Molecular characterization of TP53 gene in human populations exposed to low-dose ionizing radiation.

Ionizing radiation, such as that emitted by uranium, may cause mutations and consequently lead to neoplasia in human cells. The TP53 gene acts to maintain genomic integrity and constitutes an important biomarker of susceptibility. The present study investigated the main alterations observed in exons 4, 5, 6, 7, and 8 of the TP53 gene and adjacent introns in Amazonian populations exposed to radioactivity. Samples were collected from 163 individuals. Occurrence of the following alterations was observed: (i) a missense exchange in exon 4 (Arg72Pro); (ii) 2 synonymous exchanges, 1 in exon 5 (His179His), and another in exon 6 (Arg213Arg); (iii) 4 intronic exchanges, 3 in intron 7 (C → T at position 13.436; C → T at position 13.491; T → G at position 13.511) and 1 in intron 8 (T → G at position 13.958). Alteration of codon 72 was found to be an important risk factor for cancer development (P = 0.024; OR = 6.48; CI: 1.29-32.64) when adjusted for age and smoking. Thus, TP53 gene may be an important biomarker for carcinogenesis susceptibility in human populations exposed to ionizing radiation.

[Radiation biology of structurally different Drosophila melanogaster genes. Report I. The vestigial gene: molecular characteristic of "point" mutations].

The screening of PCR-detected DNA alterations in 9 spontaneous and 59 gamma-ray-, neutron - or neutron + gamma-ray-induced Drosophila vestigial (vg) gene/"point" mutations was carried out. The detected patterns of existence or absence of either of 16 overlapping fragments into which vg gene (15.1 kb, 8 exons, 7 introns) was divided enable us to subdivide all mutants into 4 classes: (i) PCR+ (40.7%) without the detected changes; (ii) "single-site" (33.9%) with the loss of a single fragment; (iii) partial detections (15.2%) as a loss of 2-9 adjacent fragments and (iv) "cluster" mutants (10.2%) having 2-3 independent changes of(ii) and/or (iii) classes. All spontaneous mutants except one were found to be classified as (ii) whereas radiation-induced mutants are represented by all 4 classes whose interrelation is determined by the dose and radiation quality. In particular, the efficacy of neutrons was found to be nine times as large as that of gamma-rays under the "cluster" mutant induction. Essentially, the distribution of DNA changes along the gene is uneven. CSGE-assay of PCR+-exon 3 revealed DNA heteroduplexes in 5 out of 17 PCR+-mutants studied, 2 of which had small deletions (5 and 11 b) and 3 others made transitions (A --> G) as shown by the sequencing. Therefore, gamma-rays and neutrons seem to be significant environmental agents increasing the SNP risk for the population through their action on the germ cells. The results obtained are also discussed within the framework of the track structure theory and the notion of quite different chromatin organization in somatic and germ cells.

Sun-induced nonsynonymous p53 mutations are extensively accumulated and tolerated in normal appearing human skin.

Here we demonstrate that intermittently sun-exposed human skin contains an extensive number of phenotypically intact cell compartments bearing missense and nonsense mutations in the p53 tumor suppressor gene. Deep sequencing of sun-exposed and shielded microdissected skin from mid-life individuals revealed that persistent p53 mutations had accumulated in 14% of all epidermal cells, with no apparent signs of a growth advantage of the affected cell compartments. Furthermore, 6% of the mutated epidermal cells encoded a truncated protein. The abundance of these events, not taking into account intron mutations and mutations in other genes that also may have functional implications, suggests an extensive tolerance of human cells to severe genetic alterations caused by UV light, with an estimated annual rate of accumulation of ∼35,000 new persistent protein-altering p53 mutations in sun-exposed skin of a human individual.

LET and ion-species dependence for mutation induction and mutation spectrum on hprt locus in normal human fibroblasts.

We have been studying LET and ion species dependence of RBE in mutation frequency and mutation spectrum of deletion pattern of exons in hprt locus. Normal human skin fibroblasts were irradiated with heavy-ion beams, such as carbon- (290 MeV/u and 135 MeV/u), neon- (230 MeV/u and 400 MeV/u), silicon- (490 MeV/u) and iron- (500 MeV/u) ion beams, generated by Heavy Ion Medical Accelerator in Chiba (HIMAC) at national Institute of Radiological Sciences (NIRS). Mutation induction in hprt locus was detected to measure 6-thioguanine resistant colonies and deletion spectrum of exons was analyzed by multiplex PCR. The LET-RBE curves of mutation induction for carbon- and neon-ion beams showed a peak around 75 keV/micrometers and 155 keV/micrometers, respectively. On the other hand, there observed no clear peak for silicon-ion beams. The deletion spectrum of exons was different in induced mutants among different ion species. These results suggested that quantitative and qualitative difference in mutation occurred when using different ion species even if similar LET values.

p53 mutations in tumors derived from irradiated human thyroid epithelial cells.

