Characteristics of γ-ray-induced chromosomal aberrations in mutagen-sensitive mutants of L5178Y cells

We have examined the chromosomal radiosensitivities of an ionizing-radiation- and MMS-sensitive mutant (M10), and a UV- and 4NQO-sensitive mutant (Q31), isolated from mouse lymphoma L5178Y cells, with regard to killing effects. In the first mitoses after 100 R γ-irradiations, it was found that M10 cells were highly radiosensitive in terms of chromosomal aberrations accompanying longer mitotic delay (3 h); the frequencies of both chromatid-type and chromosome-type aberrations were, respectively, about 7 and 4 times higher than that of wild-type L5178Y cells. Furthermore, chromatid exchanges, particularly triradials, isochromatid breaks with sister union, and chromatid gaps and breaks were markedly enhanced at G₁ phase of M10 cells. In contrast, the chromosomal radiosensitivity of Q31 cells after 100 R irradiation was similar to that of L5178Y cells. On the other hand, spontaneous aberration frequencies (overall breaks per cell) of M10 and Q31 cells were, respectively, 5.1 and 2.2 times higher than that of wild-type L5178Y cells. The chromosomal hypersensitivity to γ-rays in M10 cells is discussed in the light of knowledge obtained from ataxia telangiectasia cells.     

Chromosomal instability in mutagen-sensitive mutants isolated from mouse lymphoma L5178Y cells. II. Abnormal induction of sister-chromatid exchanges and chromosomal aberrations by mutagens in an ionizing radiation-sensitive mutant (M10) and an alkylating agent-sensitive mutant (MS1)

To determine the mutual relationships between cell survival and induction of sister-chromatid exchanges (SCEs) as well as chromosomal aberrations (CAs), mutagen-induced SCEs and CAs were analyzed in an ionizing radiation-sensitive mutant (M10) and an alkylating agent-sensitive mutant (MS 1) isolated from mouse lymphoma L5178Y cells. The levels of CA induction in both mutants strictly corresponded to the sensitivity to lethal effects of mutagens, except that caffeine-induced CAs in M10 are considerably lower than those in L5178Y. The results clearly indicate that except for caffeine-induced CAs in M10, mutagen-induced lethal lesions are responsible for CA induction. In contrast, SCE induction in mutants was complicated. In M10, hypersensitive to killing by gamma-rays, methyl methanesulfonate (MMS), and 4-nitroquinoline 1-oxide (4NQO), but not sensitive to UV or caffeine, the frequency of SCEs induced by gamma-rays was barely higher than that in L5178Y, and the frequencies of MMS- and UV-induced SCEs were similar to those in L5178Y, but 4NQO- and caffeine-induced SCEs were markedly lower than those in L5178Y. MS 1, which is hypersensitive to MMS and caffeine, but not sensitive to UV or 4NQO, responded to caffeine with an enhanced frequency of SCEs and had a normal frequency of MMS-induced SCEs, but a reduced frequency of UV- and 4NQO-induced SCEs. Thus, susceptibility to SCE induction by mutagens is not necessarily correlated with sensitivity of mutants to cell killing and/or CA induction by mutagens. Furthermore, the spontaneous levels of SCEs are lower in M10 and higher in MS 1 than that in L5178Y (Tsuji et al., 1987). Based on these results, we speculate that M10 may be partially defective in the processes for the formation of SCEs caused by mutagens. On the other hand, MS 1 may modify SCE formation-related lesions induced by UV and 4NQO to some repair intermediates that do not cause SCE formation. In addition, MMS-induced lethal lesions in MS 1 may not be responsible for SCE induction whereas caffeine-induced lethal lesions are closely correlated with SCE induction. Thus, the lesions or mechanisms involved in SCE production are in part different from those responsible for cell lethality or CA production.     

