(1983). Ionizing Radiation: Sources and Biological Effects. International Journal of Radiation Biology and Related Studies in Physics, Chemistry and Medicine: Vol. 43, No. 5, pp. 585-586.

Ionizing Radiation: Sources and Biological Effects

We review the genes and proteins related to the homologous recombinational repair (HRR) pathway that are implicated in cancer through either genetic disorders that predispose to cancer through chromosome instability or the occurrence of somatic mutations that contribute to carcinogenesis. Ataxia telangiectasia (AT), Nijmegen breakage syndrome (NBS), and an ataxia-like disorder (ATLD), are chromosome instability disorders that are defective in the ataxia telangiectasia mutated (ATM), NBS, and Mre11 genes, respectively. These genes are critical in maintaining cellular resistance to ionizing radiation (IR), which kills largely by the production of double-strand breaks (DSBs). Bloom syndrome involves a defect in the BLM helicase, which seems to play a role in restarting DNA replication forks that are blocked at lesions, thereby promoting chromosome stability. The Werner syndrome gene (WRN) helicase, another member of the RecQ family like BLM, has very recently been found to help mediate homologous recombination. Fanconi anemia (FA) is a genetically complex chromosomal instability disorder involving seven or more genes, one of which is BRCA2. FA may be at least partially caused by the aberrant production of reactive oxidative species. The breast cancer-associated BRCA1 and BRCA2 proteins are strongly implicated in HRR; BRCA2 associates with Rad51 and appears to regulate its activity. We discuss in detail the phenotypes of the various mutant cell lines and the signaling pathways mediated by the ATM kinase. ATM's phosphorylation targets can be grouped into oxidative stress-mediated transcriptional changes, cell cycle checkpoints, and recombinational repair. We present the DNA damage response pathways by using the DSB as the prototype lesion, whose incorrect repair can initiate and augment karyotypic abnormalities.

Recombinational DNA repair and human disease.

Gap junction-mediated communication between contiguous cells has been implicated in the regulation of cell proliferation and differentiation. This report describes a new technique to measure cell-cell communication, gap fluorescence redistribution after photobleaching, which is based on the diffusion-dependent return of 6-carboxyfluorescein-mediated fluorescence in a photobleached cell that is in contact with other fluorescently labeled cells. Fluorescence recovery rates are interpreted as dye transport across gap junctions. Results of experiments on normal human fibroblasts and human teratocarcinoma cells show that this technique can measure rapid dye transfer and detect inhibition of communication (between teratocarcinoma cells) by the tumor promoters 12-O-tetradecanoyl-phorbol-13-acetate and the pesticide dieldrin.

http://science.sciencemag.org/content/sci/232/4749/525.full.pdf

A Fluorescence Photobleaching Assay of Gap Junction-Mediated Communication between Human Cells

A mutant of CHO cells (strain EM9) previously isolated on the basis of hypersensitivity to killing by ethyl methanesulfonate (EMS) is approx. 10-fold more sensitive than the parental line, AA8, to killing by both EMS and MMS. It is also hypersensitive to killing by other alkylating agents (ethyl nitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine), X-rays, and ultraviolet radiation. The production and repair of DNA single-strand breaks (SSB) were studied using the technique of alkaline elution of DNA from filters. After exposure to 4 Gy of X-rays at 0 degrees C and subsequent incubation at 25 degrees C, SSB were repaired within 12 min in AA8, but little repair occurred in EM9. Similarly, with doses of EMS or MMS that produced comparable numbers of SSB in AA8 and EM9 at the end of a 10-min exposure, repair of SSB occurred more rapidly in AA8 than in EM9, suggesting that individual SSB are longer lived in EM9. EM9 was found to be hypersensitive also to the induction of mutations and sister-chromatid exchanges (SCE) by EMS; per unit dose the mutant had twice as many mutations to thioguanine resistance, 3 times as many mutations to azaadenine resistance, and a 7-fold enhancement in SCE, compared to AA8. Moreover, the baseline frequency of SCE in the mutant was extraordinarily high, i.e., 8.6 +/- 0.6 vs. 107 +/- 5 SCE/cell for AA8 and EM9, respectively, with 10 microM BrdUrd in the medium. The high SCE frequency in EM9 did not vary significantly with BrdUrd concentration over the range examined from 2.5 to 20 microM, and the percentage of 5-bromouracil substitution in the DNA was the same in EM9 and AA8 under these conditions. These data, however, do not rule out the possibility that the high SCE frequency in EM9 is a consequence of an altered sensitivity to incorporated BrdUrd. Thus, EM9 may carry a pleiotropic mutation affecting some function in DNA replication and/or DNA repair and causing the variety of phenotypic properties described in this study.

