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The history of X rays in biological research began almost simultaneously with Roentgen's discovery of his new rays. The history may be unique because of the remarkable collaboration of physicists, chemists, biologists and clinicians--collaborations which have produced and are continuing to produce major contributions to both biological and medical science. These contributions include the use of X rays to investigate molecular structure and function, the first demonstration of induced mutagenesis, the delineation of the cell cycle, the initiation of in vitro and in vivo cloning of mammalian cells, and original studies in DNA repair. The following is a personal overview of the history of some of these developments and their relationship to areas of current biological research. In each case an attempt has been made to trace developments from an early observation or observations to the current day. The history has been divided into two segments, each of approximately 50 years. This division seems appropriate because the separation occurs at approximately the same time as developments which were to play a major role in determining the future of radiation research.

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It has been shown that several X-ray-sensitive Chinese hamster cell mutants defective in repair of DNA double-strand breaks (DSBs) are also impaired in the process of V(D)J recombination. The hamster mutants with this phenotype represent three distinct complementation groups, represented by the xrs series, XR-1 and V-3. The murine scid cell line also shows the same phenotype, and therefore we examined whether the scid mutant represents a new complementation group or belongs to one of the existing groups. Scid cells were fused with hamster cell mutants representing the three complementation groups. Hybrids between V-3 and scid cells were only partially complemented for X-ray sensitivity, whereas hybrids derived from fusions with the other mutants were resistant to X rays. These results suggest that V-3 and scid cells are defective in the same gene. To confirm this finding, a single human chromosome 8, which is known to carry the scid gene, was introduced into V-3 cells by microcell-mediated chromosome transfer. Nine hybrid clones derived from V-3 and carrying human chromosome 8 were obtained, and seven were found to be partially complemented for X-ray sensitivity. When human chromosome 8 was introduced into scid cells, seven of eight hybrid clones became resistant to X rays. The results indicate that the defective genes in V-3 and scid are both localized on human chromosome 8. This supports the results from the fusion analysis that V-3 and scid cells are defective in the same gene.

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We have previously reported the isolation of CHO cell lines resistant to mitomycin C under aerobic conditions of drug exposure. Here it is reported that these cell lines have the same response to mitomycin C under hypoxic conditions as do controls. The cells are shown to have lower levels of DT-diaphorase activity than controls, but similar levels of activity of NADPH:cytochrome c reductase, another enzyme involved in the metabolism of mitomycin C. Evidence for molecular defects in the DT-diaphorase gene or gene transcript is presented for the deficient cell lines. The consequences of this DT-diaphorase deficiency is further explored by testing the toxicity of menadione, an established enzyme substrate. The isolation of CHO cell lines deficient in DT-diaphorase activity and resistant to mitomycin C under aerobic but not hypoxic conditions suggests that mitomycin C reduction by this enzyme has a significant impact on cytotoxicity under aerobic but not hypoxic conditions. Similarly, DT-diaphorase metabolism of menadione does not appear to have a significant impact on cytotoxicity in CHO cells.

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Both the xrs and V-3 lines of Chinese hamster ovary cells exhibit marked sensitivity to ionizing radiation. They are also sensitive to agents such as bleomycin and H2O2 but exhibit normal responses to ultraviolet light and mitomycin C. Both cell lines are defective in split-dose repair and repair of double-strand breaks in DNA. Analysis of response to radiation as a function of age in the cell cycle indicates that both cell lines exhibit a marked sensitivity in late G1 and early S phase with more limited sensitization throughout the remainder of the cell cycle.

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Four of the most radiosensitive xrs variants of CHO-K1 cells, obtained after mutagenizing treatment with EMS, have been studied in detail over three to five decades of cell survival. Although these lines were initially reported to have very steep exponential survival curves, and to vary in sensitivity between themselves by a factor of two, we found in each case a similar biphasic response. The initial sensitivity was similar for all four lines, with a D0 of 0.5-0.7 Gy. A subpopulation, representing between 0.4 and 12 per cent of the cells, showed a resistant response, characterized by a D0 of 1.5-2.0 Gy. The previously reported variation in sensitivity seems to result from differences in the fraction of resistant cells rather than from differences in the D0. The consequence of such phenotypic variants within each cloned line is considerable, both for radiobiological studies of repair, and for molecular biology studies of the repair genes. Attempts were made to clone the sensitive and resistant subpopulations from each xrs cell line. Simple cloning from an untreated population was expected to yield pure sensitive cells, but these cells also gave biphasic responses in most cases. Only the cell line with the lowest resistant fraction (xrs5) gave a completely sensitive response in two of its subclones. Cells selected as survivors after high radiation doses were expected to yield resistant populations. However, for xrs4, 5 and 7 these subclones all gave biphasic responses. Three of the subclones from xrs6 gave biphasic responses but others gave a resistant response close to the wild type. We present a model in which transient gene expression may be seen in each individual cell if the silent copy of the xrs repair gene is temporarily hemimethylated. This transient gene transcription should occur during DNA synthesis, in the interval between synthesis of the gene and maintenance methylation. This interval may vary from cell line to cell line, resulting in different fractions of resistant cells.

