2004 Summer Meeting - Amsterdam - The Pathological Society of ...

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TSLC1 Is A Tumor Suppressor And Marker For Invasion In
Cervical Neoplasia
RDM Steenbergen 1 , D Kramer 1 , BJM Braakhuis 1 , PL Stern 4 ,
RHM Verheijen 1 , CJLM Meijer 1 , PJF Snijders 1
1 VU Medical Center, Amsterdam, Netherlands, 2 VU Medical Center,
Amsterdam, Netherlands, 3 VU Medical Center, Amsterdam, Netherlands, 4
Paterson Institute, Manchester, United Kingdom, 5 VU Medical Center,
Amsterdam, Netherlands, 6 VU Medical Center, Amsterdam, Netherlands, 7
VU Medical Center, Amsterdam, Netherlands
Cervical carcinogenesis is initiated by a high-risk HPV (hr-HPV) infection and
the progression through premalignant lesions (CIN) to invasive cancer is driven
by additional genetic alterations. Here we studied the role of the tumor
suppressor gene TSLC1 (tumor suppressor in lung cancer 1). TSLC1 was found
to be silenced in 91% (10/11) of cervical cancer cell lines. Promoter
hypermethylation appeared the major mode of TSLC1 silencing. A mechanistic
involvement of TSLC1 downregulation was supported by TSLC1’s ability to
suppress both anchorage independent growth and tumor growth of cervical
cancer cells (SiHa). Furthermore, TSLC1 promoter hypermethylation was
detected in 58% (30/52) of cervical carcinomas and 35% (7/20) of high-grade
CIN lesions, but not in low-grade CIN lesions (n=12) and normal cervix (n=9).
Interestingly, TSLC1 promoter hypermethylation could be detected in archival
cervical smears of women with cervical cancer taken up to 7 years before
cancer diagnosis.
In conclusion, these data show that TSLC1 silencing is an important and highly
frequent event in the transition of hr-HPV containing high-grade CIN lesions to
invasive cervical cancer. Hence, testing for TSLC1 silencing in cervical smears
may provide a powerful tool to identify women having CIN lesions with
invasive potential.
198
Cyclooxygenase-2 inhibition decreases colon cancer cell
motility through modulating epidermal growth factor receptor
transactivation
N A Banu 1 , A Buda 1 , S Chell 2 , D Qualtrough 2 , D Elder 3 , M
Moorghen 1 , C Paraskeva 2 , M Pignatelli 1
1 Division of Histopathology, School of Medical Sciences and Bristol
Royal Infirmary, Bristol, United Kingdom, 2 Department of Pathology &
Microbiology, University of Bristol, Bristol, United Kingdom, 3
Department of Biochemistry, School of Medical Sciences, University of
Bristol, Bristol, United Kingdom
Overexpression of cyclooxygenase (COX)-2 and increased prostaglandin (PG)
levels promote tumour progression in colorectal cancer. PGs have been shown
to increase motility through transactivation of the epidermal growth factor
receptor (EGFR) in vitro.
We investigated the effect of the selective COX-2 inhibitor NS-398
on the migration of colorectal cancer cells and examined whether this effect
was associated with changes in EGFR activation. The effect of NS-398
treatment on migration of HT29, HCA7, HCT116 cell lines was studied using a
trans-well filter assay. Expression of COX-2 and PGE 2 receptors was assessed
by Western blot and RT-PCR respectively. PGE 2 concentrations after NS-398
treatment were estimated by ELISA. EGFR phosphorylation levels were
analysed by immunoblotting. Treatment with 5 and 10 µM of NS-398 (24
hours) reduced PGE 2 levels and decreased cell migration in the COX-2 positive
cell lines (HT29, HCA7). PGE 2 addition abolished the effect of NS-398 and
restored cell motility. COX-2 protein levels were unaltered following NS-398
treatment. NS-398 reduced EGFR phosphorylation and AG1478 treatment
reduced PGE 2-stimulated motility of COX-2 positive cells demonstrating that
PGE 2 acts via EGFR signalling pathway.
