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62 Mustafa Yatin 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
Page 1 and 2: STANBUL 1998 VOLUME 1 • NO. 2 •
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Page 5 and 6: 46 Gülriz Bayçu Cd Contamination
Page 7 and 8: 48 Gülriz Bayçu Figure 2: Optimiz
Page 9 and 10: 50 Gülriz Bayçu PC production was
Page 11 and 12: 52 Gülriz Bayçu plants and fungi
Page 13 and 14: 54 Gülriz Bayçu References Abraha
Page 17 and 18: cells (cancer), inflammatory sites
Page 19: 1991, Russel and Synder, 1968; Pegg
Page 23 and 24: chemotherapy and lead to a very lar
Page 25 and 26: Thomas T and Thomas T. Polyamines i
Page 27 and 28: 70 Tülin Aktaç and Elvan Bakar Ma
Page 29 and 30: 72 Tülin Aktaç and Elvan Bakar Ak
Page 31 and 32: 74 Seyhan Altun et al. Altun (1996)
Page 33 and 34: 76 Seyhan Altun et al. increased a
Page 35 and 36: 78 Seyhan Altun et al. PH ratio and
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Page 55 and 56: 102 Narçin Palavan-Ünsal et al. I
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Page 63 and 64: 110 Ayfle TABAKO⁄LU-O⁄UZ, Anima
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Page 67 and 68: 114 Volume 1, No. 2 Review articles
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