustafa Yatin 

Harvard Medical School, Massachusetts General Hospital, Department of Radiology, Division of 

Nuclear Medicine, Boston MA, 02114, USA 

Received 30 June 2002; Accepted 10 July 2002 

Abstract 

Natural polyamines, putrescine, spermidine and spermine are ubiquitous cell components essential for normal 

cellular functions and growth. Chemically these compounds are very simple organic aliphatic cations and fully 

protonated under physiological conditions. There is a strong correlation between proliferation rate of the cells and 

their polyamine contents. Adjustments of intracellular concentrations of polyamines to physiological requirements 

are orchestrated by de novo synthesis, polyamine uptake and catabolic reactions. De novo synthesis can in principle 

be substituted by polyamine uptake from extracellular environment. Over accumulation of polyamines is controlled 

by release and by a feedback regulation system that involves synthesis of a protein, antizyme that leads to 

degradation of ornithine decarboxylase and repression of polyamine uptake. The development of specific 

polyamine biosynthesis inhibitors and structural analogues of polyamines have revealed that maintaining 

polyamine levels are a prerequisite for animal cell proliferation to occur. The interruption of polyamine 

biosynthesis or minimizing the uptake of exogenous polyamines via the polyamine transport system offers 

meaningful targets for treatment of certain hyperproliferative diseases, most notably cancer. The polyamines 

influence confusingly large number biological processes, yet despite several decades of intensive research work, 

their exact functions in living organisms remains obscure. In this review, the current state of scientific knowledge 

regarding polyamines, their functions and their metabolism in mammalian cells is presented. 

KKeeyy wwoorrddss:: Polyamine, ODC, AdoMetDC, polyamine analogues, cancer 


Özet 

Do¤al poliaminler putresin, spermidin ve spermin hücrenin normal ifllev ve büyümesi için esas olan yayg›n 

hücresel bilefliklerdir. Bu bileflikler kimyasal yönden çok basit organik alifatik katyonlard›r ve fizyolojik koflullarda 

tamamen protonlanm›fl durumdad›rlar. Hücrenin ço¤alma h›z› ile poliamin içeri¤i aras›nda çok yak›n bir iliflki 

vard›r. Poliamin konsantrasyonlar›n›n fizyolojik ihtiyaca göre ayarlanmas› yeni sentez, poliamin al›nmas› ve 

katabolik reaksiyonlar aras›ndaki uyumlulu¤a ba¤l›d›r. Yeni sentez prensip olarak hücre d›fl› ortam›ndan poliamin 

al›nmas› ile sa¤lan›r. Poliaminlerin fazla birikimi, sal›nmas› ve protein sentezi, ornitin dekarboksilaz y›k›m›na 

neden olan antizim ve poliamin al›m›n›n bask›lanmas›n› içeren feedback regulasyon sistemi taraf›ndan kontrol 

edilir. Spesifik poliamin biyosentez inhibitörlerinin ve poliaminlerin yap›sal analoglar›n›n geliflimi, hayvan hücre 

ço¤almas›n›n meydana gelmesinin poliamin düzeyine ba¤l› oldu¤unu ortaya koymufltur. Poliamin biyosentezinin 

kesintiye u¤rat›lmas› veya poliamin tafl›nma sistemi vas›tas› ile d›fl ortamdan poliaminlerin al›nmas›n›n azalt›lmas›, 

çok h›zl› hücre ço¤almas›n›n söz konusu oldu¤u belirli hastal›klar›n, en önemlisi kanserin, tedavisinde anlaml› 

sonuçlar vermifltir. Poliaminler çok fazla say›daki biyolojik olaylar› etkiler, onlarca y›ld›r yap›lan yo¤un 

araflt›rmalara ra¤men, canl› organizmalardaki tam ifllevleri hala aç›k de¤ildir. Bu derlemede poliaminlerle ilgili son 

bilimsel veriler, ifllevleri ve memeli hücrelerindeki metabolizmalar› sunulmufltur. 

AAnnaahhttaarr ssöözzccüükklleerr:: Poliamin, ODC, AdoMetDC, poliamin analoglar›, kanser 

57

58 Mustafa Yatin 

What are polyamines? 

The natural polyamines (PA), putrescine (Put: 1,4diaminobutane), 

spermidine (Spd: N-(3 aminopropyl)- 

1,4-butadiamine) and spermine (Spm: N,N’-bis 

(3-aminopropyl-1,4-butanediamine), are very simple 

aliphatic multivalent cations with a primary amine 

functional group that are fully protonated under 

physiological conditions as essential constituents of 

all mammalian cells. One or more of these 

compounds are present in every living cell. All 

prokaryotic and eukaryotic cells synthesize Put and 

Spd. Spm synthesis is, however, is largely confined to 

nucleated eukaryotic cells. There are important 

interspecies differences in polyamine metabolism, 

especially between eukaryotic cells, plants, and some 

bacteria and protozoa. In some prokaryotes, only Put 

and Spd are synthesized, while in other cases, such as 

certain thermophilic bacteria, polyamines with chains 

longer than Spm are found. Additionally, in some 

parasitic organisms, there exist additional enzymes 

that are not present in the host cells and, as such, 

provide a target for the design of specific antiparasitic 

agents. In general, prokaryotes have higher 

concentration of Put than Spd and lack Spm. 

Eukaryotes, on the other hand, usually have little Put, 

but high concentrations of Spd and Spm (Thomas and 

Thomas, 2001; Marton and Pegg, 1995). Historically, 

the major pathway for the synthesis of Put and Spd 

was first established in microorganisms but was later 

found to be similar in animal cells (Tabor and Tabor, 

1984). 

Polyamine functions 

The interesting names putrescine refers to 

grow-rotten, the decomposition of organic matter by 

bacteria and fungi which results in intense odorous 

products, and spermidine and spermine recall the 

historical discoveries of these compounds in 

putrefying meat and seminal fluid. It has been 

reported that, rats immediately bury the dead rat 

bodies when in natural decay process bacterial 

by-products has perfumed the cadaver with Put; the 

same rats also bury wood rat toys that are sprinkled 

with Put, indicating olfactory detection of Put is 

sufficient enough to trigger this behavior (Coffino, 

2001; Pinel et al., 1981). Spm was first PA observed 

in human seminal fluids in 1678 by Leeuwenhoek A. 

van (1632-1723, extraordinary Dutch scientist who 

first ever recorded microscopic observations on living 

bacteria on plaques from teeth of two old man who 

had never cleaned their teeth in their entire lives) 

(http://www.ucmp.berkeley.edu/history/leeuwenhoek. 

html). Spm re-discovered several times during the 

next 200 years before the chemical structure 

[NH2(CH2)3HN(CH2)4NH(CH2)3NH2] was finally 

confirmed by its synthesis in 1962. Until recent 

decades, relatively few references could be found for 

these compounds in biochemical literature, indeed, in 

1950, there were only two citations to Spd and four 

citations to Spm in chemical abstracts. In contrast to 

only six citations in 1950, in the period 1988 to 2001 

there were 3500 citations to Spd and 2250 citations to 

for Spm in MedLine. During the last few decades 

there have been a large number of important studies in 

many laboratories (mostly in mammalian systems) 

indicating that PA are necessary requirements in 

rapidly growing normal and neoplastic cells. 

Paradoxically, although more than 300 years have 

elapsed since the first written document about the 

existence of spermine phosphate crystals in human 

semen no one can definitely describe the function of 

seminal spermine (Janne et al., 1991). 

In a universal classification, the PA belong to a 

broader group of biologically active amines together 

with the so-called biogenic amines such as serotonin, 

histamine, and tryptamine, which are monoamines 

having important physiological functions (Tabor and 

Tabor, 1984). Although the full repertoire of 

biological effects of PA are not fully known; they 

influence cellular processes at all stages from gene 

transcription to protein synthesis, and are central to 

regulation of cell growth and differentiation. There is 

a positive correlation between the proliferate activity 

of cells and their content and utilization of PA 

(Marton and Pegg, 1995; Pegg and McCann, 1994). 

Recent studies has revealed that PA levels are 

increased in both proliferating cells and extra-cellular 

tissue fluids under various inflammatory conditions, 

as consequence of excretion during tissue 

regeneration and release from damaged or dying cells. 

Increased levels of PA biosynthetic enzymes, 

polyamine uptake and thus elevated levels of PA have 

been demonstrated at highly profilerative neoplastic

cells (cancer), inflammatory sites of infection, 

trauma, neurodegenerative conditions and 

autoimmune diseases (Igarashi and Kashiawagi, 

2002; Zhang et al., 2000). 

As it is stated previously in this text, PA are fully 

protonated under physiological pH (7.2) and act as 

counter-ions for negative charges on RNA and DNA. 

Then, the question may be asked as whether all of the 

effects of polyamines in cell metabolism can be 

explained by simple cationic interactions with 

macromolecules. Polyamines regulate nucleic acid 

conformation in vitro and may have similar role in 

vivo. Why, if so, would the cells need to produce 

energetically very expensive polyamines through 

highly sophisticated pathways, when two Ca 2+ or two 

Mg 2+ would have the same number of positive 

charges? Possibly, the major advantage of the 

polyamine pathway is that cell can control both the 

synthetic production and degradation of the 

polyamines as needed independently of the 

availability from extracellular environment, a 

situation quite different from which is relevant to 

other cations having only extracellular origins. Unlike 

the point charges of Mg or Ca, the positive charge in 

PA distributed along the flexible carbon chain which 

may enable the PA uniquely to bridge critical 

distances. This unique molecular topography and 

distribution of positive charge in PA allow specific 

counter-ion interactions that neutralize the negative 

charges of phosphates in DNA helices (Thomas and 

Thomas, 2001; Vijayanathan et al., 2001; Feurerestein 

et al., 1991; Wang et al., 2001). PA’s high positive 

charge also prevents them from crossing biological 

membranes by simple diffusion (Bergeron et al., 

1995). Further progress in polyamine nucleic acid 

interaction in the regulation of transcription or 

synthesis depends on multidisciplinary studies 

involving cell biologists, biochemists, and physical 

and theoretical chemists. 

Polyamine metabolism 

Cellular polyamines accumulate via coordinated 

interactions between de novo synthesis and 

transmembrane uptake (Janne et al., 1991; Marton 

and Pegg, 1995; Seidenfeld, 1985; Quemener et al., 

1994). As shown in Figure 1, the complete 

Polyamines 59 

Figure 1: Polyamine biosynthesis. The natural PA in 

mammalian and plant cells are Put, Spd and Spm. Some 

microorganisms, including trypanasomes, contain only 

trace of Spm or may lack it completely. The four key 

enzymes making up the PA pathway in mammalian cells are 

ornithine decarboxylase (ODC) that forms Put from 

L-ornithine; s-adenosylmethionine decarboxylase 

(AdoMetDC) that forms decarboxylated (dcAdoMet), 

which act as an aminopropyl donor; spermidine synthase 

that transfers the aminopropyl group from dcAdoMet to 

putrescine; and spermine synthase that transfers the 

aminopropyl group from dcAdoMet to spermidine. In some 

plants and bacterial arginine decarboxylase (b-ADC) 

initiates an alternative two-step pathway to putrescine. The 

retroconversion of Spm back to Put can be accomplished 

by the sequential action of two enzymes, polyamine 

oxidase (PAO) and spermidine-spermine acetyltransferase 

(SSAT). SSAT-PAO pathway arranges PA pool 

composition and becomes particularly important in 

preventing PA levels from getting too high after excess 

synthesis or uptake as highyl inducible SSAT leads to a 

rapid conversion of PA to N 1 -acetylspermine (N-acetylSpm) 

and N 1 -acetylspermidine (N-acetylSpd), which are readily 

excreted from cells. 

metabolisms of the polyamines involve some seven 

enzyme reactions, each of which precisely regulated 

in order to maintain optimum intracellular 

concentrations in accordance with cellular needs. In 

addition, there are also polyamine transport processes 

both into (influx) and out (efflux) of the cell (Marton 

and Pegg, 1995; Pegg and McCann, 1994). Again, 

these polyamine accumulation activities are highly 

controlled with a strong link to the up and down


regulation of cell growth depending on need. The 

polyamine requirement of a given cell may be 

covered by de novo synthesis or by uptake from its 

environment. Under physiologic conditions, the 

relative importance of uptake and de novo synthesis is 

a cell-typic character (Marton and Pegg, 1995; Seiler 

and Dezeure, 1990; Seiler et al., 1990). The capacity 

of cells for de novo synthesis and uptake is an 

expression of their ability to adapt to environmental 

changes. 

The use of polyamine levels in body fluids as 

diagnostic markers or as indices of novel therapeutic 

effects has also been subject to extensive study but the 

results have, with a few exceptions, been 

disappointing. The extreme complexity of blood 

compartment carrying free polyamines in the plasma 

caused significant problems in the clinical 

interpretation of circulating PA levels. By the end of 

1970s the unclear potential role of PA as markers for 

cancer led to an almost total disinterest in their 

diagnostic use (Moulinoux et al., 1996). However, 

studies of polyamine metabolism in a number of 

pathogenic parasites (Seiler and Atanassov, 1994), 

inflammatory, infectious conditions (Yatin and 

Fischman, 2002) and under oxidative stress (Gilad 

and Gilad, 1999), have led to identification a number 

therapeutic targets and the development of novel 

chemotherapeutic agents (Pegg and McCann, 1994; 

Wallace and Morgan, 1990; Hebby, 1981; Seiler and 

Atanassov, 1994). Polyamines are also modulators of 

synaptic functions and play important roles in central 

nervous system (CNS) (Gilad and Gilad, 1999; Yatin 

et al., 2001; Williams, 1997). Some of the 

non-peptide venoms of spiders and wasps are natural 

polyamine analogues that are selective inhibitors of 

the glutamate receptors of the CNS (Nihei et al., 

2001; Palma et al., 1998; Albensi et al., 2000). 

Many plant compounds contain polyamine 

residues, including several families of alkaloids (Hoet 

and Nemery, 2000; Thomas and Thomas, 2001). 

Resveratrol, a natural polyphenolic phytoalexine, 

present abundantly in red wine, has been reported to 

be an anti-proliferative agent on human cancer cells, 

also caused significant decreases on ODC activity, 

indicating that PA might represent one of several 

beneficiary effects of moderate consumption of red 

wine (Nigdigar et al., 1998; Scheneider et al., 2000). 

Spm, exceptionally high in skin (human skin 

concentration - epidermis is 850 mg/g tissue 

compared to muscle - skeletal, 30 mg/g tissue), has 

been identified as a potent antioxidant and 

anti-inflammatory agent against UVB irradiation and 

oxidative stress and suggested to be an important 

anti-inflammatory antioxidant of epidermis. 

Currently, the use of Spm as a dermatologic 

antioxidant is under patent protection (Lovaas, 1995). 

Biosynthesis and regulation of cellular polyamines 

The biosynthesis pathway of polyamine accumulation 

in the cells is kinetically rate-limited by ornithine 

decarboxylase (ODC; EC 4.1.1.17), a pyridoxal 

phosphate dependent enzyme, which synthesize Put 

by decarboxylation of L-ornithine. The remarkable 

elevation in de novo polyamine biosynthesis rate that 

takes place in rapidly growing cells has led to much 

effort to develop therapeutically useful antineoplastic 

agents that would interfere with, or regulate, these 

processes in hyperproliferative diseases (Seiler and 

Atanassov, 1998). Investigation of several specific 

inhibitors of ODC (i.e. difluoromethylornithine, 

DFMO, a suicidal irreversible specific inhibitor of 

ODC) as an experimental antineoplastic strategy 

causes to reduction in total amount of cellular 

polyamines (except spermine) with only cytostatic 

effects. The lack of cytotoxicity may be due to 

polyamine interconversion from a spermine reservoir 

in the cells and/or polyamine repletion by 

transmembrane uptake from extracellular sources 

(Pegg and McCann, 1994; Hebby, 1981; Seiler and 

Atanassov, 1998). 

Many microorganisms and higher plants are able 

to biosynthesize Put from agmatine produced by 

decarboxylation of arginine, however all mammalian 

cells and many lower eukaryotes lack arginine 

decarboxylase (ADC), leaving the only route to 

produce Put to utilize the ODC. Ornithine is available 

for this enzymatic reaction from the circulating 

plasma and can also be formed by the action of 

arginase. Arginase is much more widely distributed 

than any other enzymes of the urea cycle in living 

organisms and present in extrahepatic tissues, and 

ensures the availability of ornithine in PA production 

line. In a broad sense, arginase can, therefore be 

assumed of as an initial step in the PA biosynthesis 

pathway. 

Ornithine decarboxylase was discovered in 1968 

simultaneously and independently in two laboratories 

in the United States and one in Finland (Janne et al.,

1991, Russel and Synder, 1968; Pegg and Williams, 

1968). Ornithine decarboxylase is a unique enzyme in 

many respects. First, it is one of the enzymes having 

extremely short life with a very rapid turnover rate 

(t1/2 is from 10 to 20 minutes) and it is present in very 

small amounts in normal growing cells. Its activity 

can be increased many folds within a few hours of 

exposure to trophic stimuli (Janne et al., 1991). Such 

stimuli include hormones, various drugs, tissue 

regeneration and growth factors commonly found in 

serum. Even after such stimulation, ODC remains 

only a very small fraction of the total cellular protein 

ranging from 0.01% of the cytosolic protein in 

androgen - stimulated mouse kidneys to 0.00012 % in 

thioacetamide stimulated rat liver. It turned out that 

by any definition this enzyme is a low abundance 

Polyamines 61 

Figure 2: Cellular PA pool is regulated by three influences: production, transport and catabolism. Antizyme has a negative 

feedback control on production and transport. Antizyme is a key factor in cellular PA homoestasis and its production depends 

on PA level. When cellular PA levels rise, +1 frame-shifting in AZ ribosomal mRNA occurs causing read-through of the 

internal stop codon to produce the full length antizyme protein. ODC is active and stable enzyme, if only, it forms a 

homodimer in trans form across the face of the active dimer. Higher affinity of AZ towards ODC, generates an AZ-ODC 

heterodimer exposing the C-terminus of ODC, which is then immediately recognized and degraded by the 26S proteosome. 

In contrast with all other cellular proteins degraded by this proteolytic pathway, the degradation of ODC is not triggered by 

ubiquitination. AZ also reversibly binds to some functional component of the cytoplasmic membrane PA uptake transporter and 

thereby preventing utilization of extracellular sources of PAs. AZ by itself can be bound by an AZ inhibitor, which is an 

ODC-like protein that does not posses ODC activity but which does bind AZ more strongly than the AZ-ODC complex, and so 

consequently sequesters the AZ protein. In this negative feedback loop; cellular PA rise to excessive amounts; PA induce more 

AZ; AZ inhibits and diminishes ODC and PA uptake; PA accumulation in cells decline. 

protein representing only about 3 ppm (part per 

million) of the soluble proteins of a mammalian cell. 

Second, treatment of cells with exogenously added 

polyamines cause negative feedback and result in a 

rapid and profound fall in enzymatic activity of ODC. 

Cannelakis (1989) found that the loss of ODC activity 

coincided with the appearance of another enzyme 

activity that was inhibitor of ODC. This activity was 

called anti-enzyme for ODC or more briefly antizyme 

(Coffino, 2001; Heller et al., 1976). The low 

abundance of both proteins presented tremendous 

challenge in studying their isolation and biochemical 

nature, physiological behavior and chemical 

mechanism of their interaction. Shin-Ichi Hayashi, 

addressed the problem by purifying ODC to 

homogeneity, a very difficult task at that time, to


show that pure ODC enzyme form stoichiometrically 

1:1 complex that is enzymatically inactive, but then 

can be reversed to dissociate to regenerate ODC 

activity (Figure 2) (Hayashi et al., 1996). The cell 

culture studies followed this study to come with 

finding that the antizyme:ODC ratio was strongly 

correlated with the degradation rate of ODC (Coffino, 

2001). 

Following Hayashi’s achievement of cloning of 

the antizyme cDNA and gene, it was promptly 

showed that transfection of cells to overexpression of 

antizyme significantly caused ODC activity and ODC 

protein to fall. The protease that is responsible from 

this destructive process was shown to be proteosome. 

This was later established definitely that antizyme 

promotes the destruction of ODC by the 26 S 

proteasome in an in vitro system (by using only 

purified components) (Murakami et al., 1992). The 

complete mechanism of antizyme action were then 

mechanistically confirmed with +1 translational 

frameshifting in antizyme mRNA by polyamines 

(Coffino, 2001). 

