Evidence for the inactivation of multiple replicative lifespan genes in immortal human squamous cell carcinoma keratinocytes

Abstract

Human keratinocyte immortality is genetically recessive to the normal phenotype of limited replicative lifespan and appears to require the dysfunction of p53 and the cyclin D-Cdk inhibitor p16. In order to test for the inactivation of other candidate replicative lifespan genes in the immortal cells of human tumors, we developed a series of mortal and immortal keratinocyte cultures derived from neoplastic lesions of the head and neck which were amenable to molecular genetic analysis by the loss of heterozygosity (LOH) technique. The results indicate that keratinocyte immortalization in head and neck squamous cell carcinoma (SCC-HN) development involves the inactivation of at least two further pathways to senescence and four in all. Chromosomes 1, 4 and 7 carry genes representing immortality complementation groups C, B and D respectively and immortal keratinocytes showed LOH at either 4q32-q34 between D4S1554 and D4S171 (group B) or 7q31 (group D) but never 1q25 (group C). These results tentatively suggest that the genes responsible for the immortality complementation groups encode proteins on the same pathway to senescence. In addition, all of the immortal keratinocyte lines possessed high levels of telomerase activity and a suppressor of telomerase activity has been mapped to the short arm of chromosome 3p. Five out of eight lines showed LOH at 3p21.2-p21.3, a region which may carry a gene capable of suppressing SCC-HN telomerase. However, alternative mechanisms of telomerase reactivation were also suggested by our results. None of the above genetic alterations were seen in seven senescent neoplastic keratinocyte cultures. Other loci harbouring antiproliferative genes implicated in replicative lifespan showed few or no alterations and any alterations seen were additional to those described above.

Characterization of the cyclin-dependent kinase inhibitory domain of the INK4 family as a model for a synthetic tumour suppressor molecule

“…The very low turnover of p16 mRNA and the observations of high levels of p16 in late passage number ®broblasts and keratinocytes are both in line with the idea that once p16 expression is induced it will lead to an irreversible exit from the cell cycle as part of the senescence mechanism Loughran et al, 1996;Alcorta et al, 1996). This hypothesis is supported by data suggesting the existence of a gene involved in senescence mapping to the 9p21 locus (Loughran et al, 1994) and the loss of senescence in p16 7/7 mouse embryo ®broblasts .…”

Analysis of the CAVEOLIN-1 gene at human chromosome 7q31.1 in primary tumours and tumour-derived cell lines

“…However, there appears to be no correlation between the presence of wild-type Caveolin-1 and allele loss on chromosome 7 since the cell lines COLO357, MDA-MB-231, OVCAR5, and TMSG all demonstrate chromosome 7 LOH which encompasses the CAVEO-LIN-1 locus (unpublished data), but with the exception of COLO357 express wild-type Caveolin-1. This ®nding has also been extended to a series of head and neck squamous cell carcinoma derived cell lines (unpublished data) that we have established and analysed for allele loss (Loughran et al, 1997).…”

“…Here, we report the mapping of the gene encoding Caveolin-1 to human chromosome 7q31, in proximity to the marker D7S522, which has been mapped to q31.1. A number of studies has revealed a high incidence of loss of heterozygosity (LOH) at 7q31 in a broad range of human tumours, including carcinomas of the breast (Bieche et al, 1992;Zenklusen et al, 1994a), colon (Zenklusen et al, 1995a), kidney (Shridhar et al, 1997), ovary (Kerr et al, 1996;Koike et al, 1997;Zenklusen et al, 1995b), pancreas (Achille et al, 1996), prostate (Takahashi et al, 1995;Zenklusen et al, 1994b), and stomach (Kuniyasu et al, 1994), as well as squamous cell carcinomas of the head and neck (Zenklusen et al, 1995a, Loughran et al, 1997. The highest frequency of LOH was observed at D7S522.…”

Accumulation of p16INK4a in mouse fibroblasts as a function of replicative senescence and not of retinoblastoma gene status

“…On the other hand, p16 has many of the attributes expected of a senescence mediator and it has recently been reported that ®broblasts from p16 nullizygous mice immortalize at a very high frequency, presumably indicative of a failure to senesce (Serrano et al, 1996). Moreover, the higher frequency of p16 deletions and mutations observed in human tumour cell lines as opposed to primary tumours likely re¯ects selection in culture, as a means of escaping senescence, and several studies have provided good evidence for a correlation between loss of p16 function and immortalization (Loughran et al, 1994(Loughran et al, , 1996Rogan et al, 1995;England et al, 1996;Noble et al, 1996;Rezniko et al, 1996). Although the current data imply that this selective pressure may be less critical for mouse cells, there is evidence that the mouse p16 gene also acts as a tumour suppressor.…”