Effect of ultraviolet light, methyl methanesulfonate and ionizing radiation on the genotoxic response and apoptosis of mouse fibroblasts lacking c-Fos, p53 or both.

c-Fos and p53 are DNA damage-inducible proteins that are involved in gene regulation, cell cycle checkpoint control and cell proliferation following exposure to genotoxic agents. To investigate comparatively the role of c-Fos and p53 in the maintenance of genomic stability and the induction of apoptosis, we generated mouse fibroblast cell lines from knockout mice deficient for either c-fos (fos -/-) or p53 (p53-/-) or for both gene products (fosp53-/-). The sensitivity of these established cell lines was compared with the corresponding wild-type cells as to the cytotoxic, clastogenic and apoptosis-inducing effects of ultraviolet (UV-C) light and methyl methanesulfonate (MMS). Additionally, we analysed the frequency of apoptosis of the cell lines after treatment with ionizing radiation (IR). We observed c-fos-/-, p53-/- and fosp53-/- cells to be more sensitive than wild-type cells with respect to cell death, as measured in a cytotoxicity (MTT) assay. Regarding apoptosis, all deficient cell lines displayed hypersensitivity to UV-C light, MMS and IR. With chromosomal aberrations as the endpoint, the sensitivity of the double-knockout cells was between wild-type and single-knockouts. The results indicate that both c-Fos and p53 play an important role in protecting fibroblasts against a broad range of genotoxic agents. The results also show that, in fibroblasts, apoptosis induced by UV-C light, MMS and IR does not require p53 and that, in this cell type, p53 rather protects against DNA damage-induced apoptotic cell death.

Primary mouse fibroblasts deficient for c-Fos, p53 or for both proteins are hypersensitive to UV light and alkylating agent-induced chromosomal breakage and apoptosis.

The important regulatory proteins, c-Fos and p53 are induced by exposure of cells to a variety of DNA damaging agents. To investigate their role in cellular defense against genotoxic compounds, we comparatively analysed chromosomal aberrations and apoptosis induced by ultraviolet (UV-C) light and the potent alkylating agent methyl methanesulfonate (MMS) in primary diploid mouse fibroblasts knockout for either c-Fos or p53, or double knockout for both genes. We show that c-Fos and p53 deficient fibroblasts are more sensitive than the corresponding wild-type cells as to the induction of chromosomal aberrations and apoptosis. Double knockout fibroblasts lacking both c-Fos and p53 are viable and were even more sensitive, showing additivity of the chromosomal breakage effects observed in the single knockouts. Regarding the endpoint apoptosis, double knockout fibroblasts displayed a sensitivity similar to c-Fos and p53 deficient cells. The data indicate that (a) both c-Fos and p53 are involved in cellular protection against the clastogenic effect of genotoxic agents, (b) p53 is not required for induction of apoptosis by UV light and MMS, but rather prevents fibroblasts from undergoing apoptotic cell death upon DNA damage, and (c) c-Fos and p53 seem to act independently in determining genotoxic resistance, which is hypothesized to be achieved by impaired DNA repair or differential cell cycle check point control.

Involvement of the Fanconi anemia protein FA-C in repair processes of oxidative DNA damages.

Fanconi anemia (FA) is an autosomal recessive disorder characterized by skeletal abnormalities, pancytopenia and a marked predisposition to cancer. FA cells exhibit chromosomal instability and hypersensitivity towards oxygen and cross-linking agents such as diepoxybutane and mitomycin C. An increased level of reactive oxygen intermediates and an elevation of 8-oxoguanine in FA cells point to a defective oxygen metabolism in FA cells. We investigated the repair activity of oxidatively damaged DNA in lymphoblastoid cells from FA patients of complementation groups A-E. The repair activity for oxidatively damaged DNA was significantly reduced in lymphoblastoid cell lines of complementation groups B-E. Complementation of the FA-C cell line with the wild type FA-C gene restored the repair activity to normal. This indicates that the FA-C protein participates in the repair of oxidatively damaged DNA.