The next regulatory enzyme in the polyamine 

metabolic pathway is s-adenosyl-methionine 

decarboxylase (AdoMetDC; EC 4.1.1.50) that 

provides aminopropyl groups for the synthesis of 

higher polyamines, spermidine and spermine, from 

first polyamine putrescine. The aminopropyl moiety 

is derived from methionine, which is first converted 

into s-adenosylmethionine (AdoMet) and is then 

AdoMetDC produces decarboxylated adenosylmethione 

(dcAdoMet). The half life of AdoMetDC is relatively 

longer than that of ODC but is still only about 30 to 

60 min. The substrate AdoMet is an important methyl 

donor (for DNA methylation via methyl transferase) 

in eukaryatic cells and AdoMetDC act as a regulatory 

control point in AdoMet netabolism. dcAdoMet is 

essentially inactive as a methyl donor, so once 

AdoMetDC converts AdoMet to dcAdoMet, it is 

away from all other metobolic paths and can be 

utilized only in PA biosynthetic pathway (Stanley and 

Shantz, 1994). 

Since excessive PA accumulation in cells is 

harmful for normal cell function, both PA 

biosynthesis and PA transport are expected to be 

tightly feedback-regulated by a common mechanism 

(Morris, 1991). In cell culture studies, 

polyamine-deprived cells rapidly internalize 

exogenously administered PA until intracelleular PA 

levels are replenished fully, generally within 1-3 

hours of PA addition, and then uptake is abruptly 

terminated (He et al., 1994). The red flag signal for 

this feedback response requires active protein 

synthsesis and it is strongly correlated to presence of 

excess spermidine and spermine and several PA 

analogues. Negative regulation of PA transport by 

antizyme was demonsrated in ODC overproducing 

cells and DFMO treated hepatoma cells transfected 

with antizyme cDNA (He et al., 1994). Although 

substantial progress has been made in understanding 

feedback regulation of ODC, much less is known 

about antizyme mediated feedback repression of PA 

transport (Mitchell et al., 1994). 

Polyamine transport 

Although a tightly regulated biosynthetic pathway 

largely produces intracellular polyamines, a large 

number of cell types from different species (both 

prokaryotic and eukaryotic) have been shown to 

posses polyamine uptake system, which, under 

needed conditions, can substitute for de novo 

synthesis. In evolutionary terms, PA transport serves 

as an adaptational response of cells to changes in PA 

requirement. Cellular uptake mechanisms usually 

salvage polyamines from diet and intestinal 

microorganisms (Figure 3). In mammalian organism, 

PA are taken up from gastrointestinal tract, and 

released with the urine (Quemener et al., 1994). 

Transmembrane transport of extracellular 

polyamines can be enhanced by growth factors and 

hormones, as well as by inhibition of intracellular 

biosynthesis (i.e. ODC inhibition by DFMO) 

(Seidenfeld, 1985; Lessard et al., 1995; Byers and 

Pegg, 1989). The mechanisms by which polyamine 

uptake is induced are not clear, although it is known 

that uptake of polyamines is generally low in 

quiescent cells, in contrast to cells rapidly 

proliferating or in cells that have been induced to 

differentiate and PA utilization is greatly enhanced. 

As a general rule, increases in cellular growth is not 

only accompanied by enhanced rates of intracellular 

de novo synthesis, but also by enhanced rates of 

uptakes of exogenous PA (Seiler and Dezeure, 1990; 

Seiler et al., 1996). 

PA are incorporated by a process that is energy 

requiring, temperature-dependent, capable of 

accumulating against a substantial concentration 

gradient (not via a simple diffusion), saturable and

Figure 3: Several sources of PAs are available for rapidly 

growing cells in vivo. The endogenous sources arise from a 

highly regulated intracellular metabolism and the exogenous 

sources arise principally from the gastrontestinal (GI) tract. 

Intestinal and colonic mucosa absorb PA released from food 

and from microfloral biosynthesis as well. PA 

originating from different source are finally transported by 

circulating blood, particullarly in red blood cells (RBC) in 

normal conditions or PA can be accumulated at situ under 

inflammatory conditions via migration of activated white 

blood cells (WBC). When needed, a membrane transport 

system allows the host cells to uptake PA from 

extracellular compartments. 

carrier-mediated (Seiler and Dezeure, 1990; Seiler et 

al., 1996; Aziz et al., 1994; Toursarkissian et al; 

1994). Although, not studied in most cell types, a 

human gene for polyamine transport has been 

expressed in polyamine deficient Chinese-hamster 

ovary (CHO) mutant cell line (Byers and Pegg, 1989; 

Hyvonen et al., 1994). Many cells posses single 

transmembrane carrier capable transporting Put, Spd 

and Spm. However, in one quarter of all cell types 

examined (reports from competition studies) suggests 

that more than one carrier (or in general terms, more 

than one transporter for polyamine uptake into the 

cells; one for Put only and one for Put, Spd and Spm) 

is present (Aziz et al., 1994; Toursarkissian et al; 

1994; Minchin et al., 1991). Different cells posses 

both saturable, and non-saturable uptake systems. 

Polyamine uptake by saturable systems is temperature 

dependent and carrier-mediated. The presence of 

unsaturable components in the polyamine transport 

system in some cell types has been reported, but the 

evidence is based on the results of statistical non-linear 

regression analysis and computer-fitting of the data to 

the appropriate equations (Minchin et al., 1991). 

In general, the affinity of the transporter carrier 

increases from Put, Spd and Spm. In all cell types 

investigated PA transport is independent and distinct 

from amino acid transport systems and in some cells 

it appears to be sodium-dependent (Palacin et al., 

1998). Although uptake is observed, in the absence of 

Na + , the exogenously increase of sodium in the 

medium to physiological concentrations (120 mM) 

usually increase the transport rate by 60%. 

Modulation of the Na-dependent portion by 

ionophores (gramicidin or monensin) inhibits 

sodium-dependent portion of PA uptake (Khan et al., 

1992). In addition, natural polyamines, Put, Spd, 

Spm, a large number of polyamine analogues can also 

be taken up by this system. This lack of specificity of 

polyamine uptake systems in the cells have been 

made possible exploitation of polyamine-like drugs 

for use in cancer chemotheraphy or in other 

chemotherapeutic approaches to various diseases 

(Kramer et al., 1993). 

The structural tolerances of the PA transport 

systems allowed the selection of cells resistant to the 

cytotoxic action of MGBG, which after limited 

mutagenesis had lost their ability to take up PA from 

the environment. Uptake-deficient mutants have 

widely been used to characterize the uptake systems 

or non-specific uptakes used by analogs with 

structural relationships to PA. One example to these 

uptake-deficient mutant CHO cells is CHO-MGBG 

cells. Studies with CHO-MGBG cells also revealed 

that PA uptake and export mediated by different 

transport systems (Put release was observed in 

uptake-deficient CHO-MGBG cells) (Hyvonen et al., 

1994). This may not necessarily imply that the 

transporter proteins for uptake and release are 

different. Uptake deficient mutants may not simply 

lack an active transporter protein, but another 

important constituent activating uptake part only 

(Seiler et al., 1996). 

ODC and proto-oncogenes 

Polyamines 63 

Various carcinogens, mitogen stimuli and tumor 

promoters may cause transient increases in ODC 

activity, while rapidly proliferating tissues, including 

tumors, activated macrophages and the cells of gut 

mucosa have constantly elevated ODC activities and


enhanced rates of PA uptakes that leads to elevated 

levels of PA. ODC also exhibits properties of 

oncogens, like c-myc, c-fos, c-jun, and expression of 

oncogens like src, neu, and Ha-ras result in 

significant increases of ODC activity. Similarly, 

inhibition of polyamine biosynthetic enzymes by 

specific inhibitors and depletion of PA levels is 

strongly associated with decreased transcription of 

the c-myc, c-fos and ODC gene. Inserting a partial 

cDNA coding for ODC under the control of a strong 

viral promoter and by transfecting the plasmid to cells 

caused to stable ODC over-expression (Thomas and 

Thomas, 2001; Janne et al., 1991; Marton and Pegg, 

1995). These transfected ODC over-expressed cells 

showed malignant transformation and upon 

inoculation to nude mice, they produced extensively 

vascularized, aggressive tumors (Thomas and 

Thomas, 2001). Thus, these evidences from in vitro 

and in vivo studies suggested that oncogenes may 

enhance the transcription of the ODC gene and certain 

oncogens induce ODC and enhance the formation of 

PA, and vice versa, PA induce expression of 

oncogens. 

Polyamine synthesis inhibitors 

Blockade of ODC enzyme activity by DFMO causes 

a time dependent decrease of the cellular Put level, 

followed by a decrease of Spd, in contrast, Spm 

concentrations are usually not much effected and even 

may increase. The decreases in Put concentration is 

obvious due to impairment of its producing enzyme 

ODC. The depletion of Spd has mainly three reasons: 

first, decreased formation due the limited availability 

of Put as substrate of Spd synthase. Second, dilution 

of PA pool due to cell division and decrease in the 

amount per cell. Third, enhanced formation of Spm 

due to elevation in AdoMetDC activity. The reason 

for the induction of AdoMetDC in DFMO treated 

cells (thus Spd depleted) is the unavailability of Put as 

substrate. This consequently causes to excessive 

availability of dcAdoMet and leads to accumulation 

of Spm in the cells. 

AdoMetDC is effectively inhibited by 

methylglyoxal bis (guanylhydrazone) (MGBG) and 

this drug can be also be to inhibit PA synthesis (to 

decline Spd and Spm levels) in vivo. However, 

MGBG is not specific to AdoMetDC, it also inhibits 

diamine oxidase (DAO) and has chemical structre 

with considerable resemblance to PA and is taken up 

by the same transport system as PA. Therefore, it is 

difficult to prove the antiproliferative effects of 

MGBG are due to PA depletion as its effects can be 

reversed by exogenous addition of Spd could simply 

be due to displacement of MGBG from intracellular 

sites by structuraly similar Spd or competitive 

interference with drug transport. Additionaly, 

therapeutic combination of MGBG with DFMO 

causes to abnormal accumulation in the cells and 

non-specific cytoxicities. In recent years, industrial 

research programs (Ciba-Geigy) developed structural 

derivatives of MGBG to minimize non-specific 

effects. The bicyclic analog of MGBG, 

4-(aminoiminomethyl)-2,3-dhydro-1 Hinden-1-onediaminomethylenehydrazone 

(CGP-48664) fulfilled 

this criterion, eliminating the severe toxicities 

associated with parent compound. Mutant cell lines 

lacking PA transport are resistant to MGBG, but they 

remain sensitive to CGP-48644 (Thomas and Thomas, 

2001). Thus, CGP-48644, not like MGBG, does not 

share the same uptake mechanism with PA, allowing 

combination with DFMO to deplete all three PA levels, 

has a much broader therapeutic window than MGBG. 

The back conversion of Spm to Spd and to Put is 

mediated by dual actions of N1-acetylation (SSAT) 

followed by oxidative removal acetamidopropanal by 

polyamine oxidase (PAO) to maintain the proper 

balance of PA pools to ensure cell growth (Figure 1). 

Although no inhibitor is developed to specifically 

inactivate SSAT, a series of compounds have been 

designed to inhibit PAO very potently and efficiently 

(Bolkenius et al., 1985). The most widely used of 

these inhibitors is the polyamine analogue, N1-N2-bis 

(2,3-butadienyl)-1,4-butanediamine (MDL-72527). 

Cells treated with MDL-72527 alone are not growth 

inhibited, indicating that the back conversion pathway 

is not a critical step for cell growth under normal 

conditions. However, when MDL-72527 was 

administered in combination with other PA enzyme 

inhibitors (i.e. DFMO) or PA analogues, a much greater 

depletion of PA pools were achieved and caused 

significant growth inhibition than either agent given 

alone (Claveria et al., 1987; Prakash et al., 1990). 

Polyamine analogues 

Porter and Bergeron(1988) were the first to suggest 

the use of polyamine analogues as a new approach in

chemotherapy and lead to a very large number of 

structural analogues and homologs with the general 

formula: R1-NH-(CH2)a-NH-(CH2)b-NH-(CH2)-NH- 

R2, where R1 and R2 are alkyl residues, and a and b is 

any integer number. In general, polyamine analogues 

competitively use the same transport system with PA 

to incorporate cells, interferes with PA metabolism, 

lacks the necessary physiological functions of PA in 

normal cellular functions, but suppress ODC and 

AdoMetDC activities and induce SSAT activities, 

leads to rapid depletion of PA levels (Bergeron et al., 

1997). 

Starting in mid 1980s efforts to synthesize and 

identify specific PA inhibitors have began in several 

academic and industrial laboratories. The structural 

characteristics of these PA transport inhibitors (i.e. 

compounds which deter or compete with natural PA 

for transport) have been analyzed by QSAR and by 

COMFA (comparative molecular field analysis) as 

well as simple charge to chain length correlation 

produced a preliminary theoretical predictive model 

to design new analogues (Xia et al., 1997; Li et al., 

1997; Burns et al., 2001). 

References 

Albensi BC, Alasti N and Mueller AL. Long-term 

potentiation of in the presence of NMDA receptor 

antagonist arylalkylamine spider toxins. J Neurosci Res. 

62: 177-185, 2000. 

Austin TX, Moulinoux JP, Quemener V, Delcros JG and 

Cippolla B. Circulating polyamines as biological 

markers for cancer. In: Polyamines in Cancer: Basic 

Mechanisms and Clinical Approaches. Nishioka K 

(Ed). Chapman Hill. 233-249, 1996. 

Aziz SM, Olson JW and Gillespie M. Multiple polyamine 

transport pathways in cultured pulmonary artery smooth 

muscle cells: Regulation by hypoxia. Am J Respir Cell 

Mol Biol. 10:160-166, 1994. 

Bergeron R J, Feng Y, Weimar WR, Mcmannis JS, Dimova 

H, Porter C, Raisler B and Phanstiel O. A comparison 

of structure-activity relationships between spermidine 

and spermine analogue antineoplastics. J Med Chem. 

40: 1475-1494, 1997. 

Bergeron RJ, McManis JS, Weimar WR, Scherier KM, 

Gao F, Wu Q, Ortiz-Ocasio J, Luchetta GR, Porter C 

and Vinson JRT. The role of charge in polyamine 

analogue recognition. J Med Chem. 38: 2278-2285, 1995. 

Bolkenius F, Bey P and Seiler N. Specific inhibition of 

polyamine oxidase in vivo is a method for the 

elucidation of its physiological role. Biochem Biophys 

Acta. 828: 69-76, 1985. 

Polyamines 65 

Burns MR, Carlson CL, Vanderwerf SM, Ziemer JR, 

Weeks RS, Cai F, Webb HK and Graminski GF. Amino 

acid/spermine conjugates: Polyamine amides as a 

potent spermidine uptake inhibitors. J Med Chem. 

44: 3632-3644, 2001. 

Byers TL and Pegg AE. Properties and physiological 

function of the polyamine transport system. 

Am J Physiol (Cell Physiol 26). 257: C545-C553, 1989. 

Canellakis E and Hayashi SI. Ornithine decarboxylase 

antizymes. In: Ornithine Decarboxylase Biology, 

Enzymology and Molecular Genetics. Shin-‹chi 

Hayashi (Ed). Pergamon Press. 1989. 

Claveria N, Wagner J and Knogden B. Inhibition of 

polyamine oxidase improves the antitumor effect of 

ornithine decarboxylase inhibitors. Cancer Res. 

7: 765-772, 1987. 

Coffino P. Regulation of cellular polyamines by antizyme. 

Nature Reviews Molecular Cell Biology. 2: 188-194, 2001. 

Coffino P. Antizyme, a mediator of ubiquitin-independent 

proteasomal degradation. Biochimie. 83: 319-323, 2001. 

Feuerestein BG, Williams LD, Basu HS and Marton LJ. 

Implications and concepts of polyamine - nucleic acid 

interactions. J Cell Biochem. 46: 37-47, 1991. 

Gilad GM and Gilad VH. Novel polyamine derivatives as 

neuroprotective agents. J Pharmacol Exp Therap. 

291: 39-43, 1999. 

Hayashi S and Murakami Y. Rapid and regulated 

degradation of ornithine decarboxylase. Biochem J. 

306: 1-10, 1995. 

Hayashi S, Murakami Y and Matsufuji S. Ornithine 

decarboxylase antizyme: A novel type regulatory 

protein. Trends Biochem Sci. 21: 27-30, 1996. 

He Y, Suzuki T, Kashiwagi K and Igarashi K. Antizyme 

delays the restoration by spermine of growth of 

polyamine-deficient cells through its negative 

regulation of polyamine transport. Biochem Biophys 

Res Com. 203:608-614, 1994. 

Hebby O. Role of Polyamines in the control of cell 

proliferation and differentiation. Differentiation. 

19: 1-20, 1981. 

Heller JS, Fong WF and Cannelakis ES. Induction of a 

protein inhibitor to ornithine decarboxylase by the end 

products of its reaction. Proc Natl Acad Sci USA. 

73: 1858-1862, 1976. 

Hoet PHM and Nemery B. Polyamines in the lung: 

polyamine uptake and polyamine-linked pathological or 

toxicological conditions. Am J Phsiol Lung Cell Mol 

Physiol. 278: L417-L433, 2000. 

http://www.ucmp.berkeley.edu/history/leeuwenhoek.html 

Hyvonen T, Seiler N and Persson L. Characterization of 

a COS cell line deficient polyamine transport. Biochim 

Biophys Acta. 1221, 279-285, 1994. 

Igarashi K and Kashiawagi K. Polyamines: Mysterious 

modulators of cellular functions. Bioche Biophys Res 

Com. 271: 559-564, 2000. 

Janne J, Alhonen L and Leinonen. Polyamines: From 

molecular biology to clinical applications. Trends in 

Mol Med. 23: 241-259, 1991.


Kramer DL, Miller JT, Bergeron Khomutov R, Khomutov 

A and Porter CW. Regulation of polyamine transport by 

polyamines and polyamine analogues. J Cell Physiol. 

155: 399-407, 1993. 

Khan N, Quemener V and Moulinoux JP. Phorbol esters 

augment polyamine transport by influencing Na + -K + 

pump in murine leukemia cells. Exp Cell Res. 199: 378- 

372, 1992. 

Lessard M, Zhao C, Singh SM and Poulin R. Hormonal and 

feedback regulation of putrescine and spermidine 

transport in human breast cancer cells. J Biol Chem. 

270: 1685-1694, 1995. 

Li Y, Mackerrel AD, Egorin MJ, Ballesteros MF, Rosen 

DM, Wu YY, Blamble DA and Callery PS. Comparative 

molecular field analysis-based predictive model of 

structure function of polyamine transport inhibitors in 

L1210 cells. Cancer Res. 57: 234-239, 1997. 

Lovaas E. Hypothesis: Spermine may be an important 

epidermial antioxidant. Medical Hypothesis. 45: 59-67, 

1995. 

Marton LJ and Pegg AE. Polyamines as targets for 

therapeutic intervention. Ann Rev Pharmacol Toxicol. 

35, 55-91, 1995. 

Minchin RF, Raso A, Martin RL and Ilett KF. Evidence for 

the existence distinct transporters for the polyamines 

putrescine, spermidine and spermine in B16 melanoma 

cells. Eur J Biochem. 200: 457-462, 1991. 

Mitchell AJLA, Diveley RR and Bareyal-Leyser A. 

Feedback repression of polyamine uptake into 

mammalian cells requires active protein synthesis. 

Biochem Biophys Res Com. 186: 81-88, 1992. 

Mitchell JL, Judd GG, Leyser-Bareyal A and Ling SY. 

Feedback repression of polyamine transport is mediated 

by antizyme in mammalian tissue culture cells. 

Biochem J. 299: 19-22, 1994. 

Murakami Y. Ornithine decarboxylase is degraded by the 

26 S proteasome without ubiquitination. Nature. 360: 

597-599, 1992. 

Morris DR. A new perspective on orninhine decarboxylase 

regulation: Prevention of polyamine toxicity is the 

overriding theme. J Cell Biochem. 46: 102-105, 1991. 

Nihei KI, Kato MJ, Yamane T, Palma MS and Konno K. 

An efficient and versatile syntheis of acylpolyamine 

spider toxins. Biorganic Med Chem Lett. 75: 431-436, 

2001. 

Nigdikar SV, Williams NR, Griffin BA and Howard AN. 

Consumption of red wine polyphenols reduces the 

susceptibility of low density lipoproteins to oxidation 

in vivo. Am J Clin Nutr. 68: 258-265, 1998. 

Palacin P, Estevez R, Bertran J and Zorzano A. Molecular 

biology of mammalian plasma membrane amino acid 

transporters. Physiological Reviews. 78: 969-1054, 1998. 

Palma MS, Itagaki Y, Fujiata T and Nakajima T. Structural 

characterization of a new acylpolyaminetoxin from the 

venom of Brazilian garden spider Nephilegenys 

Cruentata. Toxicol. 36. 485-493, 1998. 

Pegg AE and Williams-Ashman SH. Biosynthesis of 

putrescine in the prostate gland of the rat. Biochem J. 

108: 533-539, 1968. 

Pegg AE and McCann PP, Polyamine metabolism and 

function. Biochim Biophys Acta. 1221, C212-C221, 1994. 

