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Title: An integrated positional and functional approach for identifying ovarian cancer tumour suppressor genes on chromosome 11p
Author: Blenkiron, Cherie
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2003
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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.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available