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RSU-1069 is a highly effective hypoxic cell cytotoxin in KHT sarcomas treated in vivo. However, relative to the hypoxic cells, the oxic cells in the tumor appear more sensitive to the drug than would have been predicted on the basis of results with CHO (AA8-4) cells treated in vitro with the drug under oxic and hypoxic conditions. To examine possible reasons for this difference, suspensions of KHT cells were prepared from tumors growing in vivo, and treated with RSU-1069 in vitro under oxic or hypoxic conditions. The sensitivity of the KHT cells was similar to that of AA8-4 cells, regardless of whether the cells were obtained from untreated tumors or from tumors given 15 Gy in vivo just prior to the preparation of the cell suspension. We observed, however, that the sensitivity of both AA8-4 cells and KHT cells to drug treatment under hypoxic conditions increased with the density of the cells in the treated suspension. This result suggests the possibility that a diffusible toxic product may be released from cells. Such a product could contribute to the toxicity of the drug for oxic cells in tumors in situ.

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Mitomycin C (MMC), a bifunctional alkylating agent, requires metabolic reduction to become biologically active. We have identified a series of genetically related Chinese hamster ovary cell lines which span approximately three orders of magnitude in the concentration of MMC required for cell killing. Many mechanisms, including drug transport, drug activation, drug detoxification, and the elimination, or repair, of drug-induced lesions, may contribute to the level of drug resistance in cells. By exploring each of the above mechanisms in the various Chinese hamster ovary cell lines, we have been able to classify these cell lines into four categories. Proceeding from least resistant to most resistant to MMC, the cell lines are: (a) proficient in the bioreduction of MMC and deficient in DNA excision repair; (b) deficient in some aspects of MMC bioreduction and deficient in repair; (c) bioreduction and repair proficient; and (d) bioreduction deficient and repair proficient.

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Recently, two human DNA-repair genes have been cloned which complement the defects in complementation groups 1 and 2 of the CHO mutants which are sensitive to ultraviolet light and deficient in the incision step of excision repair. Here we report human gene transfer-mediated complementation of a group 4 CHO mutant sensitive to ultraviolet light and mitomycin C (MMC). The transfectants generated by transfecting human DNA into the repair-deficient cell line demonstrate the repair-proficient phenotype, as they have wild-type levels of resistance to UV light and MMC and are competent in performing the incision step of excision repair in response to UV irradiation. 3 of the 8 transfectants isolated display no detectable human repetitive sequences, while the other 5 contain varying amounts of human repetitive DNA. As the evidence suggests that all of the transfectants are repair-proficient as a result of the uptake of human DNA, we conclude that the human gene that complements the repair defect in group 4 CHO mutants contains no highly abundant human repetitive sequences. This imposes the necessity of developing cloning strategies involving the identification of sequences that flank the gene.

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Genetic mapping studies in bacterial, lower eukaryotic, and mammalian systems have demonstrated that the enzyme or enzyme complex involved in the initial incision step of the DNA excision repair pathway is coded for by more than one genetic locus. This paper reports the results of complementation studies that were performed with a number of DNA repair deficient Chinese hamster ovary cell lines. Complementation abilities were measured by comparing the survival of selected mutant X mutant hybrids with that of the tetrapoloid wild type after exposure to a number of physical and chemical agents. In all cases studied, hybrids formed from two different mutant lines showed resistance similar to that of the wild-type line. These results not only demonstrate that the mutant lines were in different complementation groups, but that complementation of a DNA-repair defect associated with one particular agent can complement the defect for another DNA-damaging agent.

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Reaction between GSH and the hydroxylamine derivative of SR-2508 results in the formation of two stable conjugates identified as 2-amino-4-S-glutathionyl and 2-amino-5-S-glutathionyl imidazoles. These stable conjugates are apparently formed from a reactive derivative of the hydroxylamine that is sufficiently stable to be isolated after HPLC separation. The physical and chemical properties of this derivative are consistent with it being a GSH conjugate in which the glutathionyl residue is attached to the 2-amino nitrogen of the imidazole moiety through sulphur. With excess GSH, under physiological conditions, it forms a mixture of the two stable GSH conjugates. In CHO cells exposed to SR-2508 under hypoxic conditions, this unstable GSH conjugate has been detected and suggests the possibility of GSH functioning as a carrier of a toxic metabolite of 2-nitroimidazoles under certain conditions.