Pinel JPJ, Gorzalka BB and Ladak F. Cadaverine and 

putrescine initiate burial of dead conspecifics by rats. 

Physiol Bhhav. 27: 819-824, 1981. 

Porter CW and Bergeron RJ. Enzyme regulation as an 

approach to interference with polyamine biosynthesis - 

an alternative to enzyme inhibition. Adv Enzyme Regul. 

27: 57-79, 1988. 

Prakash NJ, Bowlin TL, Adwards ML, Sunkara PS and 

Sjoerdsma A. Antitumor activity of a novel synthetic 

polyamine analogue, N,N’-bis[3-(ethylamino)-propyl]- 

1-7-heptane diamine: Potentiation by polyamine 

oxidase inhibitors. Anticancer Res. 10: 1281-1288, 1990. 

Russel D and Snyder SH. Amine synthesis in rapidly 

growing tissues: Ornithine decarboxylase activity in 

regenerating rat liver, chick embryo, and various 

tumors. Proc Natl Acad Sci USA. 60: 142-1427, 1968. 

Scheneider Y, Vincent F, Duranton B, Badolo L, Gosse F, 

Bergmann C, Seiler N and Raul F. Anti-proliferative 

effect of resvetrol, a natural component of grapes and 

wine, on human colonic cancer cells. Cancer Lett. 158: 

85-91, 2000. 

Seidenfeld J. Effects of difluoromethylornithine on 

proliferation, polyamine content and plating efficiency 

of cultured human carcinoma cells. Cancer Chemother 

Pharmacol. 15: 196-202, 1985. 

Seiler N, Bolkenius FN and Rennert OM. Interconversion, 

catabolism and elimination of polyamines. Med Biol. 

59: 334-346, 1981. 

Seiler N and Dezeure N. Polyamine transport in mammalian 

cells. Int J Biochem. 22: 211-218, 1990. 

Seiler N and Atanassov CL. The natural polyamines and the 

immune system. Progress in Drug Research. 43: 87- 

141, 1994. 

Seiler N, Delcros JG and Moulinoux JP. Polyamine 

transport in mammalian cells. An update. Int J Biochem 

Cell Biol. 28: 843-861, 1996. 

Seiler N, Atanassov CL and Raul F. Polyamine metabolism 

as target for cancer chemoprevention. Int J Oncol. 

13: 993-1006, 1998. 

Stanley BA and Shantz LM. S-adenosylmethionine 

decarboxylase structure - function relationships; 

Polyamines in clinical and basic science. Biochem Soc 

Trans. 22: 845-898, 1994. 

Quemener V, Blanchard Y, Chamaillard R, Havouis R, 

Cippola B and Moulinoux J. Polyamine Deprivation: 

A new tool in cancer treatment. Anticancer Research. 

14: 443-448, 1994. 

Tabor CW and Tabor H. Polyamines. Ann Rev Biochem. 

53: 749-790, 1984.

Thomas T and Thomas T. Polyamines in cell growth and 

cell death: Molecular mechanisms and tharapeutic 

applications. Cell Mol Life Sci. 58: 244-258, 2001. 

Toursarkissian B, Endean ED and Aziz SM. 

Characterization of polyamine transport in rat aortic 

smooth muscle cells. J Surgical Res. 57: 401-407, 1994. 

Vijayanathan V, Thomas T, Shirahata A and Thomas TJ. 

DNA condensation by polyamines: A laser light 

scattering study of structural effects. Biochem. 

40: 13644-13651, 2001. 

Wallace HM and Morgan DML. Polyamines: Cellular 

Regulators. Biochem Soc Trans. 18: 1079-1095, 1990. 

Wang L, Price HL, Juusula J, Kline M and Phanstiel O. 

Influence of polyamine architecture on the transport and 

topoisomerase II inhibitory properties of polyamine 

DNA-intercalator conjugates. J Med Chem. 44: 3682- 

3691, 2001. 

Williams K. Interactions of polyamines with ion channels. 

Biochem J. 325: 289-297, 1997. 

Xia CQ Yang JY, Ren S and Lien E. QSAR analysis of 

polyamine transport inhibitors in L1210 cells. 

J Drug Target. 6: 65-77, 1997. 

Yatin SM, Yatin M, Varadarajan S, Ain KB and Butterfield 

DA. Role of spermine in amyloid beta-peptide 

associated free radical - induced neurotoxicity. 

J Neurosci Res. 63: 395-401, 2001. 

Yatin M and Fischman A J. Involvement of polyamines in 

E. coli infected rats. Submitted article to J Nuc Med. 2002. 

Zhang M, Wang H and Tracey KJ. Regulation of 

macrophage activation and inflammation by spermine: 

A new chapter in an old story. Crit Care Med. 

28: N60-N66, 2000. 

Polyamines 67



The histopathological changes in the mouse thyroid depending on the 

aluminium 

Tülin Aktaç* and Elvan Bakar 

University of Trakya, Faculty of Arts and Sciences, Department of Biology, 22080, Edirne, Turkey 

(* author for correspondence) 

Received 12 April 2002; Accepted 9 June 2002 

Abstract 

In this study, the effects of aluminium applied through oral way on mouse thyroid follicles were investigated. 

Aluminium was applied as drinking water, at concentrations of 0.1 %, 1 % and 5 % AlCl3. The results indicate that 

aluminium caused structural tissue degeneration and cellular injury of thyroid follicles, depending on the dose. 

KKeeyy wwoorrddss:: Thyroid, AlCl3, mouse, histopathological changes 


Özet 

Bu çal›flmada, oral yolla uygulanan alüminyumun fare tiroid bezi folikülleri üzerindeki etkileri araflt›r›ld›. 

Alüminyum % 0.1, % 1 ve % 5 lik AlCl3 fleklinde içme suyu olarak verildi. Sonuçlar alüminyumun doza ba¤l› 

olarak tiroid foliküllerinde yap›sal doku hasar›na ve hücre hasar›na neden oldu¤unu iflaret etmektedir. 

AAnnaahhttaarr ssöözzccüükklleerr:: Tiroid, AlCl3, fare, histopatolojik de¤ifliklikler 

Introduction 

Although aluminium is found in very small amounts 

in living organisms it is an abundant element in the 

environment. It is always present in foods, drinking 

water, drugs, cigarette ashes (Miller et al., 1984; 

Lione, 1985; Pennington, 1987). At neutral pH, 

aluminium exists mainly as Al(OH)3, and therefore, 

the level of aluminium in surface waters is always 

very low. With the development of modern industry, 

atmospheric deposition of sulfur dioxide, nitrogen 

oxide and nitrogen dioxide produced by burning of 

coal and petroleum products make the soil solution 

acidic. As a results, large amounts of Al 3+ ions are 

released from the water-insoluble aluminium 

compounds (Gong, 1988; Kloppel et al., 1997; van 

Landeghem et al., 1998). 

The importance of the aluminium entering to 

organism with food and drinking water have rapidly 

increased from the point of view of human health. 

Harmfull effects of aluminium have mainly been 

reported from organisms that are inconstant contact 

with natural waters, e.g. plants (Foy and Flemming, 

1978; Fiskesjö, 1983; Liu and Jiang, 1991; Liu et al., 

1993), fish (Driskol et al., 1980; Grahn, 1980; Muniz, 

1983; Exley et al., 1996; Nadeenko et al., 1997) and 

fish-eating birds (Nyholm, 1981). Also, it was 

established that aliminium applied through oral way 

was caused degenerations in the mouse liver and 

kidney tissues (Bakar and Aktaç, 2001; Aktaç et al., 

2001a) and adrenal glands (Aktaç, 2001; Aktaç et al., 

2001b). In addition, Waring et al. (1996) reported that 

plasma triiodothyronine (T3) and thyroxine (T4) 

concentrations were increased in sublethally 

Al-stressed brown trout (Salmo trutta). In this study, 

we aimed to investigated the effects of aluminium on 

the mouse thyroid gland entered by gastrointestinal 

route. 

69

70 Tülin Aktaç and Elvan Bakar 

Material and methods 

The adult mice (Balb/C, Albino), raised at 

Experimental Medicine Research and Application 

Center-University of ‹stanbul were used. Animals 

were divided by four groups (3 experiment and 1 

control) for experiments. Each group contained three 

animals. Different concentrations of AlCl3 (0.1 %, 1 

% and 5 %) prepared in drinking water were given to 

experiment groups while only drinking water was 

given to the control group. The amounts of water 

consumed by the animals during the experiment are 

shown in Table 1. Animals were killed by cervical 

dislocation at the day 10. For the ligth microscopic 

examinations, tiroid glands were fixed in formalin (10 

% solution) for 24 hours and then embedded in 

paraffin wax. Sections of 5 µm thick were cut and 

stained with hematoxylin-eosin. 

Table 1: The amounts of water consumed by the animals 

during the experiment. 

A1C13 doses Animal no. Water amounts (ml) 

0.1 % 1 20 

2 18 

3 22 

1 % 1 15 

2 12 

3 10 

5 % 1 21 

2 18 

3 16 

Control 1 22 

2 17 

3 20 

Results 

The results of present study showed that progressive 

degenerative changes in thyroid gland were depended 

on the dose of aluminium. There was no significantly 

degenerative changes in the groups of 0.1% and 1% 

AlCl3 compared the control group. It was observed 

that the general histological structure of the gland was 

disappeared in some sections (Figure 1-3). 

The most abundant degenerative changes were 

observed in 5 % AlCl3 group. In the thyroid follicles, 

destruction, distortions of thyroglobulin were 

Figure 1: The thyroid tissues of control group. f: follicles, 

c: colloid, s: stroma (interstitiel tissue), bar representes 

20 µm. 

Figure 2: 0.1% AlCl3 group. The general histological 

structure of the gland was disappeared, bar representes 

20 µm. 

Figure 3: 1% AlCl3 group. The general histological 

structure of the gland was disappeared, bar representes 

10 µm.

determined (Figure 4-5). Furthermore, it was 

observed that some of the cells were lost of their 

nuclei and the cytoplasm (Figure 5). Damaged nuclei 

within follicle lumen and increased fibers within 

dispersed stroma were also observed (Figure 4-6). 

Discussion 

The literature contains numerous references to the 

toxic effects of aluminium. However, studies on the 

histopathological effects of aluminium on the 

endocrine tissues are limited. Waring et al. (1996) 

applied lethal and sublethal aluminium doses in 

Salmo trutta to investigate the relationship between 

aluminium and plasma cortisol concentrations. Also, 

it was revealed that the aluminium was found a higher 

concentrations in adrenal and parathyroid glands 

(Ifl›mer et al., 1998) and that caused to defect of 

structure and function in adrenal glands (Aktaç, 2001; 

Aktaç et al., 2001b). Waring et al. (1996) obtained 

significant increasing plasma T3 and T4 

concentrations in sublethally Al-stressed brown trout, 

Salmo trutta. In their study, however, they were not 

clarified the histopathological effects of aluminium 

on the thyroid gland. In the present study, it was 

showed that aluminium (in particularly 5 % AlCl3 

concentration) caused degenerative changes in 

thyroid gland. These changes were irreversible. It was 

indicated that increased fibers within dispersed 

stroma caused more destructive changes in tissue. 

Finally, it was indicated that the exposure to 

aluminium for a long time caused degenerative 

changes in important endocrine organ such as thyroid. 

However, it may be profitable to attempt further 

studies to demonstrate the mechanism of the effects of 

aluminium in thyroid cells. 

References 

Aktaç T. Histological changes in adrenal cortex of male 

mice fed by aluminium. Univ ‹stanbul Fac Sci Journal 

of Biology. 64: 1-7, 2001. 

Aktaç T, Hüseyinov G and Kabo¤lu A. The ultrastructural 

changes in the mouse liver, depending on the 

aluminium. Univ ‹stanbul Fac Sci Journal of Biology. 

64: 51-60, 2001a. 

fi 

fi 

Aluminium effect on thyroid 71 

Figure 4: 5% AlCl3 group. Destruction of follicles 

(big arrows), damaged nuclei within follicle lumen 

(small arrows), bar representes 10 µm. 

fi 

Figure 5: 5% AlCl3 group. Destruction of follicles 

(*) and follicular cells (big arrows), damaged nuclei 

within follicle lumen (small arrows), bar representes 

10 µm. 

Figure 6: 5% AlCl3 group. Dispersed stroma (s), bar 

representes 10 µm. 

*

72 Tülin Aktaç and Elvan Bakar 

Aktaç T, Hüseyinov G and Kabo¤lu A. Aluminium - induced 

ultrastructural changes on the secretion granules of 

adrenal medullary chromaffin cells. Almanac Nature. 1: 

180-186, 2001b. 

Bakar E and Aktaç T. The histopathological changes in the 

mouse kidney and liver tissues depending on the 

aluminium. Marmara Univ Fen Bilimleri Dergisi. 

in press. 

Driskol CT, Baker JP, Bisogni JJ and Schofield CL. Effect 

of aluminium speciation on fish in dilute acidified 

waters. Nature. 284: 161-164, 1980. 

Exley C, Wicks AJ, Hubert RB and Birchall JD. Kinetic 

constraints in acute aluminium toxicity in the rainbow 

trout (Oncorynchus mykiss). J Theor Biol. 179: 25-31, 

1996. 

Fiskesjö G. Nucleolar dissolution induced by aluminum in 

root cells of Allium. Phsiol Plant. 59: 508-511, 1983. 

Foy CD and Fleming AL. The physiology of plant tolerance 

of excess available aluminum and manganese in acid 

soils. In: Crop Tolerance to Suboptimal Land 

Conditions. Jung GA (Ed). ASA Special Publication 

No32. American Society of Agronomists, Madison, 

Wisconsin. 301-328, 1978. 

Grahn O. Fish kills in two moderately acid lakes due to high 

aluminium concentration. Proceedings International 

Conference on the Ecological Impact of Acid 

Precipitation, Olso Norway SNSF project. 310 - 311, 

1980. 

Gong SG. Problems on the acid rain. Agro-environmental 

Protection. 7: 47-48, 1988. 

Ifl›mer A, fiahin G, Ayd›n A, Baydar T and Benli K. 

Alüminyum Toksisitesi. GATA Bas›mevi, Ankara. 1-45, 

1998. 

Kloppel H, Fliedner A and Kordel W. Behavior and 

ecotoxicology of aluminium in soil and water- review 

of the scientific literature. Chemosphere. 35: 353-363, 

1997. 

Lione A. Aluminum toxicology and the aluminum-containing 

medications. Pharmacol Ther. 29: 255-285, 1985. 

Liu D and Jiang W. Effects of Al 3+ on the nucleolus in root 

tip cells of Allium cepa. Hereditas. 115: 213-219, 1991. 

Liu D, Jiang W and Li D. Effects of aluminium ion on root 

growth, cell division, and nucleoli of garlic (Allium 

sativum). Environment Pollution. 82: 295-299, 1993. 

Miller RG, Kopfler FC, Kelty KC, Stober JA and Ulmer 

NS. The occurrence of aluminium in drinking water. 

J Amer Water Works A s. 76: 84-91, 1984. 

Muniz IP. The effects of acidification or Norwegian 

freshwater ecosystems. In: Ecological Effects of Acid 

Deposition. National Swedish Environmental 

Protection Board - Report PM. 1636. 299-322, 1983. 

Nadeenko VG, Gol’dina IR, D’iachenko OZ and Pestova 

LV. Comparative informative value of chromosome 

aberrations and sister chromatid exchange in the 

evaluation of metals in the environment. Gig Sanit. 

3: 10-13, 1997. 

Nyholm NEI. Evidence of involvement of aluminium in 

causation of defective formation of eggshells and of 

impaired breeding in wild passerine birds. Environ Res. 

26: 363-371, 1981. 

Pennington JAT. Aluminum content of foods and diets. 

Food Addit Contam. 5: 61-232, 1987. 

Van Landeghem GF, De Broe ME and D’Haese PC. Al and 

Si: Their speciation, distribution and toxicity. 

Clin Biochem. 31: 385-97, 1998. 

Waring CP, Brown JA, Collins JE and Prunet P. Plasma 

prolactin, cortisol and thyroid responses of the brown 

trout (Salmo trutta) exposed to lethal and sublethal 

aluminium in acidic soft water. Gen Com Endocr. 

102: 377-385, 1996.



Inhibitory effect of 57% hepatectomized mice serum on the growth 

of L-cells 

Seyhan Altun*, Mehmet Topçul and Gül Özcan Ar›can 

University of ‹stanbul, Faculty of Science, Department of Biology, 34459, Vezneciler, ‹stanbul, Turkey 

(* author for correspondence) 


Abstract 

Adult mammalian liver cells, which are in cell cycle’s G0 phase, differentiate to accomplish specific functions and 

do not divide. Only as a result of damage or partial hepatectomy (PH), the cells multiply in a rapid way and cause 

the organ’s regeneration. In one of our previous studies, we observed that sera obtained from 35% partially 

hepatectomized mice has an inhibitory effect on the growth of the cells after the fourth day. In this study, sera are 

obtained from 57% hepatectomized mice and added to medium of L-cells. In the different days, viable cell number 

and labelling index were investigated. According to the results obtained from the experiments it was determined 

that sera obtained from 57% partially hepatectomized mice has an inhibitory effect on the growth of the L-cells in 

early days. 

KKeeyy wwoorrddss:: Partial hepatectomy, serum, mouse, L-cells, humoral factor 

%57 hepatektomi uygulanm›fl fare serumunun L-hücrelerinin ço¤almas›n› bask›lay›c› etkisi 

Özet 

Eriflkin memeli karaci¤er hücreleri, hücre siklusunun G0 faz›nda olup, belli fonksiyonlar› yapmak üzere 

farkl›laflm›fllard›r ve bölünmezler. Ancak, yaralanma veya parsiyal hepatektomi (PH) sonucu, hücreler h›zl› bir 

flekilde ço¤alarak organ›n rejenere olmas›n› sa¤larlar. Daha önceki bir çal›flmada, %35 PH yap›lan farelerden elde 

edilen serumun, L-hücrelerinin büyümesini dördüncü günden sonra inhibe etti¤i tesbit edilmifltir. Bu çal›flmada ise, 

%57 hepatektomi uygulanan farelerden serum elde edilmifl ve bu serum L-hücrelerinin yaflama ortamlar›na ilave 

edilmifltir. Farkl› günlerde hücreler toplanarak, canl› hücre say›lar› ve iflaretlenme indeksleri saptanm›flt›r. Elde 

edilen sonuçlara göre, %57 PH uygulanan hayvanlardan elde edilen serumun L-hücrelerinin büyümesi üzerinde ilk 

günlerde inhibisyon etkisi meydana getirdi¤i tesbit edilmifltir. 

AAnnaahhttaarr ssöözzccüükklleerr:: Parsiyal hepatektomi, serum, fare, L-hücreleri, humoral faktör 

Introduction 

Adult mouse liver hepatocytes are differentiated cells 

which are in cell cycle’s G0 phase under normal 

conditions and execute rather important functions of 

the organism. Mitotic index (MI) in liver cells of such 

characteristics is in such a low level that it is not even 

worth of attention. But when PH surgery is applied to 

the livers of adult mice, the diffentiated liver cells 

gain their dividing characteristics and start to multiply 

(Higgins and Anderson, 1931; Bucher, 1963; Bucher 

and Farmer, 1998; Fausto, 2001). This regenerative 

growth observed in the liver, continue until the liver 

reaches its dimension to the one prior to the surgery. 

73

74 Seyhan Altun et al. 

Altun (1996) observed that after the performance of 

PH in a ratio of 35%, the regeneration occured rapidly 

up to the third day and slowed down during the 

following days. It was determined that as a 

consequence of applying PH in a ratio of 57%, 

regeneration was faster and in a higher ratio than that 

of the application of 35% (Altun and Özalpan, 1998). 

It is asserted that a factor, present in the serum, is 

effective for the liver’s reaching its previous 

dimension by starting cell multiplication following 

hepatectomy. Parabiotic rat couples were produced in 

order to determine this factor named as humoral 

factor (Bucher et al., 1951; Moolten and Bucher, 

1967; Sakai, 1970). It was reported that from the rat 

couples whose blood circulations were connected to 

each other, even the liver of the rat which had not 

undergone PH grew. Besides, Moolten and Bucher 

(1967) observed that this growth, which occured in 

the parabiotic pairs, was related to the portion of 

removed liver. 

Stimulative effects of humoral factors on some 

tumors were also found. Paschkis et al. (1955) who 

investigated different growths such as PH, unilateral 

nephrectomy and leg-fracture determined that in rats 

with PH, some tumors grew better. The investigators 

did not observe a relation between nephrectomy and 

leg-fracture and tumors. Consequently, they put forth 

that humoral factor may cause the rapid growth of the 

tumors and this factor behaved selectively. Although, 

Ono et al. (1986) expressed that tumors of the 

animals, administered X-5563 and Ehrlich ascites 

tumor cells (EAT) were not affected by PH, in the 

MH-134 cells the response showed a difference 

depending on the administration time. In a similar 

way, Udintsev and Shakhov (1989) indicated that the 

inhibition of EAT and Pliss’ lymphosarcoma in 

animals with PH was produced by humoral factor. 

