Ovarian cancer represents the most lethal gynaecological malignancy in the UK.
Numerous tumour suppressor genes (TSG) are postulated to be involved in the
aetiology of epithelial ovarian cancer (EOC). Cytogenetic analyses of cancer cells by
methods such as LOH and CGH, have identified regions of genomic aberration.
Allele loss on chromosome 1 lp has frequently been implicated in ovarian cancers,
suggesting the presence of TSGs in these regions.Ovarian cancer cell line OVCAR3 has lost a whole copy of chromosome 11. The
remaining copy is fragmented, rearranged and duplicated. Transfer of normal
chromosome 11 into OVCAR3 by Microcell Mediated Chromosome Transfer
(MMCT) produced microcell hybrids that display suppression of growth and
cellular migration in vitro and inhibition of tumour growth in vivo. Analysis of
revertant clones was unable to further minimise regions harbouring candidate
TSGs.Subsequently, mRNA populations from OHN, a clonal derivative of the OVCAR3
parent line, and from 110H2.1, a growth suppressed microcell hybrid, were used
for expression difference analysis by Differential Display RT-PCR (DDRT-PCR),
cDNA-Representational Difference Analysis (cDNA-RDA) and cDNA high density
filter array (HDFA). In all, these techniques identified 159 up and 162 down
regulated genes with respect to growth suppression.Quantitative real time RT-PCR was used to validate expression differences in 178
transcripts. We identified, in total, 12 validated upregulated products and 4
validated downregulated products.Of the 12 upregulated products associated with growth suppression, 4 were
localised on chromosome 11, three at llpl5. These were cathepsin D (CTSD),
proteasome subunit PSMD13, ribosomal subunit RPL27A on 11 p 15 and aB
crystallin (CRYAB) on llq23. All were shown to have decreased expression in
several ovarian cancer cell lines and primary tumours. Furthermore, a tight
correlation was observed between the expression of PSMD13 and RPL27A in cell
lines and primary ovarian tumours. Low expression of CTSD and CRYAB were
associated with adverse survival in patients with ovarian cancers.The genes downregulated in association with growth suppression, and therefore of
potentially oncogenic function, were RALDH2, IGFBP2 and 2 novel cDNAs.
When examined on cell line and primary tumour panels, these genes did not
however appear to demonstrate a global increase in expression over that of normal
OSE.An extensive LOH analysis of 87 ovarian tumours and their matched normal
samples was then performed. Thirty-nine microsatellite markers spanning 19.8Mb
on 1 lp 15 were used in the most comprehensive analysis in ovarian cancer to date.
Loss of the complete region was common (24%) and peaks of high LOH (>35%)
were seen for 12 markers. Six microsatellite markers showed an association with
one or more clinicopathological variables (p<0.01). Nine minimal regions of LOH
were found.PSMD13 and CTSD were both found within these regions of LOH as characterised
by the markers D11S2071 and D11S922. RPL27a resides on llpl5.4 near the
marker D11S932 which was not located within a minimal region of loss but LOH
of that marker was significantly associated with advanced FIGO stage (p=0.0001).
This approach has demonstrated that the integration of functional and positional
molecular genetic techniques can co-operate in the identification of candidate
ovarian cancer TSGs