UV- and X-ray-sensitive double mutants of mouse L5178Y cells are synergistically more sensitive to 4-nitroquinoline-1-oxide than is either of the single mutants

The X-ray-sensitive mutant M10 and the UV-sensitive mutant Q31 of mouse lymphoma L5178Y cells are both sensitive to killing by 4-nitroquinoline-1-oxide (4NQO). Since cell hybridization experiments showed that the 4NQO sensitivities in M10 and Q31 cells behaved as codominant traits (Shiomi et al., 1982c), it is not possible to determine by complementation test whether the M10 and the Q31 mutations responsible for 4NQO sensitivities are allelic. We have obviated this difficulty by selecting double mutants that are sensitive to both X-rays and UV. From X-ray-sensitive M10 cells, two UV-sensitive mutants (XU 1 and XU 2) were isolated by a cell-suspension spotting method. XU 1 and XU 2 were found to belong to the same complementation group as Q31 (group I). Double mutants XU 1 and XU 2 were 30-37-fold more sensitive to 4NQO than parental L5178Y cells, whereas the single mutants M10 and Q31 were only 6-8-fold more sensitive to 4NQO than L5178Y cells in terms of D10 values (dose required to reduce survival to 10%). These results show that the M10-Q31-double mutations enhance 4NQO sensitivity synergistically, indicating that the M10 and the Q31 mutations relevant to 4NQO sensitivities are non-allelic. The implications of this finding are discussed.     

Studies on three mutagen-sensitive mutants of mouse L5178Y cells. I. Characterization of the hybrids between L5178Y and mutagen-sensitive mutants

Three mutagen-sensitive mutants, MS-1, M10 and Q31, have been isolated from mouse L5178Y cells. MS-1 cells are sensitive to methyl methanesulfonate (MMS), M10 cells are cross-sensitive to X-rays, MMS and 4-nitroquinoline 1-oxide (4NQO), and Q31 cells are cross-sensitive to UV and 4NQO. Lines resistant to 6-thioguanine (TGr) and 5-bromo-2'-deoxyuridine (BUr) were isolated from L5178Y and these three mutagen -sensitive mutants. All the TGr lines were sensitive to 5-bromo-2'-deoxyuridine and HAT medium and all the BUr lines were sensitive to 6-thioguanine and HAT medium. The hybrids homozygous for the mutagen-sensitive markers showed nearly the same sensitivity to UV, 4NQO, X-rays and MMS as their parental TGr and BUr lines. The hybrids constructed by fusing L5178Y BUr and TGr lines from each of MS-1, M10 and Q31 displayed the normal UV, X-ray and MMS resistancy of L5178Y cells. Thus the UV-, X-ray- and MMS-sensitive markers in MS-1, M10 and Q31 were recessive in somatic cell hybrids. The 4NQO-sensitive phenotype, however, behaved codominantly in somatic cell hybrids.     

Studies on three mutagen-sensitive mutants of mouse L5178Y cells. II. Complementation analyses between two methyl methanesulfonate-sensitive mutants and between two 4-nitroquinoline-1-oxide-sensitive mutants

Three mutagen-sensitive mutants, MS-1, M10 and Q31, were isolated from mouse L5178Y cells. MS-1 cells are sensitive to methyl methanesulfonate (MMS), M10 cells are cross-sensitive to X-rays, MMS and 4-nitroquinoline-1-oxide (4NQO); and Q31 cells are cross-sensitive to UV and 4NQO. MMS-, X-ray- and UV-sensitive markers in these mutants behaved recessively in hybrids between pairs of these mutants as in hybrids between L5178Y and these mutants as reported before (Shiomi et al., 1982b). Complementation analyses were carried out by forming hybrids between two MMS-sensitive mutants (MS-1 and M10) and between two 4NQO-sensitive mutants (M10 and Q31). MMS and 4NQO survivals were measured in these hybrid cells. MS-1 and M10 were found to belong to different complementation groups for MMS-sensitive phenotypes. The hybrid clones between M10 and Q31 were as sensitive to 4NQO as each of the mutants, indicating codominance of 4NQO sensitivity in these mutants. The hybrids constructed with L5178Y and three mutants were stable as to their chromosome constitution for 100 days of cultivation without selective pressure. From the segregation studies on these hybrids, it is concluded that neither the X-ray-sensitive mutation in M10 nor the UV-sensitive mutation in Q31 is located on the X chromosome.     