By investigating the effects of blood sera, 

obtained from animals with PH under in vivo and 

in vitro conditions more detailed information about 

humoral factor was attempted to be attained. It was 

observed that in the in vivo studies the area of 

drawing blood from which serum was obtained, 

affected the result, and sera obtained from hepatic 

portal vein or cardiac right atrium had a stimulatory 

effect on normal and regenerated liver (Adibi et al., 

1959; Moya, 1963; Levi and Zeppa, 1972). Besides, it 

was shown that serum with PH increased tumor 

growth in the in vitro tumors (Ramantanis and 

Deliconstantinos, 1985; Asaga et al., 1991; de Jong et 

al., 1995, 1998-1999). In the in vitro cultures of some 

tumors whose growth was observed to increase in the 

form of a stimulation by serum with PH, it was found 

that the effect produced changed depending on the 

time of serum administration or cell concentration 

(Asaga et al., 1991; de Jong et al., 1995). Sakai and 

Kountz (1975) determined that serum with PH 

increased DNA synthesis in lymphocyte and 

hepatocyte cultures. Chen et al. (1996) in their 

experiments with cirrhosis and non-cirrhosis rats, 

with two different PH ratios, observed that serum 

obtained from these animals increased DNA synthesis 

of hepatocyte cultures. It is asserted that together with 

liver dissection, during the first 24 hours a signal 

protein having a molecular weight of 5,000-10,000 

was responsible for the production of these effects 

and also this protein contained a factor which 

increased cell growth (Takahashi et al., 1992). 

Nevertheless, Makowka et al. (1983) informed that 

liver’s cytosole contained stimulatory and inhibitory 

factors. 

In our previous studies, it was observed that serum 

obtained from mice with 35% PH, inhibited the 

growth of L-cells in vitro following the fourth day of 

its administration (Altun et al., 2002). Besides, 

regeneration was in proportion with the portion 

removed during hepatectomy, in paralel to PH ratio 

regeneration also increased (Altun and Özalpan, 

1998). The aim of this research was to determine 

mode of effect of the serum obtained from mice with 

hepatectomy ratio increased to 57%, on L-cells in vitro. 


Animals and the preparation of sera 

The animals used were 2.5 months old male Balb/C 

strain inbred mice (Mus musculus) whose body 

weights varied between 20-30 g (n=23) The animals 

were placed in polycarbonate cages and fed with 

pellet mouse food (Hipodrom Ltd.) and tap-water 

given ad libitum. 

PH operation (57%) was carried out by removing 

the left lateral and median lobe of the liver of mice 

under ether anesthesia (Higgins and Anderson, 1931). 

Three days after the operation, blood was drawn from 

the carotid artery of hepatectomized mice. The blood

was centrifuged and the serum was separated. After 

the serum were sterilized, they were used freshly 

without freezing. 

Cells and experimental groups 

In the experiments, tumor L-cells obtained from mice 

subcutaneous connective tissue in 1943 were used 

(Shannon, 1972). The cells were propagated in 10% 

Fetal bovine serum and Medium 199. 

Cells were removed from the surface of culture 

bottles by addition of 0.25% trypsin and centrifuged 

for 3 minutes (1,500 cycle/min). With the addition of 

Medium-199 on the cell precipitate, the cells became 

ready for seeding. 

Control 1: 10% Fetal bovine serum and 90% 

Medium-199 

Control 2: 10% Fetal bovine serum, 5% normal 

mouse serum and 85% Medium-199 

PH-57: 10% Fetal bovine serum, 5% 

hepatectomized (57%) mouse serum and 

85% Medium-199. 

Growth rate 

L-cells were seeded on sterile microplates having 24 

wells in a concentration of 10 4 cells in each well. 

Microplate wells were divided into the above 

mentioned experimental groups and 1.5 ml of 

medium of each experimental group was added on 

L-cells in each well. During the experimental period, 

the microplates were kept in an atmosphere of 5% 

CO2 and 95% air at 37°C with pH 7.2 in a dessicator. 

The growth rate of L-cells were determined on 

2nd, 4th and 6th days. For this process, the cells 

multiplying in microplate were collected separately 

with trypsin and by the application of viability test 

(Phillips, 1973), L-cells count for each group and day 

were made. 

Labelling index 

In order to determine the labelling index of L-cells at 

the end of 2nd, 4th and 6th days, the cells were kept 

in a medium containing 5 mCi/ml 3 H-thymidine 

( 3 H-TdR) for 30 minutes and they were then fixed 

with 1:3 w/w acetic acid:ethanol. By the use of K2 

emulsion (Ilford) the autoradiography of the 

preparations was made. At the end of 10 days of the 

exposition period, the autoradiograms were 

developed with D19b developer and stained with 

Giemsa. The labelling index was determined by 

counting 900-1,300 cells of each group and day. 

Statistical evaluation 

The values of growth rate and labelling index 

obtained in the experiments are given as arithmetic 

means and standard error of each mean. The 

significance of the variation was determined by 

Student-t test. 

Results 

The multiplication rate of L-cells propagated in 

medium to which 5% serum obtained from mice on 

the third day following 57% PH ratio performance is 

shown in Figure 1. As seen in the Figure, two days 

after initially seeding 10 4 cells, 34,545 cells were 

counted and in the forth day, the cell number reached 

46,153. After the fourth day, the multiplication rate 

Cell number (log.) 

10 6 

10 5 

10 4 

Effect of mice serum upon L-cells 75 

Control 1 

Control 2 

PH - 35 

PH - 57 

2 4 6 

Time (day) 

Figure 1: The growth rate in the L-cells.


increased a little and on the sixth day the cell number 

was found to be 110,750. 

When the cells which were propagated in a 

medium containing normal and with PH serum were 

examined under the microscope, a layer similar to an 

oily one at the surface of the medium was observed. 

Besides, a large number of vacuoles in the cells 

cytoplasm were observed (Figure 2). 

The values of labelling index of L-cells 

propagated in serum with 57% PH are given in Table 

1. From these values, it was determined that the 

labelling index started with a considerable low level 

of 11.5%, increased rapidly and after reaching 44.1% 

on the fourth day, showed a little reduction later on. 

The labelling index of L-cells on the sixth day was 

found as 28.5%. 

Figure 2: L-cells in the PH-57 group ( vacuole). 

Discussion 

With the addition of serum, obtained from the carotid 

arteries of mice with 57% PH, the multiplication rate 

and labelling index of L-cells were examined. 

According to the results obtained, it was determined 

that L-cells exhibited a slow multiplication during the 

first days and after the fourth day, the multiplication 

rate increased. Even though, this characteristic was 

also observed in the values of labelling index, little 

reduction in the synthesis rate could be seen on the 

sixth day. These results were compared with those of 

the ones obtained with our previous studies with 

normal and with 35% PH serum (Figure 1, Table 1), 

(Altun et al., 2002). When Figure 1 is examined it can 

be seen that on the second day in comparison with 

Control 1, Control 2 and PH-35 groups, in the PH-57 

Table 1: The change in labelling index of L-cells, 

depending on days (±SE). 

Groups 2 

Time (day) 

4 6 

Control 1 35.4±7.6 41.3±5.0 42.7±0.7 

Control 2 37.2±8.2 32.7±5.3 31.2±4.8 

PH-35 14.4±2.1 16.8±9.7 14.4±0.8 

PH-57 11.5±3.0 44.1±9.4 28.5±6.8 

group a lower level of multiplication rate occured. 

Contrarily, it was observed that with the increase of 

PH-57 group’s multiplication rate in the following 

days, on the sixth day a higher cell number was 

reached in proportion to other groups. In respect to 

control groups during the first days, this decrease 

observed in L-cells group, propagated in serum with 

57% PH, showed a significant difference (p

hepatocytes. While liver’s mitotic indices in the 

peripheral blood serum obtained from left ventricule 

was 9.19, it was established that with the hepatic vein 

serum, mitotic indices reached 18.78. In respect with 

the results they obtained, they asserted that the factors 

which stimulated and inhibited growth were in 

balance under normal conditions, with the decrease of 

growth inhibitory concentration by PH the balance 

change towards the stimulating factor and this factor 

was released by the liver. Moya (1963) who applied 

PH in a ratio of 2/3 to rats from the sera obtained from 

their hepatic and portal veins, and from carotide 

artery observed that artery serum produce inhibition 

in normal rats and on the other one produced a much 

lesser inhibition rate in comparison to the artery 

serum. Moya (1963) disclosed this situation as the 

decrease in the inhibitory substance’s amount while 

passing into the tissues. Moya (1963), who added 

normal and PH serum to AH130 cells in vitro 

observed that while a decrease occured in cell number 

of the cultures on the 24th hour and sixth day, serum 

with PH produced an effect similar to the normal 

culture medium. 24th hour following 70% PH serum 

obtained from portal vein was also found to increase 

liver DNA synthesis amount in normal rats (Levi and 

Zeppa, 1972). Colorectal liver tumors were examined 

by de Jong et al. (1995, 1998-1999). When these 

investigators added portal and systematic sera to CC 

531 cells 24 hours and 14 days after PH, they 

observed that an effect in the form of stimulation 

when the cell amount was small and of inhibition 

when it was large occurred (de Jong et al., 1995). de 

Jong et al. (1998-1999) indicated that portal serum 

taken on the third day after 70% PH application 

increased the multiplication of CC 531 cells in a rate 

of 25-40%. In contrast to this, when the same 

investigators added systematic serum, taken on the 

14th day, to the same cells they observed an increase 

in the cell multiplication. Chen et al. (1995) applied 

PH in ratios of 33% and 70% to cirrhosis and 

non-cirrhosis rats and found that the serum which 

they obtained from the animals on the second and 

48th hours did not affect the mitosis of hepatocyte 

cultures, but produced an increase in DNA synthesis. 

Takahashi et al. (1992) asserted that in the portal 

serum, obtained from rats with 70% PH after 24 hours, 

a protein with a molecular weight of 5,000-10,000 

contained a factor which stimulated cell growth. 

The effects of hepatectomy ratios on parabiotic 

pairs produced by connecting the blood circulations 

Effect of mice serum upon L-cells 77 

of two rats one of which had PH, was investigated by 

Moolten and Bucher (1967). The investigators who 

examined three different ratios PH, 34%, 68% and 

85%, observed that a significant increase in normal 

rat liver DNA did not occur only in the group to which 

35% PH was performed. It was determines that 

following PH performance to cirrhosis and 

non-cirrhosis patients most of which had 

hepatocellular carcinoma, human hepatocyte growth 

factor (hHGF) was present in their peritoneal fluid 

and blood serum and this factor which was in relation 

with removed liver is portion increased DNA 

synthesis in rat hepatocyte cultures in vitro (Miyata et 

al., 1996a, 1996b). Higaki et al. (1999) reported that 

in patient with PH, the growth factor was in a higher 

level in portal serum than that of peripheral serum. 

Furthermore, in the human liver regeneration after 

PH, it has been determined that the activity of liver 

regeneration, mainly referring to proliferation of 

hepatocytes, is affected by a number of factors (both 

stimulatory and inhibitory) released from local or 

from other parts of the body (Wu et al., 1998). 

By inoculating tumor at the same time to animals 

with PH, whether PH had an effect on tumor growth 

or not was investigated. While an increase was 

observed in hepatoma and Walker 256 tumors, 

inoculated at the same time with PH, any change in 

Jensen sarcoma’s and Murphy lymphosarcoma’s 

growth was not observed (Paschkis et al., 1955). Ono 

et al. (1986) who reported that X-5563 and EAT 

tumors were not affected by PH, determined that an 

inhibition occured in tumor growth depending on 

transplantation time. In similar way, it was 

determined that the growth of EAT cells, inoculated at 

the same time with 35% PH was inhibited on the 10th 

day (Altun, 1996). This different behaviour of PH on 

tumors was expressed by Paschkis et al. (1955) as a 

selective behaviour of humoral factor which started 

liver regeneration. Udintsev and Shakhov (1989) also 

confirmed that the inhibition which occured in the 

growth of EAT and Pliss’ sarcoma of animals with PH 

was formed by the humoral factor. 

A study showing the effect of serum, obtained 

from mice with a lower PH ratio, on L-cells is present 

in the literature. The other studies show differences in 

the viewpoint of both cell type and serum. When all 

these investigations are taken into consideration with 

respect to L-cells, besides the area where serum was 

obtained and the selective effect of humoral factor on 

the tumors, the growth of tumors was also affected by


PH ratio and this observed inhibition may be 

produced by the effect of one or more than one of 

these factors. 

In conclusion, it was determined that even though 

mouse serum with 57% hepatectomy inhibited the 

growth of L-cells on the second day, this effect was 

alleviated during the following days. 

Acknowledgments 

This work was supported by the Research Fund of the 

University of ‹stanbul (Project number: 980). We are 

grateful to Prof. Dr. Atilla Özalpan for his helpful 

suggestions and critical remarks. 

References 

Adibi S, Paschkis KE and Cantarow A. Stimulation of liver 

mitosis by blood serum from hepatectomized rats. Exp 

Cell Res. 18: 396-398, 1959. 

Altun S. Normal rejeneratif ve tümöral büyümeler 

aras›ndaki kinetik iliflkiler. Tr J of Biology. 20: 153-173, 

1996. 

Altun S and Özalpan A. Hepatektomi oran› ile rejenerasyon 

aras›ndaki iliflki. ‹st T›p Fak Mecmuas›. 61: 485-491, 

1998. 

Altun S, Özcan Ar›can G, Topçul M. Inhibitory effect of 

hepatectomized (35%) mice serum upon the growth of 

L-cells. J Cell and Molecular Biology. 1: 31-38, 

2002. 

Asaga T, Suziki K, Umeda M, Sugimasa Y, Takemiya S and 

Okamoto T. The enhancement of tumor growth after 

partial hepatectomy and the effect of sera obtained from 

hepatectomized rats on tumour cell growth. Jpn J Surg. 

21: 669-675, 1991. 

Bliokh ZHL, Platonov AE and Somolianinov VV. 

Dynamics of initial L-cell adhesion. I. The effect of 

serum and albumin. Tsitologiia. 24: 692-698, 1982. 

Bucher NLR, Scott JF and Aub JC. Regeneration of the 

liver in parabiotic rats. Cancer Res. 11: 457-465, 1951. 

Bucher NLR and Farmer SR. Liver regeneration following 

partial hepatectomy: genes and metabolism. In: Liver 

Growth and Repair. Strain AJ and Dieht AM (Eds). 

Chapmann & Hall, London. 1998. 

Chen MF, Hwang TL and Yu HC. Effect of serum from 

partially hepatectomized cirrhotic and noncirrhotic rats 

on liver regeneration with primary hepatocyte cultures. 

Eur Surg Res. 28: 413-418, 1996. 

de Jong KP, Lont HE, Bijma AM, Brouwers MA, de Vries 

EG, van Veen ML, Marquet RL, Slooff MJ and Terpstra 

OT. The effect of partial hepatectomy on tumor growth 

in rats: in vivo and in vitro studies. Hepatology. 

22: 1263-1272, 1995. 

de Jong KP, Brouwers MA, van Veen ML, Brinker M, de 

Vries EG, Daemen T, Scherphof GL and Slooff MJ. 

Serum obtained from rats after partial hepatectomy 

enhances growth of cultured colon carcinoma cells. 

Invasion & Metastasis. 18: 155-164, 1998-1999. 

Fausto N. Liver regeneration. In: The Liver, Biology and 

Pathobiology. Arias IM, Boyer JL, Chisari FV, Fausto 

N, Schachter D and Shafritz DA (Eds). Lippincott 

Williams & Wilkins, Philadelphia. 2001. 

Freshney RI. Culture of Animal Cells A Manual of Basic 

Technique. Fourth Edition, Wiley-Liss, New York. 2000. 

Higaki I, Yamazaki O, Matsuyama M, Horii K, Kawai S, 

Hirohashi K and Kinoshita H. Portal serum human 

hepatocyte growth factor levels after partial 

hepatectomy. Hepatogastroenterology. 46: 1078-1082, 

1999. 

Higgins GM and Anderson RM. Experimental pathology of 

the liver. I. Restoration of the liver of the white rat 

following partial surgical removal. Arch Pathol. 

12: 186-202, 1931. 

Levi JU and Zeppa R. The response of normal rat 

hepatocytes when exposed to humoral (regenerating) 

factor. J Surg Res. 12:114-119, 1972. 

Makowka L, Falk RE, Falk JA, Teodorczyk-Injeyan J, 

Venturi D, Rotstein LE, Falk W, Langer B, Blendis LM 

and Phillips MJ. The effect of liver cytosol on hepatic 

regeneration and tumor growth. Cancer. 51: 2181-2190, 

1983. 

Miyata K, Taniguchi H, Takeuchi K, Koyoma H, Tanaka H 

and Takahashi T. Weight of resected liver is positively 

correlated with serum hHGF level. Hepato- 

Gastroenterology. 43: 1589-1593, 1996a. 

Miyata K, Taniguchi H, Takeuchi K, Tsubouchi H and 

Daikuhara Y and Takahashi T. Levels of human 

hepatocyte growth factor (hHGF) in peritoneal fluid 

after partial hepatectomy. Hepato-Gastroenterology. 

43: 1594-1600, 1996b. 

Moolten FL and Bucher NLR. Regeneration of rat liver: 

Transfer of humoral agent by cross circulation. Science. 

158: 272-274, 1967. 

Moya FJ. Inhibition of growth by post-hepatectomy blood 

serum. Effect on regenerating liver and on tissue 

cultured. Exp Cell Res. 31: 457-469, 1963. 

Ono M, Tanaka N and Orita K. Complete regression of 

mouse hepatoma transplanted after partial hepatectomy 

and the immunological mechanism of such regression. 

Cancer Res. 46: 5049-5053, 1986. 

Paschkis KE, Cantarow A, Stasney J and Hobbs JH. Tumor 

growth in partially hepatectomized rats. Cancer Res. 

15: 579-582, 1955. 

Phillips HJ. Dye exclusion tests for cell viability. In: Tissue 

Culture Methods and Applications. Kruse PF and 

Patterson MK (Eds). Academic Press, New York. 1973.

Ramantanis G and Deliconstantinos G. Effects of sera and 

spleen cells isolated from partially hepatectomized rats 

on the growth of Walker-256 tumour. Biomed 

Pharmacother. 39: 482-486, 1985. 

Sakai A. Humoral factor triggering DNA synthesis after 

partial hepatectomy in the rat. Nature. 228: 1186-1187, 

1970. 

Sakai A and Kountz SL. Stimulation of hepatocytes and 

lymphocytes in vitro by liver regeneration. Nature. 257: 

53-54, 1975. 

Shannon JE. The American type culture collection. Registry 

of Animal Cell Lines. Parklawn Drive Rockville, 

Maryland. 301: 881-2600, 1972. 

Takahashi N, Obata R and Sasaki T. In vitro immune 

response of splenic lymphocytes to portal serum agents 

from rats undergoing hepatic regeneration or hepatic 

carcinogenesis. Int J Tissue React. 14: 17-24, 1992. 

Udintsev SN and Shakhov VP. Decrease in the growth rate 

of Ehrlich’s tumor and Pliss’ lymphosarcoma with 

partial hepatectomy. Vopr Oncol. 35: 1072-1075, 1989. 

Wu J, Kuncio GS and Zern MA. Human liver growth in 

fibrosis and cirrhosis. In: Liver Growth and Repair. 

Strain AJ and Diehl AM (Eds). Chapmann & Hall, 

London. 1998. 

Effect of mice serum upon L-cells 79




Mehmet Topçul 1 *, Gül Özcan Ar›can 1 , Nevin Erensoy 2 , Atilla Özalpan 3 

1 University of ‹stanbul, Faculty of Science, Department of Biology, 34459, Vezneciler, ‹stanbul, Turkey; 

2 University of ‹stanbul, Faculty of Cerrahpafla Medicine, Department of Medical Biology, ‹stanbul, Turkey; 

3 University of Golden Horn, Faculty of Arts and Sciences, Department of Molecular Biology and 

Genetics, 34280, ‹stanbul, Turkey (* author for correspondence) 


Abstract 

C3H mouse mammary carsinoma-derived FM3A cell line grown in RPMI 1640+10%FBS medium as suspension 

culture have been used in tissue culture studies in recent years. Changes in labelling index were examined in the 

present study, applying in vitro doses of epirubicin, tamoxifen and epirubicin+tamoxifen to FM3A cells. The 

findings reveal that treatments of epirubicin and tamoxifen lower the percentage of the cells at S phase, while 

combined treatment of epirubicin and tamoxifen gives more successful results, being statistically significant 

(p

82 Mehmet Topçul et al. 

Tamoxifen was first studied in the 1960s and emerged 

as a nonsteroidal antiestrogen for the treatment of 

breast cancer in the 1970s. Tamoxifen is now the 

prototypic hormonal agent used in breast cancer and 

its considerable success has prompted the 

investigation and development of newer antiestrogens. 