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RSU-1069 combines an aziridine function with a 2-nitroimidazole and has been reported to exhibit extraordinary radiosensitization both in vitro and in vivo. Such sensitization appears to be at variance with the electron affinity of the compound. In addition, recent experiments suggest that the compound is highly toxic to hypoxic tumor cells in vivo. On the assumption that the observed radiosensitizing ability may be a manifestation of toxicity and because of the high in vivo toxicity, we have investigated aerobic and hypoxic toxicity, both in wild type CHO cells and in mutants sensitive to a variety of DNA damaging agents. With wild type cells under aerobic conditions, the compound is approximately 50 times as toxic as misonidazole and under hypoxic conditions, approximately 250 times as toxic. The ratio of hypoxic to aerobic toxicity is approximately 80 times. Under aerobic conditions, repair-deficient mutants are 10 times as sensitive to RSU-1069 as wild type cells and approximately 100 times as sensitive under hypoxic conditions. The ratio of hypoxic to aerobic toxicity for the mutant cells is approximately 900. Based on these observations, we suggest that under aerobic conditions the aziridine function is primarily responsible for toxicity, whereas, under hypoxic conditions, the aziridine moiety combined with a reduced 2-nitroimidazole moiety produces a bifunctional agent.

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The radiosensitizing and cytotoxic properties of the drug RSU-1069, (1-(2-nitro-1-imidazolyl)-3-(1-aziridino)-2-propanol) a 2-nitroimidazole with an aziridine ring in its side-chain, have been examined both in vivo and in vitro. Studies with the KHT Sarcoma or RIF1 tumour indicated that, at doses between 0.04 and 0.16 mg g-1 body wt, the drug was increasingly effective at killing tumour cells when combined with radiation. Cell survival in both tumours following combined RSU-1069 and radiation (1500 or 2000 cGy) treatment was similar when the drug was given 60 min before or immediately after irradiation suggesting that the effect observed was due to hypoxic cell cytotoxicity rather than radiosensitization. Studies with CHO cells in vitro indicated that RSU-1069 was equally as effective as a number of other 2-nitroimidazoles as a radiosensitizer when drug exposure and radiation treatment was given at 4 degrees C. It was substantially more toxic to hypoxic than to aerobic CHO cells (a factor of 90 in dose to give equivalent cell killing) and was much more toxic to CHO cells than misonidazole (a factor of approximately 100 in dose) at 37 degrees C. HeLa cells were more sensitive to RSU-1069 than CHO cells and, under hypoxic conditions, were approximately 20-fold more sensitive to the drug than when aerobic. Prior incubation of hypoxic CHO cells with RSU-1069 at toxic concentrations did not influence the sensitivity of the surviving cells to radiation treatment (i.e. there was no shoulder removal as is observed with misonidazole) nor did prior radiation treatment influence the sensitivity of the surviving cells to drug treatment. Overall the results indicate that RSU-1069 is a highly effective cytotoxic agent for hypoxic cells both in vivo and in vitro but, when drug exposure and radiation treatment are given at 4 degrees C, it is not a more effective sensitizer than other 2-nitroimidazoles.

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Nitroimidazoles, under hypoxic conditions, undergo reduction reactions producing a variety of reactive species; at the same time, they exert a variety of biological effects. The purpose of the present study was to investigate the reduction chemistry of 2-nitroimidazoles, the reaction of the reduced metabolites with nucleic acid constituents and the possible biological significance of these reactions. Earlier studies had demonstrated that, following reduction, 2-nitroimidazoles react with guanine derivatives to produce identical to that seen on reaction of glyoxal with the same guanine derivatives. We report here the identification of this product in the nucleic acid of cells exposed to 2-nitroimidazoles under hypoxic conditions. Using glyoxal as a model compound, we also demonstrate that cellular formation of such a product could account for a number of the biological properties of 2-nitroimidazoles under hypoxic conditions.