Mutation induction in a mouse lymphoma cell mutant sensitive to 4-nitroquinoline 1-oxide and ultraviolet radiation

The mutant mouse lymphoma cell Q31, which is sensitive to 4-nitroquinoline 1-oxide and ultraviolet radiation (UV), was compared with the parental L5178Y cell for the effect of caffeine and mutation induction after UV irradiation. Caffeine potentiated the lethal effect of UV in both cell strains to a similar extent, indicating that the defective process in Q31 cells was caffeine-insensitive. UV-induced mutation to 6-thioguanine resistance was determined in L5178Y and Q31 cells. The maximal yield of mutants was obtained 7 days post-irradiation in L5178Y cells and 14 days in Q31 cells for higher UV doses. It appears that a much longer time is required for the mutant cells than for the parental cells for full expression of the resistance phenotype even at equitoxic UV doses. A substantially higher frequency in induced mutations was observed in Q31 cells than in L5178Y cells at a given dose of UV. A plot of induced mutation frequency as a function of logarithm of surviving fraction again indicates hypermutability of Q31 cells as compared with the parental strain. In contrast, X-rays induced a similar frequency of mutations to 6-thioguanine resistance in L5178Y and Q31 cells.     

DNA repair in a UV-sensitive mutant of of mouse cell line

A UV-sensitive mutant, Q31, isolated from mouse-lymphoma L5178Y cells, was studied for excision and post-replication rerpairs. A nearly equal number of UV endonuclease-sensitive sites was induced by UV in L5178Y, Q31, and human Raji cells. L5178Y cells irradiated with 10 J/m² removed 18% of sensitive sites from DNA during incubation for 24 h, and Q31 cells removed 3% of the sites, a fraction less than the limit of detection, whereas Raji cells eliminated about 60% of the sites. These results indicate that mouse-lymphoma cells are capable of excision repair to a limited extend as compared with human cells and that mutant Q31 cells are essentially devoid of dimer excision. The newly synthesized DNA was of smaller size in UV-irradiated and unirradiated Q31 cells than that in the corresponding L5178Y cells, but the DNAs in both strains increased to comparable sizes after a 2-h chase.     

Repair of DNA single-strand breaks in radiation-sensitive mutants of mouse cells

The radiation-sensitive mutant M10 of mouse lymphoma L5178Y cells was examined for its ability to rejoin DNA single-strand breaks induced by gamma-rays. The alkaline sucrose gradient sedimentation analysis revealed that M10 cells repaired single-strand breaks but simultaneously produced increasing amounts of small DNA fragments with time of postirradiation incubation, something which was not observed in L5178Y cells. Since small fragments did not appear in M10 cells irradiated at room temperature, DNA fragmentation may result from cold treatment during irradiation followed by incubation at 37 degrees C. This indicates that the cold susceptibility is characteristic of M10 cells and is not related to radiation sensitivity of this mutant. This conclusion is supported by the finding that no DNA degradation takes place after cold treatment with a subsequent incubation in the other radiosensitive mutant LX830 that belongs to the same complementation group as M10.     

Effect of novobiocin on the frequencies of chromatid-type aberrations and sister-chromatid exchanges following gamma-irradiation

The effect of novobiocin on the frequencies of chromatid-type aberrations and SCEs was examined in Chinese hamster V79 cells which were exposed to gamma-rays and post-treated with novobiocin. While no chromatid aberrations were induced in the unirradiated cells by novobiocin, the frequency of SCEs was slightly increased by treatment with novobiocin alone. Irradiation of G2 cells produced multiple chromatid-type aberrations and post-treatment of the irradiated cells with novobiocin resulted in a significant increase of the aberrations, including chromatid gaps and breaks. In contrast, novobiocin failed to increase the frequency of SCEs induced by gamma-rays when the irradiated cells were post-treated with novobiocin.     

Human cells (normal and ataxia telangiectasia) transfected with pR plasmid are hypersensitive to DNA strand-breaking agents.

Ataxia telangiectasia (AT) cells are known to be hypersensitive to ionizing radiations and to drugs such as bleomycin and epipodophyllotoxin VP16, a topoisomerase II poison. Both of these produce DNA double-strand breaks even if through different mechanisms. In this work we analyzed the sensitivity to bleomycin and to epipodophyllotoxin of AT cells after transfection with pR plasmid. This plasmid, interacting with bacterial SOS repair pathways, expresses itself in mammalian cells conferring cell resistance to the SOS inducers UV and 4NQO and cell sensitivity to different drugs such as bleomycin. This effect is presumably due to the interaction of pR products with double-strand breaks. Our findings indicate that pR plasmid, in both AT lines tested (AT5BIVA fibroblasts and ATL6 lymphoblasts), expresses itself (increasing UV protection) and amplifies the already enhanced AT cell sensitivity to both bleomycin and VP16.     