Tamoxifen possesses both antiestrogenic and 

estrogenic activity. Newer antiestrogens currently 

being developed may have fewer estrogens-agonistic 

properties and more potent antiestrogenic activity 

than tamoxifen. About one-third of breast cancer 

patients with advanced disease respond to treatment 

with the antiestrogen tamoxifen (Mc Guire et al., 

1978) and most of them are patients with an estrogen 

positive primary tumour (Mouridsen et al., 1992; 

Rose et al., 1980). Epirubicin (Farmorubicin) is a new 

derivative of doxorubicin, synthesized with the aim of 

finding anthracycline analogues with an improved 

spectrum of antitumour activity and lower toxicity. It 

appears to have similar antitumour activity and 

toxicity as the parent drug in experimental and human 

tumours. Epirubicin has been used alone or in 

combination with other cytotoxic agents. In vitro cell 

culture studies show that epirubicin enters the cell 

nuclei, inhibits nucleic acid synthesis and arrests cell 

division (Di Marco, 1984; Skladannowski and 

Konopa, 1994). In the present study, we investigated 

the effect of epirubicin and tamoxifen on labelling 

index of FM3A cells in other to increase chance of 

this drugs in clinical applications. 


Cells 

We used estrogen receptor (ER) positive FM3A cell 

line in our experiments. C3H mouse mammary 

carsinoma-derived FM3A cell line grown in RPMI 

1640+10% Fetal Bovine Serum as suspension culture 

have been used in tissue culture studies in recent 

years. Test cells were divided into one control and 

three test groups. Microplate wells were covered with 

10 4 cells/ml and incubated 5% CO2 and 95% air for 

24 hours. 

Estrogen receptor assay 

ER levels were studied by the methods of Lippman 

and Huff (1976) and Raynaud et al. (1978) with minor 

modifications. ER activity as demonstrated by the 

dextran-coated charcoal tecnique is closely correlated 

with the clinical ability of tamoxifen to inhibit tumour 

growth. 

Drug application 

We used optimum doses of epirubicin and tamoxifen. 

At first, we investigated optimum doses for FM3A 

cells. These doses were found 0.01 mg/ml for 

epirubicin and 0.001 mg/ml for tamoxifen. At the end 

of the 24 hour incubation, the medium was replaced 

with a medium containing epirubicin, tamoxifen and 

epirubicin+tamoxifen doses and reincubated 0, 4, 8, 

16 and 32 hours. 

Labelling index 

For labelling index determination, cells were then 

incubated with RPMI 1640 containing 5 µCi/ml 

3 H-TdR(Amersham). Both control and treated cells 

were labelled for 1 hour. Smeared cells were than 

prepared on glass-slides. Slides were rinsed with 2% 

perchloric acid twice at 4°C for 30 minutes to remove 

dissolved radioactive material. They were then coated 

with K2 emulsion (Ilford), kept at 4°C for 3 days, and 

developed. Labelling index was calculated by 

counting at least the 3000 cells from each slide, 

stained with Giemsa for 3 minutes. 

Statistical analysis 

Student-t test was used to evalute the results (n=150). 

Results 

Figure 1 indicates labelling index values of Control, 

0.01 mg/ml epirubicin, 0.001 mg/ml tamoxifen and 

0.01 mg/ml epirubicin + 0.001 mg/ml tamoxifen 

groups after an application of 0, 4, 8, 16 and 

32 hours. 

After 4 hours treatment, when various 

concentrations of epirubicin, tamoxifen and

epirubicin + tamoxifen were added to the cultures, 

DNA synthesis decreases minimum with respect to 

the control cells (p

84 Mehmet Topçul et al. 

Now, 106 years after Beatson’s pioneering work 

with oophorectomy, endocrine therapy has been 

shown to have a major role in the adjuvant setting. 

Hormonal therapy may eventually prove to have its 

greatest impact in breast cancer preventation. 

Newer hormonal agents, especially the 

antiestrogens, have opened up additional doors for 

trials of endocrine therapy (Muss, 1992). 

Epirubicin, an anthracycline antibiotic, inhibits 

DNA replication and transcription by intercalation 

between DNA strands which are the ultimate 

intracellular target of anthracyclines (Aglietta et al., 

1993; Mouridsen, 1992). 

Cell culture studies exhibit epirubicin enters the 

cells rapidly is localized in nuclei and inhibits nucleic 

acid synthesis and cell division (Robert and Gianni, 

1993). 

In vitro studies showed that epirubicin has 

maximal cytotoxic effects in the S and G2 phases 

(Greg, 1993). 

Although the precise mechanism of epirubicin is 

not fully understood, performed studies suggest that it 

forms a complex with DNA by intercalation between 

DNA strands, thus inhibiting DNA replication and 

transcription (Sinha and Porti, 1990; Skladanowski 

and Konopa, 1994). 

Epirubicin has been shown to be cytotoxic, with 

an increase in both the degree and rate of induction of 

DNA strand breakage (Cantoni et al., 1990). 

Adding tamoxifen to epirubicin treatment induced 

S phase arrest in FM3A cells. In the majority of trials, 

chemotherapy combined with endocrine therapy has 

given improved results compared with chemotherapy 

alone (Boccardo et al., 1990; Mouridsen, 1992). 

Thus, the results of our study seem to be 

concordant with the above mentioned studies, 

suggesting that combinations of drugs are superior to 

single agents. 

As a result, we think that in this experiments, 

some interactions between cytotoxic agents and 

tamoxifen is synergistic and additive. 

In ER-positive and ER-negative breast cancer cell 

lines, the growth, cytotoxic, and cell-cycle effects 

must be examined and compared. 

References 

Aglietta M, Monzeglio C, Pasquino P, Carnino F, Stern AC 

and Gavosto F. Short-term administration of 

granulocyte-macrophage colony stimulating factor 

decreases hematopoietic toxicity of cytostatic drugs. 

Cancer. 72: 2970-2973, 1993. 

Beatson GT. On the treatment of inoperable cases of 

carsinoma of mammal: Suggestions for a new method 

of treatment, with illustrative cases. Lancet. 2: 104- 

107, 1896. 

Boccardo F, Rubagotti A, Bruzzi P, Cappellini M, Isola G, 

Nenci I, Piffanelli A, Scanni A, Sismondi P and Santi L. 

Chemotherapy versus tamoxifen versus chemotherapy 

plus tamoxifen in node-positive, estrogen receptorpositive 

breast cancer patients: Results of a 

multicentric Italian study. J Clin Oncol. 8: 1310- 

1320, 1990. 

Cantoni O, Sestili P, Cattabeni F, Geroni C and Giuliani F. 

Comparative effects of doxorubicin and 4´epidoxorubicin 

on nucleic acid metabolism and 

cytotoxicity in a human tumour cell line. Cancer 

Chemotherapy and Pharmacology. 27: 47-51, 1990. 

Clarke RB, Laidlaw IJ, Jones LJ, Howell A and Anderson E. 

Effect of tamoxifen on Ki67 labelling index in human 

breast tumours and it’s relationship to oestrogen 

progesterone receptor status. Br J Cancer. 67: 606- 

11, 1993. 

Cuzick J and Baum M. Tamoxifen and contralateral breast 

cancer. Lancet (i). 282, 1985. 

Di marco A. Epirubicin: Mechanism of action at the 

cellular level. In: Advances in Anthracycline 

Chemotherapy. Epirubicin. Bonadonna G (Ed). Masson, 

Milano - Italia. 41-47, 1984. 

Douglas KT. Anticancer drugs, DNA-intercalation and free 

radical attack. Chemistry and Industry. 766-771, 1984. 

Greg L, Faulds P and Faulds D. Epirubicin. A review of its 

pharmacodynamic and pharmacokinetic properties, and 

therapeutic use in cancer chemotherapy. Drugs. 

45: 788-856, 1993. 

Gullino PM, Pettigrew HN and Grantham FH. N- 

Nitrosomethylurea as mammary gland carcinogen in 

rats. J Natl Cancer Inst. 54: 401-414, 1975. 

Huggins SC, Grand LC and Brillantes FP. Mammary 

cancer, induced by a single feeding of polynuclear 

hydrocarbons and their suppression. Nature. 189: 204- 

207, 1962. 

Lippman M and Huff KA. Demonstration of androgen and 

estrogen receptors in a human breast cancer using a 

new protamine sulfate assay. Cancer. 38: 868-874, 

1976. 

Mc Guire WL, Horwitz KB, Zava DT, Garola RE and 

Chamness GC. Hormones in breast cancer: Update 

1978. Metabolism. 27: 487-501, 1978. 

Mouridsen H, Palshof T, Paterson J and Battersby L. 

Tamoxifen in advanced breast cancer. Cancer Treat 

Rev. 5: 131-141, 1978. 

Mouridsen HT. Systemic therapy of advanced breast 

cancer. Drugs. 44 Suppl 4: 17-28, 1992.

Murphy B and Muss HB. Hormonal therapy of breast 

cancer: State of the art. Oncology. 11, Supp. 4: 7-13, 

1997. 

Muss HB. Endocrine therapy for advanced breast cancer: 

A review. Breast Cancer Res Treat. 21: 15-26, 1992. 

Raynaud JP, Martin PM, Bouton MM and Ojasoo T. 11-bmehoxy-17-ethynyl-1,3,5 

(10)-estratriene-3, 17-b-diol 

(Moxestrol), a tag for estrogen receptor binding sites 

in human tissues. Cancer Res. 38: 3044-3050, 1978. 

Robert J and Gianni L. Pharmacokinetics and metabolism 

of anthracyclines. Cancer Surv. 17: 219-252, 1993. 

Rose C, Thorpe SM, Lober J, Daehnfeldt JL, Palshof T and 

Mouridsen HT. Therapeutic effect of tamoxifen related 

to estrogen receptor level. In: Recent Results in Cancer 

Research. Henningsen B, Linder F and Steichele C 

(Eds). Vol. 71 Berlin, Springer Verlag. 134-141, 

1980. 

Sinha BK and Porti PM. Anthracyclines, cancer 

chemotherapy. Biol Response Modif. 11: 45-57, 1990. 

Sipila PE, Wiebe VJ, Hubbard GB, Koester SK, Emshoff 

VD, Maenpaa JU, Wurz GT, Seymour RC and De 

Gregorio MW. Prolonged tamoxifen exposure selects a 

breast cancer cell clone that is stable in vitro and in 

vivo. Eur J Cancer. 29 A: 2138-2144, 1993. 

Skladanowski A and Konopa J. Interstrand DNA 

crosslinking induced by anthracyclines in tumour cells. 

Biochem Pharmacol. 47: 2269-2278, 1994. 

Epirubicin and tamoxifen effect upon FM3A cells 85




Gülruh Ulako¤lu 

University of ‹stanbul, Faculty of Science, Department of Biology, 34459, Vezneciler, ‹stanbul, Turkey 


Abstract 

The effect of different doses of adriamycin (ADM), was investigated in vitro and in vivo, in Ehrlich ascites tumor 

(EAT) cells which developed in the peritoneal cavity of mice. In the in vivo experiments it was found that injection 

of ADM as 10 mg/g i.p. prolonged the survival period of mice. In the in vitro experiments, treatment of 

ADM in 2, 4 and 6 mg/ml doses, it was observed that cytotoxic effect dependent on time occurred in 

tumor cells. 

KKeeyy wwoorrddss:: Ehrlich ascites carcinoma, adriamycin (adriablastina), in vivo, in vitro 

Adriamisinin iinn vviittrroo ve iinn vviivvoo koflullarda Ehrlich ascites tümör hücrelerine etkisi 

Özet 

Adriamisinin (ADM) çeflitli dozlar›n›n farelerin periton bofllu¤unda geliflen Ehrlich ascites tümör (EAT) hücrelerine 

etkisi in vivo ve in vitro olarak denendi. In vivo deneylerde farelere 10 mg/g dozda i.p. olarak enjekte edilen ADM in 

farelerin yaflam süresini uzatt›¤› saptand›. In vitro deneylerde EAT hücreleri 2, 4 ve 6 mg/ml dozda ADM ile muamele 

edildi¤inde, zamana ba¤l› olarak tümör hücrelerine sitotoksik etki meydana geldi¤i gözlendi. 

AAnnaahhttaarr ssöözzccüükklleerr:: Ehrlich ascites karsinoma, adriamisin (adriablastina), in vivo, in vitro 

Introduction 

ADM, being an anthracycline antibiotic, has a large 

spectrum of antitumor activity. Clinically, it was 

administered to various tumors singly or with other 

antitumor antibiotics (Sipahio¤lu, 1981). Different 

results have been obtained from ADM when 

administered in different doses both clinically and in 

laboratory experiments. In an experiment performed 

with HeLa cells, a significant decrease in the number 

of surviving cells occurred depending on the dose of 

applied ADM (Jagetia and Nayak, 1996). 

Experiments on the effective mechanism of ADM 

to tumor cells were also done. It was reported that it 

had the highest impact on DNA; DNA replication, 

transcription and repair were effected. It was also 

shown that it inhibited protein synthesis (Saraswathi 

et al., 1997). ADM interacts with cell membrane and 

by affecting the processes such as lectin interaction, 

phospholipid’s structure and organization, fluidity 

and transportation of small molecules and ions in the 

cell membrane causes cytotoxicity (Tritton and Yee, 

1982). 

During the application of ADM in high doses 

clinically, its side effects on the patients have been 

observed (Sipahio¤lu, 1981). In an experiment, it was 

observed that the administration of 20 mg/kg ADM 

increased the microsomal lipid peroxidation level in 

rats which was found to be related with toxicity 

induced by ADM. For this reason, when ADM is used 

at high doses, it was administered with drugs which 

decrease microsomal lipid peroxidation (Shinozawa 

87

88 Gülruh Ulako¤lu 

et al., 1993). A group of research workers, treated 

their breast carcinoma, gastric carcinoma and colon 

carcinoma patients with a single dose of 25 mg/m 2 i.v. 

ADM. After peripheral blood samples were obtained 

serially from these patients before and after drug 

injection were compared. They found that ADM had 

a cytotoxic effect on the blood cells which was its side 

effect (Arinaga et al., 1986). 

In this study, whether ADM prolonged or not the 

survival periods of mice bearing EAT cells was 

investigated. Besides the mode of effect of ADM’s 

different doses on EAT cells multiplication was also 

examined in vitro. 


EAT cells used in this study were hyperdiploid 

carcinoma cells, and they were maintained in our 

laboratory by making their transplantions every 7 

days. 

Preparation of ADM 

ADM isolated from Streptomyces peucetius by 

Farmitalia Research Laboratories for the first time. 

ADM (Carlo Erba, Turkey) used in the experiments 

was a lyophylized and stock solution obtained by 

dissolving 10 mg ADM in 5 ml sterile water. From 

this solution 2, 4, 6 mg/ml ADM concentrations were 

prepared to be used in the experiments. 

In vivo experiments 

In the experiments 3 months old male albino mice of 

strain Balb/c weighing between 25-30 g were used. 

The mice were first inoculated intraperitonally with 

3X10 6 EAT cells and they were separated into control 

and experimental groups. There are 10 mice in the 

control group and 29 mice in the experimental group. 

In the in vivo experiment were used total 39 mice. 4 

days after the inoculation of EAT cells, 10 mg/g ADM 

was injected i.p. in a single dose to the experimental 

mice. The control group’s mice were injected with 

physiological serum. The status of all the mice were 

controlled every day and the death dates of both the 

experimental and control mice were determined. 

In vitro experiments 

In these experimental series also EAT cells were 

drawn from the stock mice with an injector and a cell 

suspension was prepared in BSS. From this 

suspension, 3X10 5 cells/ml were transferred into steril 

tubes containing medium (Eagle + 2.5% Fetal calf 

serum + 10 mM Hepes). After the tubes were closed 

with steril rubber stoppers, they were incubated in an 

incubator at 37°C. EAT cells were separated into 

experimental and control tubes, 24 hours after being 

taken in vitro condition. ADM in 2, 4 and 6 mg/ml 

concentrations were added separately into the 

experimental tubes (Çerçi and Ulako¤lu, 2000). At 

the end of 3, 5, 24, 30 and 48 hours of drug addition, 

cell counted were done. Both the control and 

experimental cells were treated with trypan blue, 

counted under the light microscope and the amount of 

dead and surviving cells were determined. 

According to the data obtained, by the application 

of paired t-test their significance at p

Number of mice 

Average Cell Number 

25 

20 

15 

10 

5 

1 

Survival time (days) 

Figure 1: Survival periods of control and experimental mice bearing EAT cells. 

3 5 24 30 48 

Time (hours) 

cells were treated with trypan blue and the numbers of 

dead and living cells were determined. The surviving 

cell counts averages of both groups are given in 

Figure 2 shows the histogram drawn according to 

these values. According to the data obtained paired ttest 

was applied in order to find out whether a 

significantly occured among them. 

According to the counts of EAT cells made 3 

hours after the treatment with ADM in concentration 

of 2, 4 and 6 mg/ml a statistical significance in the 

cells treated with the drug according both to the 

control and among each other was not observed. 

Adriamycin effect upon EAT cells 89 

Control 

2 µg/ml 

4 µg/ml 

6 µg/ml 

Figure 2: Histogram of average survival cell number of each group after treatment of various concentration of ADM. 

Control 

Experiment 

According to counts made after 5 hours, p

90 Gülruh Ulako¤lu 

According to these results 2, 4 and 6 mg/ml 

concentrations of ADM 24 hours after administration 

of EAT cells, all concentrations caused a quite 

definite cytotoxic effect in comparison to the control, 

the most effective concentration was 6 mg/ml. The 

cytotoxic effect at 24 hours continued at after 30 

hours and after 48 hours the cytotoxic effect of all the 

concentrations increased. According to this 

observation, ADM induces a cytotoxic effect on EAT 

cells depending on concentration and time. 

Discussion 

EAT cells survive in acidic fluid present in the 

peritoneal cavity of mice in the form of a suspension. 

It was known that, after the transplantation into 

peritoneal cavity of mice the number of cells increase 

exponentially up to the 9. day and reach the 

plato-phase following 9. and 10. days (Lazebnik et al., 

1991; Song et al., 1993; Vinuela et al., 1991). For this 

reason, during the in vivo experiments of this study 

ADM was injected to mice at the 4. day following 

EAT transplantation to them. The reason why ADM 

was given in the middle of the multiplication period 4. 

day to see how it affected the exponential growth of 

EAT cells. 

In a study similar to this one, to DBA/2 mice 

bearing acidic murine lymphocytic leukemia, ADM 

and daunorubicin were administered separately and 

their survival periods were controlled. In this study 

0.25-2.00 mg/kg ADM was injected to tumor bearing 

mice for 5 days. The survival of mice, receiving a 

high dose prolonged 140 % that of the controls and 20 

% of the experimental mice survived up to to 50. day 

(Schwartz and Grindey, 1973). Even though different 

kind of tumor bearing mice and different 

concentrations of ADM were used, the results of this 

experiment also supports theirs. 

Experiments on the survival time’s prolongation 

started in 1978. Extracts prepared from muscle and 

liver tissues of mice were injected i.p. for a period of 

12 days to EAT bearing mice. It was found that these 

mice survived longer that the control ones. In 

contrast, a positive results were not obtained from 

spleen, kidney, lung, serum, skin and small intestine 

extracts (Tong et al., 1978). 

Different doses of ADM was investigated in 

various cells. It was shown that ADM in 0.2 mg/ml 

concentration exerted cytotoxic effect on human 

peripheral lymphocytes. In the same study, it was also 

observed that this concentration decreased the 

multiplication of V79 (Chinese hamster’s lung 

fibroblast cells) cells 50 % (Szabona, 1996). In our 

study, it was also observed that ADM’s 2, 4 and 6 

mg/ml concentrations exerted a cytotoxic effect on 

EAT cells which increased depending on time. When 

K562 (human erythroleukemia) cell, propagated in 

tissue culture, were treated with 5, 10 and 30 mM 

ADM, it was determined that the cell number was 

decreased in comparison to the control and the drug 

exerted a cytotoxic effect depending on the dose used 

(Ciaccia et al., 1994). It was observed that allogenic 

tumor cells and mouse spleen cells, propagated in cell 

culture, when treated with ADM in an in vitro 

medium, ADM exerted a cytotoxic effect on them 

(Ehrke et al., 1984; Tomozic et al., 1980). It was also 

found that when HeLa cells, propagated in an in vitro 

medium, were treated with 5, 10, 25, 50 and 100 

mg/ml ADM, their survival ratio decreased depending 

on the dose (Jagetia and Nayak, 1996). 

