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We have compared the induction of apoptosis and cytokine release by UVB and gamma-radiation in primary (untransformed) and in two immortalized human epithelial/keratinocyte cell lines, HaCaT and KB (KB is now known to be a subline of the ubiquitous keratin-forming tumour cell line HeLa and we therefore designate it HeLa-KB). In both the primary and the immortalized cell lines apoptosis and release of the inflammatory cytokine interleukin-6 are induced rapidly following UVB irradiation. In contrast, only the immortalized cells undergo apoptosis and release interleukin-6 after gamma-irradiation and here the onset of apoptosis and cytokine release are delayed. The same distinction between primary and immortalized cells was observed when double-strand breaks were induced with the anticancer drug mitoxantrone, which stabilizes topoisomerase II-cleavable complexes. We suggest that immortalization may sensitize keratinocytes to the apoptogenic effect of ionizing radiation or mitoxantrone by deregulating normal cell cycle checkpoints. In both human keratinocytes and fibroblasts, cell killing, as assayed by loss of colony-forming ability, is not coupled to apoptosis. Immortalization increases resistance to gamma-radiation killing but sensitizes to apoptosis. In contrast, although immortalization also sensitizes to UVB-induced apoptosis, it does not affect UVB-induced cell killing. Apoptosis unambiguously indicates death at the single cell level but clonal cell survival integrates all the cellular and genetic processes which prevent or permit a scorable clone to develop.

We have selected two N-methyl-N-nitrosourea (MNU)-resistant derivatives of the SV40-transformed, alkyltransferase-deficient (Mex-) human fibroblast cell line MRC5V1. Both derivatives remain Mex-. They are cross-resistant to methylmethanesulphonate (MMS) and 6-thioguanine (6TG) but not 2,6-diaminopurine. They show increased sensitivity to the bifunctional chloroethylating agent mitozolomide (MTZ). We have transfected MRC5V1 and one of our MNU-resistant lines with the bacterial O6-methylguanine (O6-MeG)-DNA methyltransferase (ada) gene. Transfectants of MRC5V1 are significantly more resistant to MNU but exhibit only a small increase in resistance to MMS and MTZ. Transfectants of the MNU-resistant derivative exhibit only a small additional increase in resistance to MNU, no further increase in resistance to MMS and a large increase in resistance to MTZ. The pattern of resistance to cytotoxic agents of these transfectants suggests that a second mechanism of resistance to MNU, independent of alkyltransferase expression, is operating in our resistant lines. This mechanism apparently enables the cells to tolerate O6-MeG and 6TG, but not chloroethyl adducts in their DNA.