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In aqueous solution, in the presence of ammonium chloride, N1-substituted 2-nitroimidazoles are readily reduced to the corresponding hydroxylamines. In air, under neutral conditions, analogous to the reactions of aromatic hydroxylamines, 2-hydroxylaminoimidazoles are converted to the azoxy derivatives via a base-catalyzed condensation reaction between the hydroxylamine and its oxidation product, the nitroso derivative. In nitrogen, rearrangement to form the 2-amino-4(5)hydroxyimidazole derivative followed by addition of water across the C4-C5 double bond to yield isomers of a 4,5-dihydro-4,5-dihydroxy derivative appears to be a major reaction. 2-hydroxylaminoimidazoles undergo a complex series of reactions with glutathione. The initial reaction is the formation of a labile conjugate involving an N-S-linkage. Subsequently in the presence of excess GSH, under neutral conditions, two stable conjugates identified as 2-amino-4-S-glutathionyl- and 2-amino-5-S-glutathionyl imidazoles are formed. Nucleophilic attack by GSH on the imidazole ring of a nitrenium ion is postulated as the initial step in the formation of the stable GSH conjugates as well as the 2-amino-4,5-dihydro dihydroxy derivative. The results provide a molecular mechanism for many of the biological effects of N1-substituted 2-nitroimidazoles in hypoxic mammalian cells.

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The survival of the wild-type parent and two mutant lines of Chinese hamster cells, known to be defective in DNA repair, has been determined as a function of exposure to gamma rays under aerobic and hypoxic conditions. When compared to the wild-type line, one of the mutants selected for sensitivity to ethyl methyl sulfonate (EMS), and known to be defective in the repair of DNA strand breaks, exhibits a markedly enhanced sensitivity to aerobic irradiation but a reduced enhancement to hypoxic irradiation and thus an enhanced oxygen enhancement ratio (OER). In contrast, the other line, known to be defective in the incision step of excision repair, exhibits the reverse pattern of sensitivity and hence a reduced OER. The results are compared to findings in bacterial mutants and cells obtained from ataxia telangiectasia (AT) patients and heterozygotes.

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The genes and gene products involved in the mammalian DNA repair processes have yet to be identified. Toward this end we made use of a number of DNA repair-proficient transformants that were generated after transfection of DNA from repair-proficient human cells into a mutant hamster line that is defective in the initial incision step of the excision repair process. In this report, biochemical evidence is presented that demonstrates that these transformants are repair proficient. In addition, we describe the molecular identification and cloning of unique DNA sequences closely associated with the transfected human DNA repair gene and demonstrate the presence of homologous DNA sequences in human cells and in the repair-proficient DNA transformants. The chromosomal location of these sequences was determined by using a panel of rodent-human somatic cell hybrids. Both unique DNA sequences were found to be on human chromosome 19.

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Misonidazole, after reduction to the hydroxylamine derivative, reacts with glutathione (GSH) under physiological conditions. The reaction product has been identified as a mixture of two isomeric conjugates. When water soluble extracts of CHO cells exposed to misonidazole under hypoxic conditions are subjected to HPLC analysis, misonidazole derivatives, having the same chromatographic properties as the GSH-MISO conjugates, were detected. The identity of the synthetic and cellular products was further confirmed by identification of the amine derivative of misonidazole after desulfurization with Raney Nickel. When CHO cells were incubated with misonidazole in the presence of added GSH, a substantial increase in the amount of the conjugate was detected. When extracts of CHO cells exposed to misonidazole under hypoxia were subsequently exposed to GSH, an increased formation of the conjugate was observed. A rearrangement product of the hydroxylamine derivative of misonidazole is postulated as the reactive intermediate responsible for the formation of the conjugate.

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Chemical studies have indicated that, following reduction of misonidazole to the hydroxylamine derivative, reaction with guanosine leads to the formation of a 2-carbon addition product of guanosine. In this study, the formation of the guanosine product is used to detect the presence of a reactive metabolite of misonidazole in the urine of patients treated with misonidazole. Urine samples were incubated with [14C]guanosine and the guanosine product was separated by HPLC analysis. The quantities of product vary as much as 10-fold from patient to patient and it is suggested that the assay might be useful as a predictor of patients susceptible to the development of peripheral neuropathy or other effects of misonidazole.

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A misonidazole metabolite capable of reacting with guanosine has been detected in extracts of Chinese hamster ovary cells exposed to misonidazole under hypoxic conditions. A misonidazole metabolite with identical chromatographic properties and reactivity with guanosine has been detected in earlier studies with misonidazole reduced to the hydroxylamine state by chemical, radiolytic, or electrolytic means. The proposed structure of the guanosine product involves the addition of a two-carbon fragment between the N1 and N2 positions of guanosine. Rearrangement of the N-hydroxy derivative of misonidazole to a C-hydroxy derivative is postulated as the initial step in the reaction scheme.

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