Even though ADM makes cytotoxic effect in 

tumor cells, it also exerts negative effects on normal 

cells. For this reason, when ADM was administered to 

patients in the clinic, drugs which remove its side 

effects were also used (Vichi and Tritton, 1993). 

Many studies are present where ADM is applied in 

various doses to many different kinds of tumors. In 

this study, the results derived from first time this 

ADM doses applied to EAT cells are in accordance 

with those indicated in literature. 

References 

Arinaga S, Akiyoshi T and Tsuji H. Augmentation of the 

generation of cell-mediated cytotoxicity after a single 

dose of adriamycin in cancer patients. Cancer Research. 

46: 4213-4216, 1986. 

Ciaccia M, Tesoriere L, Pintaudi AA, Re R and Cardillo SV. 

Vitamin A preserve the cytotoxic activity of adriamycin 

while counteracting its peroxidative effects in human 

leukemic cells in vitro. Biochemistry and Molecular 

Biology International. 34: 329-335, 1994. 

Çerçi B and Ulako¤lu G. The effects of adriamycin on 

L-Strain cells. Chimica Acta Turcica. 28: 25-27, 2000. 

Ehrke JM, Ryoyama K and Cohen AS. Cellular basis for 

adriamycin-induced augmentation of cell-mediated 

cytotoxicity in culture. Cancer Research. 44: 2497- 

2504, 1984. 

Jagetia CG and Nayak V. Micronuclei-induction and its 

correlation to cell survival in HeLa cells treated with

different doses of adriamycin. Cancer Letters. 110: 123- 

128, 1996. 

Lazebnik YA, Medvedeva DN and Zenin VV. Reversible 

G2 Block in the cell cycle of Ehrlich ascites carcinoma 

cells. Exp Cell Res. 195: 247-254, 1991. 

Saraswathi V, Subramanian S, Ramamoorthy N, Mathuram 

V and Govindasamy S. In vitro cytotoxicity of 

echitamine chloride and adriamycin on Ehrlich ascites 

carcinoma cell cultures. Med Sci Res. 25: 167-170, 

1997. 

Schwartz SH and Grindey BG. Adriamycin and 

daunorubicin: A comparison of antitumor activities and 

tissue uptake in mice following immunosuppression. 

Cancer Research. 83: 1837-1844, 1973. 

Shinozawa S, Gomita Y and Araki Y. Protective effects of 

various drugs on Adriamycin (Doxorubicin) induced 

toxicity and microsomal lipid peroxidation in mice and 

rats. Biol Pharm Bull. 16: 1114-1117, 1993. 

Sipahio¤lu H. Onkoloji. Hacettepe, Tafl Ltd. fiti. 

1981. 

Song Z, Varani J and Goldstein IJ. Differences in cell 

surface carbohydrates and in laminin and fibronectin 

synthesis between adherent nonadherent Ehrlich ascites 

tumor cells. Int J Cancer. 55: 1029-1035, 1993. 

Szabona E. Comparison of the in vitro effect of 

adriablastina on induction of SCEs in V79 cells and 

human peripheral blood lymphocytes. Neoplasma. 

43: 407-409, 1996. 

Tomozic V, Ehrke JM and Mihinch E. Modulation of the 

cytotoxic response against allogenic tumor cells in 

culture by adriamycin. Cancer Research. 40: 2748- 

2755, 1980. 

Tong C, Bergevin P, Appelbaum J, Parmar D. and Grob D. 

Inhibition of growth of mouse Ehrlich ascites by normal 

tissue extracts. Chemotherapy. 24: 34-38, 1978. 

Tritton RT and Yee G. The anticancer agent adriamycin can 

be actively cytotoxic without entering cells. Science. 

217: 248-250, 1982. 

Vichi JP. and Tritton RT. Protection from adriamycin 

cytotoxicity in L 1210 cells by brefeldin A. Cancer 

Research. 53: 5237-5243, 1993. 

Vinuela JE, Rodriguez R, Gil J, Coll J, Concha E and 

Subiza JL. Antigen shedding vs. development of 

natural suppressor cells as mechanism of tumor escape 

in mice bearing Ehrlich tumor. Int J Cancer. 47: 86-91, 

1991. 

Adriamycin effect upon EAT cells 91



Camalexin is not required for the function of RRPPPP11 and RRPPPP1133 resistance 

genes in AArraabbiiddooppssiiss tthhaalliiaannaa inoculated with PPeerroonnoossppoorraa ppaarraassiittiiccaa 

Figen Mert-Türk 1 *, and Eric B. Holub 2 

1 University of Çanakkale Onsekiz Mart, Faculty of Agriculture, Department of Plant Protection, 17100, 

Çanakkale, Turkey; 2 Department of Plant Genetics and Biotechnology, Horticulture Research 

International, Wellesbourne, Warwickshire, CV35 9EF, England (* author for correspondence) 


Abstract 

Peronospora parasitica is an obligate biotrophic pathogen and causes downy mildew in Arabidopsis thaliana. The 

Col-pad3 mutant of A. thaliana accession Col-0 was previosly selected in a screen for phytoalexin deficiency 

following inoculation by the strains of Pseudomonas syringae. The mutant does not accumulate detectable levels 

of camalexin (the phytoalexin of A. thaliana) following inoculation with P. parasitica or abiotic inducer AgNO3. 

RPP1 and RPP13 resistance genes lie at the bottom arm of chromosome three of accession Nd-1. Inoculation of 

Nd-1 plants with RPP1- and RPP13-avirulent isolates of P. parasitica result pitting necrosis on the cotyledons and 

flecking chlorosis of the seedlings with no pathogen sporulation. In this research, this only known possible 

phytoalexin biosynthetic mutant was employed to investigate whether the mutation in PAD3 locus would have any 

effect on the recognition of certain isolates of P. parasitica by RPP1 and RPP13 disease resistance genes in 

A. thaliana accession Nd-1. According to the results camalexin is not required for the function of these two disease 

resistance genes. 

KKeeyy wwoorrddss:: Arabidopsis thaliana, Peronospora parasitica, pad mutants, resistance, phytoalexins, camalexin 

Kamaleksin PPeerroonnoossppoorraa ppaarraassiittiiccaa ile inokulasyonundan sonra AArraabbiiddooppssiiss 

tthhaalliiaannaa’n›n RRPPPP11 ve RRPPPP1133 dayan›kl›l›k genlerinin ifllevleri için gerekli de¤ildir 

Özet 

Peronospora parasitica bir zorunlu biyotrof patojen olup Arabidopsis thaliana’da mildiyöye sebep olur. A. 

thaliana ’n›n bir ekotipi olan Col-0’dan izole edilen Col-pad3 mutant bitkisinin, daha önce Pseudomonas 

syringae’nin ›rklar›yla inokulasyonundan sonra kamaleksin üretmedi¤i bulunmufltur. Bu mutant bitki P. parasitica 

ve AgNO3 ile inokulasyondan sonra da tespit edilebilir miktarda kamaleksin üretmemektedir. Hastal›klara 

dayan›kl›l›k genleri olan RPP1 ve RPP13 genleri ekotip Nd-1’de üçüncü kromozomun alt kolunda yer almaktad›r 

ve bu genleri tan›yan P. parasitica’n›n avirulent ›rklar› ile inokulasyonu takiben, fidelerde nekrotik lekeler 

oluflmakta ve patojenin sporulasyonuna rastlanmamaktad›r. Bu çal›flmada bilinen tek fitoaleksin biyosentetik 

mutant› olan Col-pad3’ü kullanarak, PAD3 lokusunda meydana gelen mutasyonun, A. thaliana ekotip Nd-1’de 

bulunan RPP1 ve RPP13 dayan›kl›l›k genleri üzerindeki etkisini araflt›rmak hedeflenmifltir. Elde edilen sonuçlara 

göre, kamaleksinin bu iki dayan›kl›l›k geninin ifllevleri için gerekli olmad›¤› bulunmufltur. Pad3 mutasyonunun 

Nd-1’de bulunmas› sonucu bu genotiplerde belli izolatlara karfl› duyarl›l›k gözlenmedi¤inden, yani hastal›k 

oluflumu meydana gelmedi¤inden dolay›, kamaleksinin bu iki dayan›kl›l›k geninin ifllevleri için gerekli olmad›¤› 

bulunmufltur. 

AAnnaahhttaarr ssöözzccüükklleerr:: Arabidopsis thaliana, Peronospora parasitica, pad mutantlar›, dayan›kl›l›k, fitoaleksinler, 

kamaleksin 

93

94 Figen Mert-Türk and Eric B. Holub 

Introduction 

The response of plants to pathogen attack can be 

associated with a complex of metabolic responses 

including accumulation of phytoalexins. These 

secondary metabolites, which are synthesized by 

plants in response to diverse forms of biotic and 

abiotic stress, are part of a plant’s chemical and 

biochemical defense mechanisms (Mansfield, 1982; 

Kuc, 1995; Dixon et al., 1996; Mansfield, 1999). 

Phytoalexins are absent in healthy (non-stressed) 

tissues and accumulate after infection with fungal and 

bacterial pathogens in both monocotyls and dicotyls. 

Camalexin was first isolated from the leaves of 

Camelina sativa in response to infection by 

Alternaria brassicae (Browne et al., 1991). Tsuji et al. 

(1992) reported that camalexin was also synthesised 

by Arabidopsis thaliana (L.) Heynh. where it 

accumulates to high levels after infection with 

avirulent strain of Pseudomonas syringae pv. 

syringae. 

Glazebrook and Ausubel (1994) isolated three 

phytoalexin mutants (pad) of A. thaliana accession 

Col-0 to help elucidate the role(s) of phytoalexins in 

plant-pathogen interaction. Infection by P. syringae 

pv. maculicola strain ES4326 (PsmES4326) induced 

camalexin in the Col-pad1, Col-pad2 and Col-pad3 

mutants to 30, 10 and

pad3 locus from the Col-pad3 mutant to investigate 

whether PAD3 is involved in defence responses 

mediated by the RPP1 or RPP13 resistance genes. 


Isolates of P. parasitica 

All isolates of P. parasitica known to be recognised 

by RPP1 and RPP13 genes in accession Nd-1 were 

used. These isolates are Emco5, waco5, Maks9, 

Aswa1, Goco1, Edco1, Emoy2 and Hiks1. 

Interaction phenotypes 

The seeds were sown according to Holub et al. 

(1994). The seedlings were inoculated as described by 

Holub et al. (1994). Seven days old seedlings were 

inoculated with appropriate isolate of P. parasitica 

and incubated in a growth room. The interaction 

phenotypes were recorded at 3 and 7 days after 

inoculation (dai) according to host response and 

pathogen sporulation. Pitting necrosis on the 

cotyledon was recorded as "P" and this was always 

associated with no pathogen sporulation, thus it was 

abbreviated as "PN". Clorotic flecks (abbreviated as 

F) were also seen on the host, but sporulation of the 

pathogen may occur although very low. In A. 

thaliana-P. parasitica research, pathogen growth was 

almost always scaled into five categories according to 

pathogen sporulation in each cotyledon (Holub et al., 

1994; Tör et al., 1994; Botella et al., 1998; Bittner- 

Eddy et al., 1999). These categories are abbreviated to 

letters as follows: "N" is for none sporulation, "R" is 

for rare (less than 1 conidiophore for per cotyledon), 

"L" is for low sporulation (1-5 conidiophores per 

cotyledon), "M" is for medium sporulation (5-20 

conidiophores per cotyledon) and "H" is for heavy 

sporulation (more than 20 conidiophores per 

cotyledon). 

Selection of genotypes carrying the RPP1, RPP13 

and pad3 loci 

The Col-pad3 mutant and the accession Nd-1 plants 

were grown to flowering and cross-pollination was 

processed. F1 was back-crossed to parent Nd-1 to 

obtain more Nd-1 effect. F1 seeds were grown for F2 

seeds. All selections were made from these F2 families. 

Camalexin and RPP1 and RPP13 resistance genes 95 

The Col-pad3 mutant does not accumulate 

camalexin following induction by P. parasitica or 

abiotic inducer AgNO3 (Mert-Türk et al., 1998; 

Mert-Türk, 2001; Mert-Türk et al., manuscript in 

preparation). Therefore, the marker for the allele of 

pad3 would be the phenotype that does not 

accumulate camalexin. Camalexin extraction and 

Thin Layer Chromotography (TLC) were carried out 

as described by Glazebrook and Ausubel (1994). F2 

genotypes were first screened for camalexin 

deficiency. The genotypes that did not accumulate 

camalexin were labelled that they carried the pad3 

allele. Then these genotypes were tested whether they 

carry RPP1 and RPP13 genes as well. As the map 

position and primers near to the RPP1 and RPP13 

genes are known all selection process were made 

using Polymerase Chain Reaction (PCR). 

DNA isolation 

PhyoPure Plant DNA Extraction Kit (Nucleon 

Biosciences, UK) was used for DNA isolation. Leaf 

samples were harvested and freeze-dried at-60°C for 

2 days 0.02 g dried tissue was added to a milling tube 

containing two ball bearings and milled for 10 min. 

The powder was transferred to a 1.5 ml sterile 

Eppendorf tube and DNA was extracted as 

Manufacture’s advice. 

Polymerase Chain Reaction (PCR) 

The amplification reaction were carried out in a 

thermocycler. Two specific primers, one from each 

end of the DNA fragment to be amplified, were used 

for selective amplification. The primers 

encompassing the RPP1 and RPP13 resistance genes 

are shown in Table 1. 

PCR reactions were carried out in 25 µl volumes 

that contained 2.5 µl 10X PCR buffer, 1 µl of 5 mM 

dNTP (equal mixture of four), 1 µl of 50 mM MgCl2, 

1 µl of each of 10 µM primers and 1 µl of DNA which 

is about 25-50 ng and 0.2 µl Taq DNA polymerase. 

Conditions for amplification were as follows: 1 min at 

95°C for 1 cycle, denaturation at 95°C for 30 seconds, 

annealing 30 seconds at 50-58°C dependent on the 

primers used, and polymerisation at 72°C for 2 min. 

This cycle was repeated 35 times. A final cycle was 2 

min at 72°C for the uncompleted cycles. The 

reactions were stopped by chilling to 4°C.


Table 1: The sequences of primers encompassing RPP1 and RPP13 resistance genes their restriction enzymes 

(Nam et al., 1989; Oppenheimer et al., 1991; Bittner-Eddy et al., 1999 and 2000; Bittner-Eddy, personel communication). 

Primers R.E. a 

Gl-1 F b 

R c 

5’-ATA TTG AGT ACT GCC TTT AG-3’ Taq1 

5’-CCA TGA TCC GAA GAG ACT AT-3’ 

Py3003 F 5’-TCC TGT GTG TAG AGA ACC GC-3’ Rsa1 

R 5’-TAG CGA GAA AAT AAT GTC TG-3’ 

pAT389 F 5’-AAT CAC CAT TAC TAA TCA GG-3’ n.e. d 

R 5’-AGA TTG GCA TCG TGA GGC AC-3’ 

m249 F 5’-CAG AGA GTG ACC AAA TCT GAA CC-3’ Aci1 

R 5’-GCA TTA TGT TAG ACC AAT GTG C-3’ 

p1C7L F 5’-CTC CCC AAG TAG GCT TCC ATT C-3’ Mse1 

R 5’-GAG TGT TGT TGG CTT TCA TGC AG-3’ 

a R.E. Restriction enzymes 

b F: Forward primers 

c R: Reverse primers 

d n.e. No Enzyme necessary as natural polymorphism is present 

Restriction enzyme digestion 

To identify restriction endonucleases that would 

reveal polymorphism in the amplified fragments, 

restriction digests were carried out in a final 20µl 

volume containing 12.5 µl of PCR product, 2 µl 10X 

enzyme buffer, 1-3 units of appropriate enzyme (0.2- 

0.4µl) and 5.1-5.3 µl sterile ddH2O as shown in Table 

1. The tubes were incubated at temperature advised 

by the manufacture. 

Electrophoresis 

Restriction fragments were assesed by electrophoresis 

on a 1-2.5% agarose gel made up in 1X TBA buffer; 

the same buffer was used as a running buffer. 1/5 

volume of loading dye was added to each tube before 

loading into the wells of the gel. Electrophoresis was 

carried out at 85-150 V for 2-3 h. The gel then 

removed from the tank and submerged in a solution 

containing 0.5 µg/ml ethidium bromide in deionized 

water. The gel was destained in deionized water for 15 

min and visulalized in UV transilluminator for 

polymorphism. 

Results 

Recombinants carrying Nd-alleles of RPP1, RPP13 

and the pad3 allelle from the Col-pad3 mutant 

After testing 177 F2 plants using TLC plate assays, 37 

F2 genotypes were identified as camalexin deficient 

when the plates were visualised under UV radiation 

suggesting that they were homozygous for pad3 

(pad3 is a single recessive mutation, Glazebrook and 

Ausubel, 1994). Among these genotypes three (671, 

678 and 848) were found homozygous for Nd-1 DNA 

across the interval containing RPP1 and RPP13. 

These genotypes were selfed until F4 seeds were 

obtained to minimise the heterozygosity in other loci. 

F4 seeds were, therefore, used for the pathogenicity 

test using the isolates of P. parasitica (Emoy2, Waco5 

and Hiks1 for RPP1 resistance gene; Edco1, Maks9, 

Aswa1, Goco1 and Emco5 for RPP13 resistance 

gene). 

All three Nd-pad3 genotypes (671, 678 and 848) 

were found to be homozygous in RPP1 and RPP13 

genes from Nd-1 and pad3 allele from the Col-pad3 

mutant. All these genotypes were included in the

Table 2: Phenotypic interaction between the F4 genotypes of Arabidospsis thaliana carrying Nd-1 resistance genes 

at RPP1, RPP13 and the pad3 allele from Col-pad3 mutant following inoculation with isolates of Peronospora parasitica. 

Lines of Predicted 

A. thaliana Genotype a 

Nd-1 RPP1/RPP13/PAD3 PN b 

experiment with a thought that all three may have 

different genotypes as a result of crossing-over 

following cross-pollination. No camalexin 

accumulation was observed in these genotypes during 

screening, as all genotypes were selected for 

camalexin deficiency. 

Interaction phenotypes (IPs) of the genotypes 

following inoculation by the isolates recognised by 

RPP1 and RPP13 genes 

Three genotypes (hereafter will be abbreviated as Ndpad3) 

containing pad3 mutation along with RPP1 and 

RPP13 resistance genes, the Col-pad3 mutant and its 

wild-type Col-0, where it was derived from, and Nd- 

1 seedlings were inoculated by the isolates of P. 

parasitica at 7 days after sowings. The seedlings were 

assessed for their phenotypes 3 and 7 days after 

inoculation (dai). The Nd-1 seedlings exhibited 

pitting necrosis and no sporulation of all three isolates 

were observed (Figure 1a). Following inoculation by 

Waco5, Emoy2 and Hiks1 (RPP1-avirulence 

isolates), the wild-type Col-0 exhibited heavy 

sporulation, low sporulation with flecking host 

response and no sporulation with flecking response, 

respectively. The Col-pad3 seedling were also as 

susceptible as wild-type Col-0 after inoculation with 

Waco5, however, exhibited medium sporulation with 

the isolate Emoy2 (Figure 1b). Although Hiks1 was 


Isolates of Peronospora parasitica 

RPP1-avirulence RPP13- virulence 

Waco5 Emoy2 Hiks1 Edco1 Maks9 Aswa1 Goco1 Emco5 

PN PN FN FN FN FN FN 

Col-0 rpp1/rpp13/PAD3 H FL FN H H H H H 

Col-pad3 rpp1/rpp13/pad3 H M N H H H H H 

Nd-pad3 (671) RPP1/RPP13/pad3 PN PN PN FN FN FN FN FN 



a Based on genetic marker assisted selection in the case of Nd-pad3 mutants numbered 671, 678 and 848. 

b Interaction phenotypes (per cotyledon). H: heavy sporulation (>20 conidiophores); M: medium sporulation 

(5-20 conidiophores); L: low sporulation (1-5 conidiophores); N: none sporulation; F: chlorotic flecks; 

P: necrotic pits. 

not able to grow in the Col-pad3 mutant, the host 

response of the mutant was different than wild type as 

no flecking response, which is associated with HR, 

was observed. All three Nd-pad3 genotypes exhibited 

a PN phenotype following inoculation with RPP1avirulent 

isolates (Emoy2, Waco5 and Hiks1) as 

shown in Figure 1c and Table 2. 

A. thaliana accession Nd-1 carries RPP13 gene 

that recognises avirulence product of at least five 

isolates of P. parasitica (Edco1, Maks9, Aswa1, 

Goco1 and Emco5). This gene has recently been 

cloned (Bittner-Eddy et al., 2000). Clorotic flecking 

host response with no sporulation of the pathogen was 

seen in the accession Nd-1 (Figure 1d). However, the 

Col-0 accession does not have this allele, therefore, 

the plants are very susceptible to the isolates which 

are recognised by RPP13 gene. The Col-pad3 mutant 

was also suseptible to these isolates (Figure 1e). All 

three genotypes were, however, showed the same 

response as seen in Nd-1 parent. The seedlings were 

resistant to the five isolates and flecking response was 

seen 7 dai. No sporulation of the pathogen was seen 

(Figure 1f and Table 2). 

