engleski

Genetic instability of FADD-deficient mouse cells

We used fibroblast cultures from the FADD-deficient mouse embryos, which were originally constructed by T.W. Mak (Toronto, Ont.) to assess directly the role of FADD, a death receptor-associated protein, in cells responses to UV radiation. These FADD-deficient fibroblasts showed much increased growth rate compared with the wild-type homozygous cells. However, the FADD - deficient fibroblasts showed the same sensitivity to UVC radiation as the wild type homozygous fibroblasts measured by MMT test. There was no difference between repair activity of UV-induced DNA damages products in the FADD - deficient and proficient cells, either. However, delay of entering S phase after UV-irradiation was reduced in the FADD-deficient cells, indicating some abnormality in the checkpoint function of the cell cycle in these deficient cells. We further analyzed changes of p53 sequences in the FADD-deficient and wild-type cells. Genomic DNA was analyzed by allele-specific polymerase chain reaction (AS-PCR) for CC to TT mutation at codons 154-155 and 175-176 in exon 5 and for C to T mutations at codons 270 and 275 in exon 8 0f the p53 gene. The mutant-specific forward primer was used for each mutation. The reverse primers for amplification of mutations were not mutant-specific. Allele-specific PCR detection of p53 in genomic DNA were analyzed by gel electrophoresis. The results showed the high frequency of changes in the mutant-specific primer for codon 270 amplified 134-base pair product from FADD deficient cell DNASs. Although frequencies of UV-induced mutations were not different between the FADD- deficient and wild-type cells, distributions and spectrum of base-substitution mutations at the p53 were different.

FADD; p53; Apoptoza; UV

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