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.

The production of fibronectin, laminin and glycosaminoglycans (GAGs) has been investigated in normal and SV40-transformed human epidermal keratinocytes. Normal keratinocytes produced small amounts of laminin and fibronectin and three classes of GAGs; hyaluronic acid, heparan sulphate and chondroitin sulphate, hyaluronic acid being the major GAG. The SV40-transformed keratinocytes retained their cell-surface laminin and fibronectin, and in some cases showed elevated levels when compared to normal keratinocytes. All the transformants showed a shift in GAG production from hyaluronic acid to heparan sulphate, so that in all SV40-transformed keratinocytes heparan sulphate was the major GAG. These results demonstrate that the loss of extracellular matrix, which normally occurs in transformed fibroblastic cells, is not a feature of SV40-transformed human keratinocytes.

No abstract

Two human cell lines derived from squamous cell carcinomas (SCCs) of the epidermis, SCC-12 clone F and SCC-13 clone Y, were made to be independent of the Swiss 3T3 feeder layer to perform somatic-cell genetic experiments. We fused these SCC lines with normal human fibroblasts, and all resulting hybrids senesced after completing 12-17 population doublings, suggesting that in part, immortalization of the keratinocyte during SCC development results from the loss of gene function. We also tested whether these two SCC lines mapped to known complementation groups for immortality by fusing them with representatives of groups A (GM847), B (HeLa), and C (143B), but most of these hybrids were indistinguishable from those derived from homotypic crosses set up as immortal hybrid controls. As reported by others, fusions of cell lines from different complementation groups-143B (group C) x HeLa (group B) or GM847 (group A) x Hela (group B)--resulted in predominantly senescent hybrids. Our results confirmed and extended previous observations by others that the phenomenon of senescence is dominant to that of immortality, but they did not allow us to assign either of the SCC lines we studied to a complementation group for immortality.

Desmoglein-3 (Dsg3), the Pemphigus Vulgaris (PV) antigen (PVA), plays an essential role in keratinocyte cell-cell adhesion and regulates various signaling pathways implicated in the pathogenesis the PV blistering disease. We show here that expression of Dsg3 may directly influence p53, a key transcription factor governing the response to cellular stress. Dsg3 depletion caused increased p53 and apoptosis, an effect that was further enhanced by UV and mechanical strain and reversed by Dsg3 gain-of-function studies. Analysis in Dsg3-/- mouse skin confirmed increased p53/p21/caspase-3 compared to Dsg3+/- control in vivo. This Dsg3-p53 pathway involved YAP since Dsg3 forms a complex with YAP and regulates its expression and localization. Analysis of PV patient samples detected increased p53/YAP with diffuse cytoplasmic and/or nuclear staining in cells surrounding blisters. Treatment of keratinocytes with PV sera evoked pronounced p53/YAP expression. Collectively, our findings establish a novel role for Dsg3 as an anti-stress protein, via suppression of p53 function, suggesting that this pathway, involving YAP-Hippo control of skin homeostasis, is disrupted in PV.