Discussion 

The existence of pad3 mutation on Nd-1 background did 

not obviously affect the function of either RPP genes.


a b 

c d 

e f 

Figure 1: The interaction phenotypes between the 

isolates of Peronospora parasitica and the lines of 

Arabidopsis thaliana. Nd-1 (a) exhibited PN (pitting with 

no pathogen sporulation phenotypes) after inoculation with 

the Emoy2 isolate. However, Col-pad3 was more 

susceptible than wild-type accession Col-0 following 

inoculation with the same isolate and exhibited medium 

sporulation (b). The response of Nd-pad3 to Emoy2 was 

same as the Nd-1 parent (c). Flecking host response with no 

sporulation was observed in Nd-1 (d) following inoculation 

with any RPP13-avirulent isolate (d). Col-pad3 was fully 

susceptible as it does not have a functional resistance gene 

to recognise the isolates (e). The Nd-pad3, however, 

exhibited the same interaction phenotype observed in Nd-1 

parent (f). 

These results indicate that PAD3 is not required for 

resistance mediated by either RPP1 or RPP13 genes 

against to the corresponding isolates of 

P. parasitica, therefore, camalexin is not an essential 

defence component for either source of downy 

mildew resistance. Therefore, phytoalexin deficient 

Nd-1 has only camalexin disability but still keeps 

other known defence molecules for protecting itself 

from pathogen. 

Because the Col-pad3 mutant was more 

susceptible than wild-type accession Col-0 following 

inoculation with the isolate Emoy2, the result might 

suggest that camalexin accumulation is important but 

not enough in protection Col-0 from Emoy2. Col-0 

recognises the avirulence product of P. parasitica 

with RPP4 resistance gene. Therefore RPP4 is likely 

camalexin-dependent. 

Thomma et al. (1999) reported that resistance of 

the Col-pad3 mutant to Botrytis cinerea is not altered 

relative to that of wild-type. When Col-pad3 

inoculated with Alternaria brassicicola, however, it 

exhibited enhanced susceptibility compared to the 

wild-type suggesting a role for PAD3 in resistance to 

A. brassicicola. This finding proved that camalexin is 

an important defence components. The same research 

group also reported that PR1, therfore SA, and also 

jasmonate/ethylene-dependent defence responses 

were not impaired in the Col-pad3 mutant following 

inoculation with A. brassicicola. Therefore camalexin 

production appears to be controlled by a pathway that 

exhibits little or no cross-talk with salicylate-, 

ethylene-, and jasmonate-dependent signalling 

events. 

Acknowledgement 

This research was supported financially by Turkish 

Higher Educational Council (YOK) and Çanakkale 

Onsekiz Mart University, Turkey. This article is 

dedicated to first author’s parents for their 

ever-lasting help and support during her education. 

References 

Aarts N, Metz M, Holub E, Staskawicz BJ,Daniels MJ and 

Parker J. Different requirements for EDS1 and NDR1 

by disease resistance genes define at least two R 

gene-mediated signalling pathways in Arabidopsis. 

Proc of the Nat Ac of Sci USA. 96: 10307-10311, 1998. 

Bittner-Eddy P, Can C, Gunn N, Pinel M, Tör M, Crute I, 

Holub E and Beynon J. Genetic and physical mapping 

of the RPP13 locus in Arabidopsis responsible for 

specific recognition of several Peronospora parasitica 

(downy mildew) isolates. Mol Plant-Microbe Int. 12: 

792-802, 1999. 

Bittner-Eddy PD, Crute IR, Holub EB and Beynon JL. 

RPP13 is a simple locus in Arabidopsis thaliana for 

alleles that specify downy mildew resistance to 

different avirulence determinants in Peronospora 

parasitica. The Plant Journal 21: 189-214, 2000. 

Botella MA, Parker JE, Frost LN, Bittner-Eddy PD, 

Beynon JL, Daniels MJ, Holub EB and Jones JDG. 

Three genes of Arabidopsis RPP1 complex resistance

locus recognise distinct Peronospora parasitica 

avirulence determinants. The Plant Cell. 10: 1847- 

1860, 1998. 

Browne LM, Conn KL, Ayer WA and Tewari JP. The 

camalexins: new phytoalexins produced in the leaves of 

Camelina sativa (Cruciferae). Tetrehedron. 47: 3909- 

3914, 1991. 

Dixon RA, Lamb CJ, Sewalt VJH and Paiva NL. Metabolic 

engineering: Prospects for crop improvement through 

the genetic manipulation of phenylpraponoid 

biosynthesis and defence responses- a review. Gene. 

179: 61-71, 1996. 

Glazebrook J. and Ausubel FM. Isolation of 

phytoalexin-deficient mutants of Arabidopsis thaliana 

and characterisation of their interactions with bacterial 

pathogens. Proc of the Nat Ac of Sci USA. 91: 8955- 

8959, 1994. 

Glazebrook J, Zook M, Mert F, Kagan I, Rogers EE, 

Crute IR, Holub EB, Hammerschmidt R and 

Ausubel FM. Phytoalexin-deficient mutants of 

Arabidopsis reveal that PAD4 encodes a regulatory 

factor and that four PAD genes contribute to downy 

mildew resistance. Genetics. 146: 381-392, 1997. 

Holub EB, Beynon JL and Crute IR. Phenotypic and 

genotypic characterisation of interactions between 

isolates of Peronospora parasitica and accessions of 

Arabidopsis thaliana. Mol Plant Mic Int. 7: 223-239, 

1994. 

Holub EH. Organisation of resistance genes in Arabidopsis. 

In: The Gene-For-Gene Relationship in Plant-Parasite 

Interactions. Crute IR, Holub EB and BurdonJJ (Eds). 

CAB International. 5-26, 1997. 

Holub EB and Beynon JL. Symbiology of mouse-ear cress 

(Arabidopsis thaliana) and Oomycetes. Adv in Bot Res. 

24: 227-272, 1997. 

Kúc J. Phytoalexins, stress metabolism, and disease 

resistance in plants. Annu Rev of Phytopathol. 33: 275- 

297, 1995. 

Mansfield JW. Role of phytoalexins in disease resistance. 

In: Phytoalexins. Mansfield JW and Bailey J (Eds). 

Blackie, Glasgow. 1982. 

Mansfield JW. Antimicrobial compounds and resistance: 

the role of phytoalexins and antianticipins. In: 

Mechanisms of Resistance to Plant Diseases. 

Slusarenko AJ, Fraser RSS and VanLoon LC (Eds). 

Kluwer, Amsterdam. 1999. 

Mert-Türk F, Bennett MH, Glazebrook J, Mansfield J and 

Holub E. Biotic and abiotic elicitation of camalexin in 

Arabidopsis thaliana. 7 th International Congress of 

Plant Pathology. Edinburgh, Scotland, UK.1998. 

Mert-Türk F. Quantification of enhanced downy mildew 

susceptibility and camalexin accumulation in the 

mutants of Arabidopsis thaliana. PhD thesis. University 

of London, Imperial College of Science, Technology 

and Medicine, UK. 2001. 


Nam HG, Giraudat J, Den Boer B, Moonan F, Loos WDB, 

Hauge BM and Goodman HM. Restriction fragment 

polymorphism lincage map of Arabidopsis thaliana. 

Plant Cell. 1: 699-705, 1989. 

Oppenheimer DG, Herman PL, Sivakumarin S, Esch J and 

Marks MD. A myb gene required for leaf trichome 

differentiation in Arabidopsis is expressed in stipules. 

Cell. 67: 483-493, 1991. 

Thomma BPHJ, Nelissen I, Eggermont K and Broekaert F. 

Deficiency in phytoalexin production causes enhanced 

susceptibility of Arabidopsis thaliana to the fungus 

Alternaria brassicola. The Plant Journal. 19: 163-171, 

1999. 

Tör M, Holub EB, Brose E, Musker R, Gunn N, Can C, 

Crute IR and Beynon JL. Map position of three loci in 

Arabidopsis thaliana associated with isolate-specific 

recognition of Peronospora parasitica (downy mildew). 

Mol Plant Mic Int. 7: 214-222, 1994. 

Tsuji J, Jackson EP, Gage DA, Hammerschmidt R and 

Somerville SC. Phytoalexin accumulation in 

Arabidopsis thaliana during the hypersensitive reaction 

to Pseudomonas syringae pv. syringae. Plant Physiol. 

98: 1304-1309, 1992.




Narçin Palavan-Ünsal 1 *, Serap Ça¤ 2 , Ergül Çetin 2 and Damla Büyüktunçer 1 

1 Golden Horn University, Faculty of Arts and Sciences, Department of Molecular Biology and Genetics, 

34280, F›nd›kzade, ‹stanbul, Turkey; 2 University of ‹stanbul, Faculty of Science, Department of Biology, 

34460, Süleymaniye, ‹stanbul, Turkey (* author for correspondence) 

Received 29 April 2002; Accepted 24 May 2002 

Abstract 

A new family of endogenous aromatic cytokinin have been discovered by Strnad et al. (1997) and named as 

meta-topolin (mT). The aim of this research is to reveal the relation of mT with senescence in excised wheat leaf 

segments. The effect of mT on protease activity, chlorophyll and soluble protein contents as senescence parameters 

have been investigated. Exogenous application of mT was effective in increasing chlorophyll content. Incubation 

of leaf segments in 0.25, 0.5 and 1.0 mM mT increased the chlorophyll a content by 14, 23, 41 % respectively 

compared to the control leaves on 10 th day of incubation. Chlorophyll b also exhibited the results in the same 

manner. Various concentrations of mT had inhibitory effect on acid and neutral protease activities, specially neutral 

protease activity decreased gradually with the increasing concentration of mT. Fresh weight and soluble protein 

content exhibited linear stimulation by the application of increasing mT concentration. mT treatments prevented the 

DNA degradation during the senecence but these determinations did not depend on variation in concentration. A 

natural aromatic cytokinin mT is very active in the retardation of senescence. mT responded to senescence 

parameters very significantly. The results of this research is exhibited mT as a promising plant growth regulator in 

physiological studies. 

KKeeyy wwoorrddss:: Chlorophyll, meta-topolin, nucleic acid, protease, senescence 

MMeettaa-topolinin bu¤day yapraklar›nda senesensi geciktirmesi 

Özet 

Yeni bir aromatik sitokinin ailesi Strnad ve ark. (1997) taraf›ndan keflfedilmifl ve meta-topolin (mT) olarak 

isimlendirilmifltir. Bu araflt›rman›n amac› bu¤day yapraklar›ndan al›nan segmentlerde mT in senesens ile iliflkisini 

ortaya koyabilmektir. Bu amaçla mT in senesens parametreleri olan klorofil y›k›m›na, proteaz enziminin 

aktivitesine ve protein içeri¤ine olan etkileri araflt›r›ld›. Haricen uygulanan mT klorofil y›k›m›n› önlemede etkin 

bulundu. Yaprak segmentlerinin 0.25, 0.5 ve 1.0 mM mT içinde inkübe edilmesi klorofil a içeri¤ini 10. günde 

kontrol yapraklar›na oranla s›ras› ile % 14, 23 ve 41 oran›nda artt›rd›. Klorofil b içeri¤inde de ayn› do¤rultuda 

sonuçlar elde edildi. mT in farkl› konsantrasyonlar›n›n asit ve nötr proteaz aktivitesini inhibe etti¤i, özellikle nötr 

proteaz aktivitesinin artan mT konsantrasyonu ile dereceli bir flekilde azald›¤› belirlendi. Taze a¤›rl›k ve protein 

içeri¤i de artan mT konsantrasyonu ile birlikte lineer bir yükselifl sergiledi. mT uygulamalar›n›n senesens s›ras›nda 

meydana gelen DNA y›k›m›n› da önledi¤i belirlendi, fakat bu saptamalar konsantrasyon de¤iflimine ba¤›ml›l›k 

göstermedi. Do¤al aromatik sitokinin mT senesensi geciktirmede çok aktif bulundu. mT senesens parametrelerine 

kayda de¤er bir flekilde cevap verdi. Bu araflt›rman›n sonuçlar› mT in fizyolojik araflt›rmalar için çok ümit verici 

bir bitki büyüme düzenleyicisi oldu¤unu ortaya koydu. 

AAnnaahhttaarr ssöözzccüükklleerr:: Klorofil, meta-topolin, nukleik asit, proteaz, senesens 

101

102 Narçin Palavan-Ünsal et al. 

Introduction 

A regulatory role of cytokinins in senescence comes 

from the senescence-delaying effects of cytokinin 

treatments. Because of the potential and realized 

benefits from delaying senescence of various tissues 

with cytokinin treatments numerous studies have 

concentrated around this subject (Naito et al., 1978; 

Gilbert et al., 1980; Nooden and Leopold, 1988; 

Kraus et al., 1993). 

Cytokinin researches had focused for a long time 

on members of the isoprenoid class represented by 

zeatin, isopentenyladenin and related compounds. 

For this reason the aromatic 6-benzylaminopurine 

(BA) and its derivatives were thought to be purely 

synthetic cytokinins. But other cytokinins with an 

aromatic side chain have also been determined and 

identified in different plant tissues. Recently Strnad et 

al. (1997) have discovered a new family of 

endogenous aromatic cytokinin and named as 

meta-topolin (6-[3-hydroxylbenzyl-amino]purine) 

(mT). This compound was first detected in poplar 

leaves and Strnad et al. (1997) adopted the name 

topolin derived from "topol" the Czech word for 

poplar. 

Plant senescence is initiated and accompanied by 

a series of degradative events. In leaves senescence is 

correlated with sharp increases in RNase and protease 

activities which may lead to nucleic acid and protein 

breakdown, followed by disintegration of chloroplast 

structure and ultimately chlorophyll loss (Thimann, 

1980; Stoddart and Thomas, 1982). 

The senescence of leaves involves changes in their 

photosynthetic apparatus. Because yellowing is so 

conspicious, chlorophyll breakdown has served as the 

major parameter for the measurement of leaf 

senescence (Gut et al., 1987; Jenkins et al., 1981; 

Young et al., 1991). Cytokinins are very effective in 

delaying this breakdown indicating that these 

hormones are somehow involved in maintaining the 

photosynthetic apparatus of plant organs. However it 

is now known that there are some exceptions that 

show poor correlation or no correlation at all between 

chlorophyll breakdown and the other characteristic 

symptoms of senescence (Thomas and Stoddart, 

1980). 

One of the early events in leaf senescence is the 

well documented rise in protease activity (Thimann, 

1980). However little is known about the regulation 

of proteolysis itself during leaf senescence. The early 

observations (Richmond and Lang, 1957; Wollgiehn, 

1967) that cytokinins exert parallel effects in 

maintaining protein and nucleic acid levels while 

inhibiting senescence has led to the generalization 

that cytokinins delay senescence by maintaining or 

promoting protein and nucleic acid synthesis. 

The aim of this research is to reveal the relation of 

mT a new aromatic cytokinin, with senescence in 

excised wheat leaf segments. For this purpose we 

have investigated the effect of mT on chlorophyll 

breakdown, protease activity, and soluble protein 

content as main senescence parameters. 


Plant material 

Six first leaf segments (3 cm each) from 10 days old 

wheat (Triticum aestivum) seedlings were floated in 

various concentrations (0.25, 0.5 and 1.0 mM) of mT 

for 10 days in plant growth chamber (8000 lux light 

intensity, 12 h light, 12 h dark photoperiod and 

25±2°C). Distilled water was used for control 

treatments. 

Measurement of chlorophyll content 

For chlorophyll determination leaf segments were 

homogenized in 80 % acetone. The samples were 

centrifuged at 4000 g for 5 min and the optical density 

of the supernatant was read at 663 and 645 nm using 

a spectrophotometer according to the Arnon (1949). 

Initial values of each analysis were measured in 

leaf segments at the start of each experiment. We have 

expressed and discussed all results according to the 

final controls which mean incubated in distilled water 

for 10 days. 

Measurement of soluble protein content 

Soluble protein content was determined by the 

method of Bradford (1976) using bovine serum 

albumin as standard. 

Determination of protease activity 

Six leaf segments (3 cm each) were homogenized in a

prechilled mortar with 2 ml of cold 50 mM phosphate 

citrate buffer (pH 6.0). The homogenates were kept 

in the cold (4°C) for 0.5 h and then centrifuged at 

12000 g for 15 min at 4°C. The clear supernatant 

fraction was assayed for protease activity using 

Azocoll (Calbiochem) as the substrate 

(Kaur-Sawhney et al., 1982). The final 1 ml reaction 

mixture contained 5 mg Azocoll, 0.8 ml of 50 mM 

phosphate-citrate buffer (pH 4.2 for acid protease and 

pH 6.6. for neutral protease) and 0.2 ml of the crude 

enzyme. The tubes stoppered, vortexed and floated in 

a water bath equipped with a shaker and maintained at 

43°C for 3 h. Controls were similarly prepared, 

without enzyme. The reaction was terminated by 

immersing the tubes in an ice bath for 1 h, and the 

tubes were centrifuged to remove the undigested 

Azocoll. All data are expressed as A (520 nm) per g 

fresh weight. The absorbance of the supernatant 

fractions was measured at 520 nm. Each assay was 

replicated five times. 

DNA extraction and electrophoresis 

Approximately 100 mg frozen leaf segments were 

homogenized in liquid nitrogen by the addition of 

extraction buffer (15 % sucrose, 50 mM Tris-HCl pH 

8.0, 50 mM EDTA, 250 mM NaCl). Initial control 

samples were not incubated in distilled water, they 

were extracted directly after excision of leaf segments 

(Walbot, 1988). These extracts were centrifuged at 

6000 g for 5 min at 4°C and pellet was suspended in 

suspension buffer (20 mM Tris HCl, pH 8.0, 10 mM 

EDTA) after that 100 ml sodium lauryl sulfate was 

added and incubated at 70°C for 15 min, then 7.5 M 

ammonium acetate was added to the tubes and left in 

ice for 30 min. After this procedure extracts were 

centrifuged at 18 000 g for 5 min and 3 ml 

isopropanol was added to the supernatants and again 

were left in ice and DNA was precipitated by the 

centrifugation at 17 000 g for 5 min. After this 

procedure pellets were dissolved in Tris-EDTA buffer 

(10 mM Tris HCl pH 8.0, 1 mM EDTA) and RNase (5 

mg/ml) was added to the samples and left at 37°C for 

5 min. Phenol-chloroform mixture was added to each 

tube and centrifuged again at 17 000 g for 10 min. 

Upper phases were transferred to the another tube and 

same procedure was repeated. Again upper phase was 

transferred to the new tube and sodium acetate (pH 

5.2) was added to adjust the final concentration to 0.3 

M and two volume cold ethanol was added to the 

extract and frozen in liquid nitrogen and left to -70°C 

for 30 min. After this process extracts were 

centrifuged at 18 000 g for 10 min and pellets were 

washed with 70 % cold ethyl alcohol, and evaporated 

after that dissolved in Tris-HCl buffer. Isolated DNA 

was determined spectrophotometrically at 260 nm. 

Isolated DNA was analyzed by electrophoresis. 

1.2 % agarose gels were prepared in tris acetate 

EDTA buffer (40 mM tris acetate, pH 8.2, 1 mM 

EDTA) Ethidium bromide (10 mg/ml) was added to 

the gels to be able to observe DNA under UV light. 

Loading buffer contained bromophenol blue (25 %) 

and sucrose (40 %). Electrophoresis was carried out 

with a current of 140 V and 70 mA for two hours. 

Results and Discussion 

Senescence and meta-topolin 103 

In the present study it was established that the fresh 

weight of wheat leaf segments increased gradually 

with the increasing concentration of mT comparing 

with the final control leaves (Figure 1). Incubation of 

leaf segments in 0.25, 0.5 and 1.0 mM mT incresed 

the fresh weight by 4, 6, and 9 % respectively. 

It was also determined that exogenous application 

of mT was effective in preventing chlorophyll 

breakdown during the senescence. There was 54 % 

total chlorophyll loss in final control leaves which 

were senesced compared to initial values on 10th day 

of incubation (Figure 2). Inhibitions of total 

chlorophyll loss by mT was increased with the 

incubation time: Decrease in chlorophyll a content 

was 5 % on 3rd day of incubation, but compared to 

initial value this percentage increased to 41 % 

compared to final control condition on 10th day 

(Figure 2). The chlorophyll b content difference 

between initial and final control leaves was found to 

be 54 %. Incubation of leaf segments (Figure 3) in 

0.25, 0.5 and 1.0 mM mT increased the chlorophyll a 

content by 18, 25, 43 % respectively compared to the 

final control leaves on 10 th day of incubation. In our 

study mT at 1.0 mM concentration has been found to 

be the most effective in the retardation of senescence. 