A CHO-cell strain having hypersensitivity to mutagens, a defect in DNA strand-break repair, and an extraordinary baseline frequency of sister-chromatid exchange

The report reviews and evaluates the current literature (about 125 primary publications) on chemically induced specific locus mutations in the V79 Chinese hamster lung cell line. The V79 cell is convenient to use for mutagenesis studies since it has a rapid growth rate, high plating efficiency, and a stable karyotype. Mutation can be easily measured at either the hypoxanthine-guanine phosphoribosyl transferase or the Na+/K+ ATPase locus, both of which have been well characterized. Other less-studied markers are also described. We discuss the protocols for quantitative mutation studies including measurements of cytotoxicity, mutant expression times, mutant selection agents, cell densities during selection, and the stability and verification of mutant phenotypes. Mutations in the V79 cells by chemicals that require activation can be tested after their metabolism by cell homogenates or by intact cells, and the results with each type of activation are compared. For purposes of analysis, we classified a compound as mutagenic if it induced a mutation frequency that is at least 3 times higher than the spontaneous mutant frequency reported for that specific experiment. By this criterion two-thirds of the chemicals analyzed were mutagenic--; 11% with and 55% without metabolic activation. Of the 191 chemicals examined; 119 were polycyclic aromatic hydrocarbons; 25 were nitro or nitroso compounds, 9 were alkyl halides; 7 were purine or pyrimidine derivatives and the remaining 31 were from other chemical classes. We also defined mutagenic potency as the concentration of a compound that increases the mutant frequency by 10 times the spontaneous frequency. Mutagenic potencies of the compounds examined varied over a range of 5 X 10(6). We have also found large interlaboratory variations in the mutagenic potencies. Such variation in potency could be reduced by normalizing the results to a standard mutagen such as N-methyl-N'-nitro-N-nitrosoguanidine. The role of the V79 assay in mutagenicity and carcinogenicity testing is discussed and recommendations are suggested for future investigation.

Mutagenesis by chemical agents in V79 chinese hamster cells: a review and analysis of the literature. A report of the Gene-Tox Program

The rates of spontaneous mutation to 6-thioguanine resistance were determined in fibroblasts derived from normal and two Bloom syndrome individuals (GM 2548 and GM 1492). Two methods were utilized to determine the rates. Method I obtained the spontaneous mutation rate from the increase in the mutation frequency of a cell population in logarithmic-phase growth over 10 days. The two Bloom syndrome strains had spontaneous mutation rates of 16 X 10(-6) and 17 X 10(-6) mutations per cell per generation, whereas two normal strains had rates of 1.5 X 10(-6) and 1.1 X 10(-6). Method II utilized fluctuation analysis to measure the rate of spontaneous mutation. This method resulted in rates of 19 X 10(-6) and 23 X 10(-6) mutations per cell per generation in Bloom syndrome cells, compared to rates of 4.6 X 10(-6) and 4.9 X 10(-6) in the control strains. These data suggest that Bloom syndrome may be a mutator mutation, a previously unrecognized phenomenon in humans, and that an elevated spontaneous mutation rate in vivo may be responsible for the clinical phenotype of primordial dwarfism and increased cancer incidence.

