Extensive genetic studies have documented frequent alterations affecting various chromosomal regions in CC. LOH and CGH analyses have consistently shown frequent loss of genomic material in a number of chromosomal regions suggesting the presence of potential tumor suppressor genes at these sites [4, 6, 15, 16]. Of these, chromosome 5p deletions have been noted to be very frequent in invasive CC [5–7]. However, the locations of exact regions of LOH are not known because of a lack of systematic and high-resolution deletion mapping of the 5p region in CC. We have previously identified that markers mapped to 5p15 regions are more frequently deleted [4, 7]. To further characterize the 5p deletions in CC, we performed a high-density LOH mapping in the present study. We found high frequency of LOH and identified two minimal regions of deletions at 5p15.3 and 5p15.2-15.3.
The 5p15.3 minimal deletion spans between 0–5.5 cM genetic distance. Consistent with this pattern, the markers D5S392 and D5S417 exhibited the highest frequency (53.8% and 53.6%, respectively) of LOH among all the tested loci. The genomic size of the 5p15.3 deletion interval is approximately 3.2 Mb. A database search has identified three candidate genes, PDCD6, TERT, and TRIP13, with suggestive tumor suppressor gene function. The programmed cell death gene, PDCD6, is a member of the family of intracellular Ca2+-binding proteins and a part of the apoptotic machinery controlled by T-cell receptor (TCR), Fas, and glucocorticoid signals [11, 17]. TERT encodes a reverse transcriptase required for the replication of chromosome termini and plays a role in telomere elongation. Although telomerase expression is a hallmark of cancer, the mice lacking the RNA component of telomerase (mTERC) exhibit progressive telomere shortening and chromosomal instability associated with epithelial tumors . Consistent with the established role of this enzyme in cell immortalization and cancer pathogenesis , we did not find expression in normal cervix while it was abundantly present in all eight cell lines studied (Fig. 3). Recently, it has been shown that the hTERT amplification and over expression is common in CC . TRIP13, thyroid receptor interacting protein 13, is a transcription factor that regulates expression of a variety of specific target genes including the human papillomavirus type 16 (HPV16) E1 protein . Similar to TERT, the TRIP13 also showed very low expression in normal cervix and highly elevated levels of expression in CC cell lines (Fig. 3). Presence of these genes at the 5p15.3 interval suggests that they may be target TSGs of these deletions in CC. However, expression analysis of these genes did not provide evidence of down-regulation in CC cell lines.
The second site of minimal deletion at 5p15.2-15.3 between 10.7 and 17.7 cM genetic distance spans approximately 2.5 Mb genomic region. A database search has identified POLS, a topoisomerase-related function protein-4-1, gene that encodes polymerase (DNA directed) sigma within the deleted region. The yeast homolog TRF4 plays a critical role in chromosome segregation by coordinating between DNA replication and sister chromatid cohesion . Analysis of expression of this gene in CC cell lines did not show evidence of down regulation. Although the genes tested so far showed no deregulated expression, it remains to be seen whether any of these genes or the others mapped to the minimal deleted regions are targets of mutations in CC. However, the present study identified two discrete sites of deletions at 5p15.3 and 5p15.2-15.3 that harbor putative TSGs important in CC development providing a basis for their identification.
5p deletions also have been reported in precursor CIN lesions with the frequencies between 14.7 and 42% [7–10]. We have previously shown, in a different geographical population, that 21% of CINs had 5p deletions. Based on the follow-up studies, we suggested that 5p genetic deletions might identify high-risk lesions for progression to invasive cancer . In a more recent study it was also shown that the 5p deletions were significantly more frequent in dysplasias that were recurrent compared to non-recurrent dysplasias . These data therefore suggest that 5p genetic alterations occur early in cervical carcinogenesis and they may be critical to the development of invasive cancer. Supporting previous reports, in the present study, we detected LOH at 5p15 in 16% of the high-grade CINs (Table 2).
The role and mechanisms involved in high-risk HPV induced genomic instability has been of a considerable speculation [reviewed in ref. ]. High-risk HPV E6 and E7 gene expression can independently immortalize various human cell types in vitro. The E6 gene interacts with p53 and an apoptotic protein BAK, followed by its degradation, which results in chromosomal instability and resistance to apoptosis [21, 22]. The E7 protein interacts with pRB followed by its degradation . The p53 and pRB proteins have been shown to play multiple roles in cell growth and in genomic integrity. There have been contradictory reports on targeted 5p deletions in relation to HPV infection [5, 24]. Ku and co-workers have shown that 5p deletions occur in significantly high proportion of CC infected with HPV 16 compared to low-risk HPVtypes . Contradictory to this, another study has shown that 5p deletions are independent of HPV16 and HPV18 infection . In the present study, we did not find any significant differences in 5p LOH between patients harboring HPV16 and other HPV types. However, we found that patients harboring HPV16 in combination with other HPV types showed significant increase in 5p loss compared to patients harboring HPV16 alone. These data, therefore, suggest that multiple high-risk HPV infections may have synergistic effect in causing genomic instability. Thus, currently it is unclear the role of high-risk HPV types in causing 5p genomic instability. Further studies are needed to address this question.