We have established the same trends for chlorophyll b 

(Figure 3). Total chlorophyll loss was 44 % in final 

control leaves according to the total chlorophyll 

content at initial leaves. The loss of total chlorophyll 

in the final control was greater than that of mT-treated


Figure 1: Effects of meta-topolin on fresh weight of wheat leaf segments. Vertical bars represent standard errors. Each value 

are average of 10 experiments. 

Figure 2: Effects of meta-topolin on chlorophyll a content of wheat leaf segments. Vertical bars represent standard errors. 

Each value are average of 4 experiments. 

Figure 3: Effects of meta-topolin on chlorophyll b content of wheat leaf segments. Vertical bars represent standard errors. 

Each value are average of 4 experiments.






Figure 6: Effects of meta-topolin on soluble protein contents of wheat leaf 5 segments. Vertical bars represent standard errors. 

Each value is average of 4 experiments.


Figure 7: The effect of meta-topolin on total genomic DNA content in excised wheat leaf segments. Each value is 

average of 4 experiments. 

Figure 8: The effect of meta-topolin on the pattern of DNA 

in excised wheat leaf segments. Lane 1: Marker, Lane 2: 

Initial control, Lane 3: 1 mM mT, Lane 4: 0.5 mM mT, Lane 

5: 0.25 mM mT, Lane 6: Final control. 

leaf segments (Figure 4). Similarly many researchers 

have reported that exogenously applied cytokinins 

retarded the loss of photosynthetic pigments during 

the senescence of leaves and cotyledons (Thimann, 

1980; Stoddart and Thomas, 1982; Chen and Kao, 

1991; Jordi et al., 1993; Durmufl and Kad›o¤lu, 1998). 

When excised wheat leaves are incubated in 

distilled water their protease activity increases 

dramatically with the advancement of senescence. 

Increased activity was observed in two commonly 

reported proteases having pH optima at 4.2 and 6.6 

with Azocoll as substrate. The rise in protease 

activities was inhibited when leaves were floated on 

mT solutions (Figure 5). Various concentrations of 

mT had inhibitory effect on acid and neutral protease 

activities, specially neutral protease activity 

decreased gradually with the increasing concentration 

of mT (Figure 5). The inhibition was greater for 

neutral than for acid proteases. The acid protease 

activity was inhibited by 41 % and neutral protease 

activity was inhibited by 49 % after the treatment of 

mT at 1 mM concentration compared to the final 

control leaves. These results indicate that protease 

activity is inhibited by mT. Besides these protease 

activites especially neutral protease activities was 

very low at initial compared to final control leaf 

segments which were already senesced. 

Similarly Anderson and Rowan (1965) have 

established the decrease in protein contents during the 

aging of tobacco leaves. It has also been shown that 

the synthesis of the proteolytic enzymes synthesis 

preceded the senescence period (Martin and Thimann, 

1972; Drivdahl and Thimann, 1977). Besides these 

Thayer et al. (1987) have revealed the necessity of 

protein synthesis to occur senescence signal and also 

the requirement of the synthesis of proteases for 

breakdown of proteins during the senescence. 

Figure 6 shows the changes of soluble protein 

contents in wheat leaf segments treated with mT 

compared to the final control leaves. Total protein 

amounts was 30 % less in final control leaves 

compared to initial protein amounts. All concentration 

of mT used in this research increased the soluble 

protein contents during retarding the senescence 

insignificantly. These results are closely correlated 

with the protease activities. It has been well 

established that cytokinins are effective in retarding

the loss of protein (Tavares and Kende, 1970; 

Lamattina et al., 1987; Kraus et al., 1993). 

Total genomic DNA amount was decreased by 83 

% in final control samples which were senesced for 

10 th days compared to initial control samples (Figure 

8). Whereas, total DNA content increased by the 

treatment with mT compared to final control samples; 

0,25, 0,50 and 1,0 mM mT applications decreased the 

total genomic DNA amounts by 43, 52 and 41 % 

respectively. All data showed that mT treatments 

prevented the DNA degradation during the 

senescence, but there was no significant differences 

depending on concentration of mT (Figure 8). 

Electrophoretic patterns of DNA showed that 

there was no detectable amount of DNA in final 

control samples (Figure 8). However, the level of 

DNA in 1 mM mT treated leaves was almost the same 

as in initial control. These results also showed that mT 

retarded the senescence by inhibiting DNA 

destruction in excised wheat leaves. 

In conclusion natural aromatic cytokinin 

mT is very active in the retardation of senescence of 

excised wheat leaves. The results of this research are 

exhibited mT as a promising plant growth regulator in 

physiological studies. 

Acknowledgement 

We thank to Dr. M. Strnad and his colleaques for the 

generous gift of aromatic cytokinins. This study was 

supported by ‹stanbul University Resarch Fund 

(Project number: B-429/13042000). 

References 

Anderson JW and Rowan KS. Activity of peptidase in 

tobacco leaf tissue in relation to senescence. Biochem 

Jour. 97: 741-746, 1965. 

Arnon DI. Copper enzymes in chloroplasts, 

polyphenoloxidase in Beta vulgaris. Plant Physiol. 24: 

1-15, 1949. 

Bradford MA. A rapid and sensitive method for the 

quantification of microgram quantities of protein 

utilizing the princible of protein - dye binding. 

Anal Biochem. 72: 248-254, 1976. 

Chen SH and Kao CH. Localised effect of polyamines on 

chlorophyll loss. Plant Cell Physiol. 24: 1463-1467, 

1991. 

Drivdahl RH and Thimann KV. The proteases of senescing 


oat leaves. I. Purification and general properties. Plant 

Physiol. 59: 1059-1063, 1977. 

Durmufl N and Kad›o¤lu A. Effect of benzyladenine on 

peroxidase activity during senescence of sunflower 

(Helianthus annuus L.) cotyledons. Phyton. 37: 253- 

261, 1998. 

Gilbert ML, Thompson JE and Dumbroff EB. Delayed 

cotyledon senescence following treatment with 

cytokinin; an effect the level of membranes. Can Jour 

of Bot. 58: 1797-1803, 1980. 

Gut H, Rutz C, Matile P and Thomas H. Leaf senescence in 

a non-yellowing mutant of Festuca pratensis: 

Degradation of carotenoids. Physiol Plant. 70: 659-663, 

1987. 

Jenkins GI, Baker NR and Woolhouse HVW. Changes in 

chlorophyll content and organisation during senescence 

of primary leaves of Phaseolus vulgaris L. in relation to 

photosynthetic electron transport. Jour of Exp Bot. 32: 

1009-1020, 1981. 

Jordi W, Dekhuijzen HM, Stoopen GM and Overbeek JHM. 

Role of other plant organs in gibberellic acid-induced 

delay of leaf senescence in alstroemeria cut flowers. 

Physiol Plant. 87: 426-432, 1993. 

Kaur-Sawhney R, Shih LM, Cegielska T and Galston AW. 

Inhibition of protease activity by polyamines. 

Relevance for control of leaf senescence. FEBS Letters. 

145: 345-349, 1982. 

Kraus TE, Hofstra G and Fletcher RA. Regulation of 

senescence by benzylaminopurine and uniconazole in 

intact and excised soybean cotyledons. Plant Physiol 

and Biochem. 31: 827-834, 1993. 

Lamattina L, Anchoverri V, Conde RD and Lezica RP. 

Quantification of the kinetin effect on protein synthesis 

and degradation in senescing wheat leaves. Plant 

Physiol. 497-499, 1987. 

Martin C and Thimann KV. The role of protein synthesise 

in the senescence of leaves. I. The formation of 

protease. Plant Physiol. 49: 64-71, 1972. 

Naito K, Tsuji H and Hatakkeeyama I. Effect of 

benzyladenine on DNA, RNA, protein and chlorophyll 

contents in intact bean leaves: Differential responses to 

benzyladenine according to leaf age. Physiol Plant. 43: 

367-371, 1978. 

Nooden LD and Leopold AC. Senescence and Aging in 

Plants. Academic Press, New York. 1988. 

Richmond AE and Lang A. Effect of kinetin on protein 

content and survival of detached Xanthium leaves. 

Science. 125: 650-651. 1957. 

Stoddart JL and Thomas H Leaf senescence. In: Nucleic 

Acids and Proteins. Boulter D and Parthier B (Eds). 

Encyclopedia of Plant Physiology, New Series, 

Springer Verlag. 592-636, 1982. 

Strnad M, Hanus J, Vanek T, Kaminek M, Ballantine JA, 

Fussell B and Hanke DE. Meta-topolin, a highly active 

aromatic cytokinin from poplar leaves (Populus x


canadensis Moench., cv. Robusta). Phytochem. 45: 

213-218, 1997. 

Tavares J and Kende H. The effect of 6-benzylaminopurine 

on protein metabolism in senescing corn leaves. 

Phytochem. 9: 1763-1770, 1970. 

Thayer SS, Choe HT, Tang A and Huffaker RC. Protein 

turnover during senescence. In: Plant senescence: Its 

Biochemistry and Physiology. Thomson WW, 

Nothnagel EA and Hufaker RC (Eds). The 

American Society of Plant Physiologists. 71-80, 1987. 

Thimann KV. Senescence in Plants. Thimann KV (Ed). 

CRC Press, Boca Raton FL. 85-115, 1980. 

Thomas H and Stoddart JL. Leaf senescence. Annual 

Review of Plant Physiol. 31: 83-111, 1980. 

Walbot V. Preparation of DNA from single rice seedlings. 

Rice Genetics Newsletter. 5: 149-151, 1988. 

Wollgiehn R. Nucleic acid and protein metabolism of 

excised leaves. Symposium of Soc of Exp Biol. 

21: 231-246, 1967. 

Young AJ, Wellings R and Britton, G. The fate of 

chloroplast pigments during senescence of primary 

leaves of Hordeum vulgare and Avena sativum. 

J Plant Physiol. 137: 701-705, 1991.

Book Reviews 

Ülkü EM‹RO⁄LU, Betül BÜRÜN, Angiospermlerde 

Efley Tipleri ve Döllenme. Mu¤la Üniversitesi 

Yay›nlar›, Mu¤la, 87 sayfa, ISBN: 975-7207-20-9, 

2001. 

Angiospermlerde efley tipleri ve döllenme biyoloji 

biliminin temel konular›n› oluflturmaktad›r. 5 bölüm 

fleklinde düzenlenen bu kitapta önce bitkilerde efley 

durumu ele al›nm›fl, daha sonra gametlerin oluflumu, 

döllenme, embriyo ve endospermin geliflimi 

incelenmifltir. Son bölümde de kendine uyuflmazl›k 

ve erkek k›s›rl›k konular› ayr›nt›l› olarak 

incelenmifltir. Bitki embriyolojisine girifl 

mahiyetindeki bu kitapta tablo ve flemalara yer 

verilerek konular›n daha iyi anlafl›labilmesi 

amaçlanm›flt›r. 

Türkiye’de bu alanlarda Türkçe kaynaklar›n 

eksikli¤i göz önüne al›nd›¤›nda, bu kitab›n biyoloji 

e¤itimi yapan ö¤rencilere faydal› olaca¤› kan›s›nday›m. 

Narçin PALAVAN-ÜNSAL 

Haliç Üniversitesi, 

Moleküler Biyoloji ve Genetik Bölümü 

Ülkü EM‹RO⁄LU, Betül BÜRÜN, Sexual Types 

and Fertilization in Angiosperms. Published by 

Mu¤la University, Mu¤la, 87 pp. ISBN: 975-7207-20- 

9, 2001. 

Sexual types and fertilization constitute basic topics 

in biological sciences. This book arranged as in 5 

chapters, first sexual position in plants was discussed, 

then formation of gametes, fertilization, development 

of embryo and endosperm were investigated. 

Self-incompability and male sterility were investigated 

in detail in the last chapter. It was designed as an 

introductory to plant embryology and subjects were 

explained with schemes and tables to be able to 

examined them in depth. This book is a good 

document in Turkey because of the missing Turkish 

109 

publication in this area, for this reason I recommend 

specially undergraduate biology students. 

Narçin PALAVAN-ÜNSAL 

Golden Horn University, 

Department of Molecular Biology and Genetics 

Ayfle TABAKO⁄LU-O⁄UZ, Hayvan Embriyolojisi. 

‹stanbul Üniversitesi Yay›nlar›, ‹stanbul, 266 sayfa, 

ISBN: 975-404-157-1, 2001. 

Kitap iki ana bölümden oluflmaktad›r. Bu 

bölümlerden birincisi temel embriyoloji bilgilerini 

içermektedir. Bunlar; embriyolojiye girifl ve tarihçe, 

ökaryotlarda genom ve iflleyifl modeli, gametogenez, 

döllenme, segmentasyon ve gastrulasyon, efleysiz 

üreme ve morfogenez bafll›klar› ile ele al›nm›fllard›r. 

‹kinci bölümde ise seçilmifl baz› omurgas›z ve 

omurgal› örneklerin embriyonal geliflimleri 

aç›klanm›flt›r. Bunlar; süngerler, hidra, Turbelleria, 

Annelida, Crustacea, Insecta, Mollusca, 

Echinodermata, Amphioxus, kemikli bal›klar, 

kurba¤a ve piliç embriyolar›n›n geliflimlerini 

içermektedir. Özellikle piliç embriyosunun 

gelifliminde organogenez olay› ayr›nt›l› bir flekilde ele 

al›nm›flt›r. Tüm gruplarda geliflme olaylar› aç›klay›c› 

flekillerle desteklenmifltir. 

Bu kitab›n ö¤retmen ve ö¤rencilere hayvan 

embriyolojisi konusunda önemli bilgiler 

kazand›rmas› aç›s›ndan çok yararl› bir rehber olaca¤› 

kan›s›nday›m. Ayr›ca Türkiye’de bu alanlarda kaynak 

oluflturacak Türkçe eserlerin say›lar› da son derece 

s›n›rl› oldu¤u için, bu kitab› biyoloji ö¤retmeni ve 

ö¤rencilerine öneririm. 



Moleküler Biyoloji ve Genetik Bölümü

110 

Ayfle TABAKO⁄LU-O⁄UZ, Animal Embriyology, 

Published by ‹stanbul University, ‹stanbul 266 pp, 

ISBN: 975-404-157-1, 2001. 

This book consist of two main parts. The first part 

contains basic embryology knowledge such as; 

introduction to embryology and historical background, 

genome and mode of action, gametogenesis, 

fecondation, cleaveage and gastrulation, asexual 

reproduction and morphogenesis. The second part 

contains embryonic development of the some 

choosen animals among both invertebrates and 

vertebrates such as; sponges, hydra, Turbellaria, 

Annelida, Crustacea, Insecta, Mollusca, 

Echinodermata, Amphioxus, bony fishes, Amphibia 

and chick. Organogenesis is given in detail in the 

chick development. All statement are supported with 

a plenty of explanatory figures. 

The book is a valuable guide of animal 

embryology for teacher and students. On the other 

hand, this book is a good document because there are 

very limited Turkish publication in this area. For this 

reason I recommend this book to the biology teachers 

and students. 


Golden Horn University, 

Department of Molecular Biology and Genetics.

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Journal of Cell and Molecular Biology, 


Fen-Edebiyat Fakültesi, 

Ahmet Vefik Pafla Cad. No :1 

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‹stanbul-Turkey 

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References: 

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al. (1990) or (Smith et al., 1990). 

2. In the list, references must be placed in alphabetical 

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cover all situations. The punctuation given must be 

exactly followed. 

Redford IR. Evidence for a general relationship 

between the induced level of DNA double-strand 

breakage and cell killing after X-irradiation of 

mammalian cells. Int J Radiat Biol. 49: 611- 620, 

1986. 

Taccioli CE, Cottlieb TM and Blund T. Ku 80: Product 

of the XRCCS gene and its role in DNA repair and 

V (D) J recombination. Science. 265: 1442-1445, 

1994. 

Ohlrogge JB. Biochemistry of plant acyl carrier 

proteins. In: The Biochemistry of Plants: A 

Comprehensive Treatise. Stumpf PK and Conn EE 

(Ed). Academic Press, New York. 137-157, 1987. 

Weaver RF. Molecular Biology. WCB/Mc 

Graw-Hill.1999. 

2. Only papers published or in press should be cited in 

the literature list. Unpublished results, including 

submitted manuscripts and those in preparation, 

should be cited as unpublished in the text. 

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throughout and checked thoroughly before 

submission. 

P roofs and offprints 

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proofs should be sent back to the Editor within 

three days of receipt, otherwise Editor reserves the 

rights to correct the proofs himself and to send the 

material for publication. 

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free of charge.

Volume contents 

Volume 1, No. 1 

Review article 

Phytoalexins: Defense or just a response to stress? 

Fitoaleksinler: Savunma m› yoksa sadece strese bir tepki mi? 

F. Mert-Türk 


The role of opsinin in phagocytosis by coelomyctes of the earthworm 

DDeennddrroobbaaeennaa vveenneettaa 

Opsininin Dendrobaena veneta’n›n sölom hücrelerinde fagositozdaki rolü 

Y. Kalaç, A. Kimiran, G. Ulako¤lu, A. Çotuk 

Cytological mechanisms of unreduced pollen formation SSoollaannuumm ttuubbeerroossuumm 

L.cv. Morfana 

Solanum tuberosum L.cv. Morfana’da indirgenmemifl polen oluflumunun sitolojik 

mekanizmalar› 

M. Ünal, O. Alp 

Efficiency of the gamma irradiation in the induction of iinn vviittrroo somatic mutations 

In vitro somatik mutasyon oluflturulmas›nda gama radyasyonunun etkisi 

S. Alikamano¤lu 

Analysis of the three STR loci (D16S539, D7S820, D13S317) in a population 

sample of Marmara region of Turkey 

Türkiye, Marmara bölgesinden bir populasyon örne¤inde üç STR lokusunun 

(D16S539, D7S820, D13S317) analizi 

A. H. Çak›r, F. fiimflek, A. Çelebio¤lu, B. Tafldelen 

Inhibitory effect of hepatectomized (35%) mice serum upon the growth of L-cells 

Hepatektomi (%35) uygulanm›fl fare serumunun L-hücrelerinin ço¤almas› üzerine 

bask›lay›c› etkisi 

S. Altun, G. Özcan, M. Topçul 

Book Reviews 


113 

1-6 

7-14 

15-18 

19-24 

25-30 

31-38 

39-42 

43-44

114 

Volume 1, No. 2 




G. Bayçu 



M. Yatin 













G. Ulako¤lu 

Camalexin is not required for the function of RRPPPP11 and RRPPPP1133 resistance genes 

in AArraabbiiddooppssiiss tthhaalliiaannaa inoculated with PPeerroonnoossppoorraa ppaarraassiittiiccaa 

Kamaleksin Peronospora parasitica ile inokulasyonundan sonra Arabidopsis 

thaliana’n›n RPP1 ve RPP13 dayan›kl›l›k genlerinin ifllevleri için gerekli de¤ildir. 





Book Reviews 



Author index 

45-55 

57-67 

69-72 

73-79 

81-85 

87-91 

93-99 

101-108 

109-110 

111-112 

113-114 

115

Author index 

Aktaç T. 69 

Alikamano¤lu S. 19 

Alp O. 15 

Altun S. 31, 73 

Bakar E. 69 

Bayçu G. 45 

Büyüktunçer D. 101 

Ça¤ S. 101 

Çak›r AH. 25 

Çelebio¤lu A. 25 

Çetin E. 101 

Çotuk A. 7 

Erensoy N. 81 

Holub EB. 93 

Kalaç Y. 7 

Kimiran A. 7 

Mert-Türk F. 1, 93 

Özalpan A. 81 

Özcan Ar›can G. 31, 73, 81 

fiimflek F. 25 

Tafldelen B. 25 

Topçul M. 31, 73, 81 

Ulako¤lu G. 7, 87 

Ünal M. 15 

Ünsal-Palavan N. 101 

Yatin M. 57 

115


CONTENTS Volume 1, No. 2, 2002 




G. Bayçu 



M. Yatin 











The effect of adriamycin on Ehrlich ascites tumor cells in vitro and in vivo 


G. Ulako¤lu 

Camalexin is not required for the function of RPP1 and RPP13 resistance genes in 

Arabidopsis thaliana inoculated with Peronospora parasitica 

Kamaleksin Peronospora parasitica ile inokulasyonundan sonra Arabidopsis thaliana’n›n 

RPP1 ve RPP13 dayan›kl›l›k genlerinin ifllevleri için gerekli de¤ildir. 


Retardation of senescence by meta-topolin in wheat leaves 



Book Reviews 



Author index 

45-55 

57-67 

69-72 

73-79 

81-85 

87-91 

93-99 

101-108 

109-110 

111-112 

113-114 

115

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