EVIDENCE FOR THE INACTIVATION OF MULTIPLE REPLICATIVE LIFE-SPAN GENESIN IMMORTAL HUMAN SQUAMOUS-CELL CARCINOMA KERATINOCYTES

Human keratinocyte immortality is genetically recessive to the normal phenotype of limited replicative lifespan and appears to require the d ysfunction 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 a nd immortal keratinocyte cultures derived from neoplastic lesions of t he 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 respe ctively and immortal keratinocytes showed LOH at either 4q32-q34 betwe en D4S1554 and D4S171 (group B) or 7q31 (group D) but never 1q25 (grou p C). These results tentatively suggest that the genes responsible for the immortality complementation groups encode proteins on the same pa thway to senescence. In addition, all of the immortal keratinocyte lin es possessed high levels of telomerase activity and a suppressor of te lomerase activity has been mapped to the short arm of chromosome 3p. F ive 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, altern ative mechanisms of telomerase reactivation were also suggested by our results. None of the above genetic alterations were seen in seven sen escent neoplastic keratinocyte cultures, Other loci harbouring antipro liferative genes implicated in replicative lifespan showed few or no a lterations and any alterations seen were additional to those described above.