Abnormal pattern of post-γ-ray DNA replication in radioresistant fibroblast strains from affected members of a cancer-prone family with Li-Fraumeni syndrome

Abstract

Summar-Non-malignant dermal fibroblast strains, cultured from affected members of a Li-Fraumeni syndrome (LFS) family with diverse neoplasms associated with radiation exposure. display a unique increased resistance to the lethal effects of -y-radiation. In the studies reported here. this radioresistance (RR) trait has been found to correlate strongly with an abnormal pattern of post-y-ray DNA replicative synthesis. as monitored by radiolabelled thymidine incorporation and S-phase cell autoradiography. In particular. the time interval between the y-ray-induced shutdown of DNA synthesis and its subsequent recovery was greater in all four RR strains examined and the post-recovery replication rate was much higher and was maintained longer than in normal and spousal controls. Alkaline sucrose sedimentation profiles of pulse-labelled cellular DNA indicated that the unusual pattern of DNA replication in irradiated RR strains may be ascribed to anomalies in both replicon initiation and DNA chain elongation processes. Moreover. the RR strain which had previously displayed the highest post-y-ray clonogenic survival was found to harbour a somatic (codon 234) mutation (presumably acquired during culture in vitro) in the same conserved region of the p53 tumoursuppressor gene as the germline (codon 245) mutation in the remaining three RR strains from other family members. thus coupling the RR phenotype and abnormal post--y-ray DNA synthesis pattern with faulty p53 expression. Significantly. these two aberrant radioresponse end points. along with documented anomalies in c-mc and c-raf-l proto-oncogenes. are unprecedented among other LFS families carrying p53 germline mutations. We thus speculate that this peculiar cancer-prone family may possess in its germ line a second, as yet unidentified, genetic defect in addition to the p53 mutation

Radioresistant MTp53-expressing rat embryo cell transformants exhibit increased DNA-dsb rejoining during exposure to ionizing radiation

“…The normalized tail moments (NTM) relating to the number of DNA double strand breaks following LDR-irradiation measured at 0 and 60 min following irradiation were determined for the MTp53-and WTp53-expressing clones. The relatively radioresistant HPVE7/ras-trans- (Cheong et al, 1993;Mirzayans et al, 1995), we found no signi®cant di erences between CLASS II and I clones in the rejoining kinetics of DNA-dsbs following 20 Gy given at high dose rate (data not shown). However, these results do indicate that MTp53-expressing cells are not de®cient at DNA-dsb break repair when irradiated during high dose-rate radiation.…”

“…Abnormal DNA replication in the form of decreased replicon initiation and chain elongation has been reported by Mirzayans et al, (1995) in radioresistant MTp53-expressing Li ± Fraumeni ®broblasts compared to normal WTp53-expressing controls. However, no di erences in DNA-ssb rejoining were reported following HDR-radiation between these cell types; unfortunately, relative DNA-dsb break rejoining was not investigated by Mirzayans and co-workers.…”

Chromosome instability is a predominant trait of fibroblasts from Li-Fraumeni families

“…Comparison of individual groups with the control group showed that resistance, now highly significant at both radiation doses (P = 0.003), was confined to the mutation carriers (groups B and C). In addition, we determined the survival of 2800T, previously described as being resistant to HDR radiation (Bech-Hansen et al, 1981;Mirzayans et al, 1995), and three mutation-carrying strains that had lost the wild-type allele (m/-), clone 163/29, 172MA and the tumour-derived strain 161 MA-F (Table 8). Taking two standard deviations of the mean of the control group as a limit of significance, strain 2800T was significantly resistant at both radiation doses and was the most resistant to LDR of all the strains tested.…”

“…Cells from three of the families had normal sensitivity to high-dose-rate (HDR) ionizing radiation (Little et al, 1987), but mutation-carrying fibroblasts from another extensively studied family with a germline mutation in codon 245 (Srivastava et al, 1990) were resistant to HDR radiation (Bech-Hansen et al, 1981). The most radioresistant strain (2800T) was derived from a blood relative of the affected proband who did not have the codon 245 mutation, but had acquired a mutation in codon 234 during growth in vitro (Mirzayans et al, 1995). Recently, we reported that LFS fibroblasts with TP53 mutations are more resistant than normal fibroblasts to low-dose-rate (LDR) ionizing radiation (Sproston et al, 1996).…”

Aberrant p21WAF1-dependent growth arrest as the possible mechanism of abnormal resistance to ultraviolet light cytotoxicity in Li–Fraumeni syndrome fibroblast strains heterozygous for TP53 mutations

“…Non-cancerous dermal ®broblast strains derived from members of the LFS kindred under study by us harbor either a germ-line (codon 245) or somatic (codon 234) mutation in one allele of the TP53 gene (Srivastava et al, 1990;Mirzayans et al, 1995). These strains exhibit an unusually complex phenotype, encompassing abnormal resistance to the killing eect of acute g irradiation (Bech-Hansen et al, 1981) and faulty DNA double-strand break rejoining (Parshad et al, 1993).…”