Chromosomal hypersensitivity in mutant MCN-151 mouse cells exposed to mitomycin C

After exposure to mitomycin C, the mouse lymphoma cell mutant MCN-151, previously shown to be sensitive to the toxic effect of the drug, demonstrates higher frequencies of chromatid-type aberrations and of aberrant cells in comparison to L5178Y cells. At any given dose of mitomycin C, the frequencies of chromosomal aberrations increased with time reaching a plateau in both cell strains, with the mutant in all cases being more sensitive than normal cells.     

Functional complementation of the radiation-sensitive mutant M10 cell line by human chromosome 5

The mouse lymphoma (L5178Y) cell mutant M10 is defective in rejoining DNA double-strand breaks and is hypersensitive to ionizing radiation. The introduction of human chromosome 5 into M10 cells by microcell mediated chromosome transfer complemented the ionizing-radiation hypersensitivity defect of this cell line. The presence of chromosome 5 in the microcell hybrids was shown using PCR with chromosome-specific primers and fluorescence in situ hybridization. From this data we conclude that the gene that corrects the radiation hypersensitivity of M10 cells is located on chromosome 5 and tentatively assigned to the 5q14 to 5pter region. We designate this gene XRCC4L.     

Different contributions of the indirect effects of γ-rays on the cytotoxicity in M10 and XRCC4 transfected M10 cells

The protective effects of dimethyl sulfoxide (DMSO) against cell killing by ¹³⁷Cs γ-rays were investigated in XRCC4-deficient cell line M10, XRCC4-complemented M10 and the parental mouse leukemia cell line L5178Y. Cell survival was determined by the colony-forming ability. M10 cells were more sensitive to γ-ray-induced cell death than L5178Y and complemented M10 cells. Cell survival was increased in both M10 and L5178Y in the presence of DMSO. However, estimation of the DMSO-protectable fraction revealed a smaller protectable fraction for M10 cells than for L5178Y cells, indicating that indirect effects contributed in a smaller extent to the cytotoxicity in M10 than that in L5178Y. This effect is due to XRCC4 deficiency, since transfection of XRCC4 cDNA into M10 cells restored the radioprotective effects of DMSO to the level seen in L5178Y. In M10 cells, the killing effects of high LET radiation (Auger electrons from ¹²⁵I-antipyrine, carbon ions with an LET of 166 keV μm⁻¹) were similar to those of low LET radiation (¹³⁷Cs γ-rays, characteristic X-rays from ¹²⁵I-bovine serum albumin). We discuss that lethal lesions produced by indirect actions in L5178Y and XRCC4-complemented M10 cells may differ, at least in part, from DNA double-strand breaks repairable by non-homologous end joining.     

Effects of X-irradiation on cell-cycle progression, induction of chromosomal aberrations and cell killing in ataxia telangiectasia (AT) fibroblasts

Survival, cumulative labeling indices, chromosomal aberrations and cell-cycle distribution by flow microfluorometry (FMF) were studied in fibroblasts from normal and three ataxia telangiectasia (AT) families after X-irradiation during density-inhibition of growth and immediate release by subculture to low density. Homozygotic AT (proband) fibroblasts were very hypersensitive to cell killing by X-irradiation (D0 = 40-45 rad). Fibroblasts from AT heterozygotes (parents) were minimally hypersensitive, with D0's (100-110 rad) slightly lower than those for normal fibroblasts (D0 = 120-140 rad). There were three different response groups for a G1 phase block induced by 400 rad of X-rays: (1) minimal or no G1 block was observed in AT homozygote cell strains; (2) 10-20% of the cells were blocked in G1 in normal cell strains; and (3) 50% or more of the cells were blocked in AT heterozygote strains. FMF profiles and cumulative labeling indices showed that homozygotic AT cells irradiated in plateau phase moved into the S-phase following subculture with no additional delay over non-irradiated controls. Homozygotic AT cells showed not only a 4-5 times higher frequency of X-ray-induced chromosomal aberrations than normal strains, but approximately 30% of these were of the chromatid-type. There were no differences in the frequency or type of X-ray-induced chromosomal aberrations between normal and heterozygotic AT cells.     

Induction and repair of DNA strand breaks in cultured mammalian cells following fast neutron irradiation.

