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Evidence for the inactivation of multiple replicative lifespan genes in immortal human squamous cell carcinoma
O Loughran1, L J Clark, J Bond
1CRC Beatson Laboratories, Beatson Institute for Cancer Research, Garscube Estate, Bearsden, Glasgow, Scotland.
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.
Insights
Human keratinocyte immortalization in head and neck cancers involves inactivating multiple senescence pathways. Genetic analysis identified new chromosomal regions linked to this process and telomerase regulation.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Human keratinocyte lifespan is limited, with immortality requiring p53 and p16 dysfunction.
- Neoplastic lesions of the head and neck provide models for studying keratinocyte immortalization.
Purpose of the Study:
- To identify additional genes and pathways involved in keratinocyte immortalization in head and neck squamous cell carcinoma (SCC-HN).
- To investigate the role of chromosomal alterations and telomerase activity in SCC-HN development.
Main Methods:
- Development of mortal and immortal keratinocyte cultures from head and neck neoplastic lesions.
- Molecular genetic analysis using loss of heterozygosity (LOH) technique.
- Assessment of telomerase activity and mapping of telomerase suppressors.
Main Results:
- Keratinocyte immortalization in SCC-HN involves at least four pathways to senescence, with inactivation of genes on chromosomes 4 (group B) and 7 (group D).
- Loci on chromosome 1 (group C) did not show alterations, suggesting genes in complementation groups B, C, and D may act in the same senescence pathway.
- All immortal lines had high telomerase activity; LOH at 3p21.2-p21.3 in five lines suggests a potential telomerase suppressor gene, though alternative reactivation mechanisms exist.
Conclusions:
- Keratinocyte immortalization in SCC-HN is a complex process involving the inactivation of multiple senescence pathways and potential telomerase regulation.
- Specific chromosomal regions (4q, 7q, 3p) are implicated in SCC-HN development, offering targets for further research.
- Senescent neoplastic keratinocytes did not exhibit the same genetic alterations, highlighting distinct mechanisms in immortalization versus senescence.