A non-tumorigenic human thyroid epithelial cell line (HTori-3) has been transformed into tumorigenic cells by exposure in vitro to alpha particles or gamma-radiation. These transformants were tumorigenic in athymic nude mice and tumors were transplantable into other nude mice. To further characterize processes involved in neoplastic progression, the tumor cell lines derived from these radiation-induced primary tumors were screened for mutations in the p53 tumor suppressor gene. p53 mutation was detected by single-strand conformation polymorphism (SSCP) analysis of exons 5 to 8 inclusive. Mutations detected by SSCP analysis were confirmed by sequencing. Mutations were detected in all four exons analysed, although there was no correlation between dose, LET or mutation position or frequency. Mutations in p53 exons 6 and 7 have been reported in the childhood papillary thyroid carcinomas in Belarus presumably as a result of radioiodine fall-out. Similarly here, p53 mutations are induced experimentally during the development of human thyroid tumors generated by irradiation of a human thyroid epithelial cell line in vitro.

Induction of multiple PCR-SSCPE mobility shifts in p53 exons in cultures of normal human urothelium exposed to low-dose gamma-radiation.

We have previously shown that primary explant cultures of human urothelium exposed to low doses of gamma-radiation subsequently accumulate a high level of stable p53 but it was not clear from those studies whether this protein stabilization occurred through an event in another gene involved in p53 protein control or possibly an epigenetic event. In these experiments, primary urothelial cultures from five different patients were exposed to either 0.5 or 5 Gy gamma-radiation from a 60 Cobalt source and allowed to grow for 7-10 division cycles to allow development of any radiation-induced, non-lethal changes in the cells. C-myc, Bcl-2 and stable p53 proteins were found to be elevated in cultures following both radiation doses. PCR-SSCPE analysis of the p53 gene was performed on cultures in order to determine whether genetic mutations could be the underlying basis for persistent increased stable p53 expression. Following 0.5 Gy exposure, the cultures also developed multiple distinct 'foci' of rapidly dividing cells which strongly overexpressed p53. These grew on a background of morphologically normal cells. When such foci were selectively analysed for their p53 mutation status by PCR-SSCPE, there was evidence that they contained cells which had developed changes to the p53 gene post-irradiation. These changes appeared to occur more frequently in focal cells than in cells of normal morphological appearance in the same culture. These results may have mechanistic importance given the controversy regarding low-dose radiation effects and p53-related genomic instability.

Oncogenic changes in murine lymphoid tumors induced by in utero exposure to ionizing radiation.

We have investigated the oncogenic alterations in murine lymphomas induced by in utero exposure to gamma-radiation. The expression of the myc oncogene increased in 23% of the tumors. Alterations in the expression of the ras oncogenes and in the p53 tumor suppressor gene were not characteristic. The p53 gene was mutated in a low percentage of the tumors (12%). Ras mutations were not detected. Loss of heterozygosity (LOH) at the p53 locus was found in 30% of the tumors, and LOH at the mts tumor suppressor gene was detected in 23% of lymphomas. Multiple oncogenic changes were infrequent in the investigated tumors. There were no essential differences in the frequency of carcinogenic alterations in spontaneous and gamma-radiation-induced lymphomas.

Carcinogen-induced de novo methylation in c-myc exon I.

During the response of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methylation occurred at the Hpa II site of c-myc exon I, which is located downstream of the P1 initiation site, as evidenced by the assays of Hpa II-PCR. The Hpa II spite of the 5' flanking region did not undergo methylation. UV-irradiation also led to methylation in exon I. The extent of methylation increased depending on the dose of MNNG and UV. The results suggested that methylation takes place in transcriptionally active c-myc responsible for carcinogens and is caused by mechanisms different from that of alkylation in a specific CpG site. Possible contribution of methylation to less repair found in c-myc is discussed.

Multiplex polymerase chain reaction-based deletion analysis of spontaneous, gamma ray- and alpha-induced hprt mutants of CHO-K1 cells.