Our results showed that functional inhibition of COX-2 by NS-398
reduces migration of colorectal cancer cells. This effect is associated with a
perturbation of the EGFR signalling pathway activated by PGE 2.
199
Expression Pattern Of DNA Double Strand Break Repair
Proteins BRCA1, BRCA2, ATM And RAD51 In Normal
Human Tissues
L Barker 1 , M Dattani 2 , D Duncan 2 , S Gray 1 , P Quirke 2 , H
Grabsch 2
1 Dept of Histopathology, Leeds General Infirmary, Leeds, United
Kingdom, 2 Academic Unit of Pathology, University of Leeds, Leeds,
United Kingdom
All cells are under constant genotoxic stress and have developed DNA repair
mechanisms to maintain the integrity of the genome. One of the most
detrimental forms of DNA damage is the double-strand break [DSB]. The
expression of BRCA1, BRCA2, ATM and RAD51, proteins that play a crucial
role in DSB repair, has not been characterised comprehensively in normal
human tissues.
We studied BRCA1, BRCA2, ATM and RAD51 by immunohistochemistry on
tissue microarrays constructed from 36 different types of normal human tissue.
These tissues originated from the gastrointestinal, urinary, and male and female
genital tracts, lymphoid system, muscle, nervous system, endocrine organs,
skin, breast, placenta, and salivary gland. The distribution of the positively
stained cells within the tissue as well as the subcellular localisation of the
protein was noted.
BRCA1 and RAD51 were expressed in all tissues. BRCA2 expression was
observed in all tissues except prostate. ATM expression was observed in all
tissues except liver. Within each tissue, all proteins were expressed in nearly all
mesenchymal and epithelial cells with no particular pattern. Subcellular
localisation of BRCA1 and ATM was predominantly nuclear, that of BRCA2
and RAD51 predominantly cytoplasmic.
The widespread expression of DSB repair proteins in human tissues is
consistent with their vital function in protecting the cell from the consequences
of DNA damage. The lack of expression of some of the proteins in liver and
prostate is unexpected and warrants further investigation. Our study provides
the necessary baseline data with which to compare the expression pattern of
these proteins in different diseases.
200
High Resolution Analysis Of DNA Copy Number Changes In
Malignancies With Chromosome 20q Gains
J. Coffa 1 , M.A.JA Hermsen 1 , B. Carvalho 1 , J.P. Schouten 2 ,
G.A. Meijer 1
1 Department of Pathology, VU University medical center, Amsterdam,
Netherlands, 2 MRC Holland, Amsterdam, Netherlands
DNA copy number changes at chromosome 20 frequently occur in solid
cancers, with a 800kb region at 20q13.2 as the locus for a putative oncogene. In
addition, also areas outside 20q13 frequently show gains. Therefore, we tested
the presence of DNA copy number changes for multiple genes in a series of
malignancies, using multiplex ligation-dependent probe amplification (MLPA).
DNA of 43 colorectal cancers, 15 gastric adenocarcinomas, 5 Barrett
carcinomas, 6 breast carcinomas, 2 oesophagal squamous cell carcinomas, 1
retinoblastoma and 5 different colon cellines was analysed with a dedicated
oligonucleotide probe set for MLPA, containing 28 genes on chromosome 20
and 13 reference genes spread over the genome was designed.
All tumours showed abnormalities on 20q, including gains (ratio>1,3) for more
than 80% of all probes in 50% of the cases. Discrete amplifications (ratio >2,5)
were found at 20q11 and 20q13.2, and sometimes at 20q13.3.
High resolution copy number analysis of different malignancies with gain of
chromosome 20q revealed that approximately 50% of the cases in addition to
gain of 20q also have amplification of smaller regions that seem to concentrate
around 20q11, 20q13.2 and 20q13.3.
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