Elevated spontaneous mutation rate in Bloom syndrome fibroblasts

Chinese hamster (V79) cells were irradiated with a fractionated regime of ultraviolet light (UV 1 + UV 2 ) The fractionation of a UV dose always increased the colony-forming ability but reduced (or it did not change) the mutation frequencies Treatment with cycloheximide between the two UV irradiations resulted in two types of effects, depending on the protocols used Long exposures to cycloheximide (ie, >6 h) for the entire period between UV 1 and UV 2 or partial treatment of cycloheximide (ie, 3h) long before UV 2 always resulted in reduced colony-forming ability and enhanced or unchanged mutation frequencies Exposure to cycloheximide for the entire period in the short fractionated regime (ie, 4h) between UV 1 and UV 2 or partial treatment of cycloheximide just prior to UV 2 tended to give the opposite effects Caffeine treatment before UV 2 , with or without UV 1 , significantly increased the mutation frequencies These results suggest that an error-free postreplication repair system exists in Chinese hamster cells which is inhibitable by particular cycloheximide or caffeine treatments

Modification of UV-induced mutation frequencies in Chinese hamster cells by dose fractionation, cycloheximide and caffeine treatments.

Through a new approach, we have sought to isolate ultraviolet light (UV)-sensitive and DNA repair mutant Chinese hamster fibroblasts. The procedure consisted of 1) mutation induction by 5-bromodeoxyuridine (Brd U)-blacklight and UV treatments; 2) incorporation of 3H-thymidine in repair-proficient cells at high temperature (38.5 degrees C) following UV damage; 3) cold holding (4.0 degrees C) of these cells to induce tritium killing; and 4) recovery and testing of repair-deficient and UV-sensitive cells which have survived and formed colonies at low temperature (34.0 degrees C). In our initial attempt at this protocol, we isolated 72 surviving colonies from 2 x 10(7) cells plated for selection. Of the 72 colonies, 20 demonstrated potential interest and four were selected for extensive study. One, identified as UVs-7, is slightly more sensitive to UV, but not sensitive to X rays or N-acetoxy-2-acetylaminofluorene (NAc-AAF). The mutant exhibits a highly reduced level of unscheduled DNA synthesis (UDS), as compared to the parental line. Two additional lines, UVs-40 UVs-44, are sensitive to UV, X ray, N-methyl-N-nitro-N-nitrosoguanidine (MNNG), and NAc-AAF, but exhibit normal UDS. A fourth line, UVr-23, has enhanced UDS, is resistant to UV, but exhibits no difference in sensitivity to x ray or NAc-AAf. These mutants aremore » all stable, and should be useful for the study of mammalians DNA repair processes and mechanisms of mutagenesis.« less

Isolation and partial characterization of mutagen-sensitive and DNA repair mutants of Chinese hamster fibroblasts.

We have previously reported the isolation and partial characterization of DNA repair and/or mutagen-sensitive mutant Chinese hamster cell strains. Here we present the results of a detailed study of the ultraviolet light (UV)-induced mutability of one of these strains, UVs-7, and provide preliminary mutability data on two additional lines, UVr-23 and UVs-40. UVs-7 in extremely deficient in unscheduled DNA synthesis (UDS) but only slightly more sensitive to UV than the parental line. When examined for the UV-inducibility of mutants resistant to ouabain, 6-thioguanine, or diphtheria toxin, UVs-7 was found to be hypermutable at all three loci as compared to the parental line. The degree of hypermutability was not the same for any two loci. UVs-40, a highly UV-sensitive strain, was also found to be hypermutable at the ouabain-resistant (ouar) locus. UVr-23, which is UV-resistant and more proficient at UDS than the parental line, appeared to exhibit a tendency toward hypomutability at both the ouabain(ouar) and 6-thioguanine--resistant (6TGr) loci. Further characterization of all these lines should aid in delineating mammalian mechanisms of DNA repair and mutagenesis.

Roger Alan Schultz

Papers

Elevated spontaneous mutation rate in Bloom syndrome fibroblasts

Modification of UV-induced mutation frequencies in Chinese hamster cells by dose fractionation, cycloheximide and caffeine treatments.

Isolation and partial characterization of mutagen-sensitive and DNA repair mutants of Chinese hamster fibroblasts.

The mutation studies of mutagen-sensitive and DNA repair mutants of Chinese hamster fibroblasts.