Induction of gamma-H2AX foci in human exfoliated buccal cells after in vitro exposure to ionising radiation.

PURPOSE: To test the gamma-H2AX (Histone 2AX phosphorylation of serine 139) foci assay for the detection of ionising radiation-induced DNA damage in buccal exfoliated cells. MATERIALS AND METHODS: Buccal mucosa cells from five individuals (three females, two males, aged 26-47 years) were exposed to 0, 0.5, 1, 2 and 4 Gy of gamma-rays. DNA damage and DNA damage removal were measured using the gamma-H2AX foci assay. Lymphocytes from one donor and the nuclear antigen H2B were used as a positive control to test the staining protocol. RESULTS: In the absence of radiation exposure, no significant differences for both H2B and gamma-H2AX signals were detected when comparing buccal cells and lymphocytes. The gamma-H2AX foci rate per cell in non-irradiated buccal cells was 0.08 +/- 0.02. The number of gamma-H2AX foci increased linearly with ionising radiation dose in the interval from 0-4 Gy, and reached a foci rate per cell of 0.82 +/- 0.22 at 4 Gy. Incubation experiments after in vitro gamma irradiation revealed that the number of gamma-H2AX foci did not show a significant decrease 5 h post exposure under the experimental conditions used. CONCLUSION: Data suggest that it is possible to apply the gamma-H2AX foci assay for the detection of ionising radiation-induced DNA damage in buccal exfoliated cells. The low removal of ionising radiation induced gamma-H2AX foci in buccal cells is a potential advantage for a biological dosimetry application.

Specific TP53 mutation pattern in radiation-induced sarcomas.

The mutagenic properties of ionizing radiation are well known, but the presence of specific mutations in human radiation-induced tumours is not established. We have studied a series of 36 secondary sarcomas arising in the irradiation field of a primary tumour following radiotherapy. The allelic status and the presence of mutations of the TP53 gene were investigated. The mutation pattern was compared with data from sporadic sarcomas recorded in the IARC TP53 somatic mutations database. A high proportion (58%) of the radiation-induced sarcomas exhibited a somatic inactivating mutation for one allele of TP53, systematically associated with a loss of the other allele. The high frequency (52%) of short deletions observed in the mutation pattern of radiation-induced sarcomas may be related to the induction of DNA breaks by ionizing radiation. The lack of hyper-reactivity of CpG dinucleotides and the presence of recurrent sites of mutation at codons 135 and 237 seem also to be specific for radiation tumorigenesis.

Non-homologous end-joining genes are not inactivated in human radiation-induced sarcomas with genomic instability.

DNA double-strand break (DSB) repair pathways are implicated in the maintenance of genomic stability. However the alterations of these pathways, as may occur in human tumor cells with strong genomic instability, remain poorly characterized. We analyzed the loss of heterozygosity (LOH) and the presence of mutations for a series of genes implicated in DSB repair by non-homologous end-joining in five radiation-induced sarcomas devoid of both active Tp53 and Rb1. LOH was recurrently observed for 8 of the 9 studied genes (KU70, KU80, XRCC4, LIG4, Artemis, MRE11, RAD50, NBS1) but not for DNA-PKcs. No mutation was found in the remaining allele of the genes with LOH and the mRNA expression did not correlate with the allelic status. Our findings suggest that non-homologous end-joining repair pathway alteration is unlikely to be involved in the high genomic instability observed in these tumors.