Independent Chinese hamster ovary (CHO)-K1 cell mutants at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus were isolated from untreated, 60Co gamma ray- and 212Bi alpha-exposed cells and the genetic changes underlying the mutation determined by multiplex polymerase chain reaction (PCR)-based exon deletion analysis. In the 71 spontaneous mutants analyzed, 77.5% of the clones showed no change in exon number or size, 15.5% showed a loss of a single exon, 4.2% showed a loss of 2-8 exons, and 2.8% showed loss of all nine hprt exons (total gene deletion). Exposure to 6 Gy of gamma rays, which reduced survival levels to 10%, produced a significantly different deletion spectrum that was shifted toward deletions with 45% of the 20 mutants analyzed showing a loss of a single exon and 30% showing a loss of all nine exons. Exposure to 2 Gy alpha radiation from 212Bi, a 220Rn daughter, a dose which also reduced survival levels to about 10%, resulted in a deletion spectrum similar to the gamma-ray spectrum in that more than 75% of the 49 mutants analyzed were deletions. The alpha spectrum, however, was significantly different from both the spontaneous and gamma spectra with 55.1% of the alpha mutants showing a loss of all nine exons, 10.2% showing loss of a single exon, and 14.3% showing loss of 2-8 exons. Thus, alpha-radiation appears to produce larger intragenic deletions than gamma radiation. The results suggest that intragenic deletion size should be considered when low- and high linear energy transfer (LET) mutation spectra are compared.

Reduced induction of P53 protein by gamma-irradiation in ataxia telangiectasia cells without constitutional mutations in exons 5, 6, 7, and 8 of the p53 gene.

Ataxia telangiectasia (AT) is an autosomal recessive disease of childhood with several phenotypic characteristics. One of the hallmarks of this syndrome is its hypersensitivity to ionizing radiation, which is believed to be due to defects in DNA repair/processing. In addition to radio-resistant DNA synthesis, both fibroblasts and lymphoblastoid cell lines derived from these patients have been shown to have an impaired G1 arrest and prolonged G2 accumulation of cells indicating a defect in the regulation of cell cycle after irradiation. Since the (tumor suppressor) p53 protein has been reported to participate in the regulation of G1 arrest after irradiation, the possibility of p53 gene mutation and deregulating cell cycle in AT needed to be examined. We used the PCR amplification and DNA sequencing methods to detect mutations in the hypermutable exons (5-8) of germline p53 in fibroblast cells from 3 AT homozygotes. No mutation was found in any of these exons. In order to determine the role of the p53 protein in G1 arrest, its levels were measured before and after gamma-irradiation by flow cytometry in both AT and normal cells. Radiation-induced p53 protein levels in the AT cells varied from 6 to 60% compared to the normal cells, indicating a reduced induction of the protein in AT. These results suggest that mutation in the AT gene affects the p53 induction by irradiation and may, thus, alter the cell cycle regulation in the AT patients.

Ultraviolet-specific mutations in p53 gene in skin tumors in xeroderma pigmentosum patients.

Mutations in the p53 gene were identified in five of eight non-melanoma skin tumors in the sun-exposed areas of xeroderma pigmentosum patients by the polymerase chain reaction and single strand conformation polymorphism analysis followed by sequencing of the DNA. All mutations occurred at the dipyrimidine sites, indicating that they were caused by UV irradiation. Two tumors had multiple mutations, and four tumors had nonsense mutations. Since xeroderma pigmentosum patients are extremely sensitive to UV, the solar UV should have caused the mutations in the p53 gene and the mutations must have played a significant role in UV tumorigenesis.

Presence of point mutations in the N-ras gene in radiation-transformed rat embryo cells.

We study the transforming ability of X-rays in multistep carcinogenesis by irradiating primary rat cells which contain transfected c-myc oncogene. X-irradiation induces fully transformed phenotypes, including anchorage-independent growth and tumor formation in nude mice. Of seven foci examined, five exhibited an A to G conversion in codon 61 of the N-ras oncogene. Another transformed isolate has a single G to A base substitution in codon 14 in the same oncogene, while no point mutation is detected in the other focus. This is the first in vitro demonstration of the association between point mutation and X-ray-transformed cells.

Carcinogen-specific mutational pattern in the p53 gene in ultraviolet B radiation-induced squamous cell carcinomas of mouse skin.

We have examined 35 epidermal tumors induced in mice of four different strains by chronic exposure to ultraviolet B radiation for the presence of aberrations in the p53 tumor suppressor gene. Polymerase chain reaction products from p53 exons 5 to 8 were screened by single-strand conformation polymorphism analysis and sequencing. Base substitutions were found in seven tumors (20%). All mutations occurred at dipyrimidine sequences; most frequent were C-->T single base and CC-->TT tandem transitions suggesting the involvement of UV radiation in the genesis of the mutations. Three base substitutions were located at codon 148, and all dipyrimidine-derived mutations occurred at sites where the sequence is present in the nontranscribed DNA strand, indicating some site and strand specificity of the ultraviolet B-induced p53 mutations.