Induction and repair of DNA breaks following irradiation with NIRS cyclotron neutrons were studied in cultured mammalian cells (L5178Y) in comparison to those following gamma-rays. The yield of the total single-strand breaks, 3'OH terminals and sites susceptible to S1 endonuclease following fast neutrons was found to be approximately 50 per cent of that following gamma-irradiation. On the other hand, the yield of double-strand breaks was slightly higher after fast neutrons than after gamma-rays. The percentage of the total single-strand breaks remaining unrejoined at 3 hours after post-irradiation incubation was found to be distinctly higher after the fast neutrons than after gamma-rays. The neutron-induced damage appears to carry a higher proportion of alkali-labile lesions compared to gamma-rays. It was concluded that the increase in the yield of double-strand breaks and of unrejoinable breaks is responsible for a high r.b.e. of the cyclotron neutrons.     

Lethal and mutagenic effects of radiation and chemicals on cultured fish cells derived from the erythrophoroma of goldfish (Carassius auratus)

GEM 199 cells derived from an erythrophoroma of goldfish (Carassius auratus), which had a high plating efficiency, were used to investigate the lethal and mutational effects of radiations (UV and gamma-rays) and chemicals (4NQO and MNNG). The cells were more resistant to gamma-rays than mammalian cells and CAF-MM1 cells derived from the normal fin tissue of goldfish. They were also more resistant to UV-irradiation than CAF-MM1 cells. Photoreactivation after UV-irradiation was present in GEM 199 cells for both survival and mutation. The initial shoulder of the survival curve of UV-irradiated cells was reduced greatly by caffeine, suggesting a high activity of the post-replication repair. The spontaneous mutation frequency to ouabain resistance was 1-5 X 10(-6) clones per viable cell. MNNG was effective in inducing ouabain-resistant mutation, while 4NQO and gamma-rays did not induce mutation.     

Response of two strains of L5178Y cells to cis-dichlorobis(cyclopentylamine)platinum(II). I. Cross-sensitivity to cis-PAD and UV light.

The response to cis-dichlorobis(cyclopentylamine)platinum(II) (cis-PAD) an antitumour platinum complex, was studied in two strains of murine lymphoma L5178Y cross-sensitive to X-rays and UV light. Dose-survival relationship, DNA synthesis formation of chromatid aberrations, progression through the cell cycle, and growth and viability changes after 1 h cis-PA; treatment at 37 degree C were examined and compared with the effects of X-rays and UV light. In both strains, cis-PAD caused immediate inhibition of progression through the cell cycle, reduced rate of DNA synthesis, delayed appearance of chromatid aberrations, and delayed death; however, there is a marked difference in sensitivity to cis-PAD between L5178Y-S strain (D0 approx. 5.8 microgram/ml) and L5178Y-R strain (D0 approx. 2.5 microgram/ml). In both strains a close resemblance was found between dose-survival relationships after cis-PAD and UV light treatment, respectively.     

The biological activity of hydrogen peroxide. I. Induction of chromosome-type aberrations susceptible to inhibition by scavengers of hydroxyl radicals in human embryonic fibroblasts

The cytogenetic effect of hydrogen peroxide (H2O2) was investigated in human embryonic fibroblasts. Chromosome-type aberrations were found together with chromatid-type aberrations in metaphase cells harvested 24 h after a single 10-min treatment with 10(-5)-10(-3) M H2O2 in 0.9% NaCl solution. The chromosome-type aberrations were observed to be predominantly dicentrics and deletions. Both types of aberration showed a dose-response relationship to the dose of H2O2 over the range of 10(-5)-1.5 X 10(-4) M H2O2. The intercellular distribution of dicentrics showed a Poisson distribution. Centric and acentric rings and abnormal monocentrics were a minor fraction of the chromosome-type aberrations. The chromatid-type aberrations observed, such as breaks, exchanges and gaps, showed no dose-response relationship. The frequency of isochromatid breaks was higher than that of chromatid breaks and approximately 70% of the isochromatid breaks were found in the centromeric or pericentromeric region. The intercellular distribution of chromatid exchanges showed an over-dispersed distribution. The generation of aberrations by H2O2 was effectively suppressed by catalase and several scavengers of hydroxyl radicals (.OH) such as ethanol, dimethyl sulfoxide (DMSO) and mannitol. This result suggest that .OH plays an essential role in the generation of the chromosome aberrations by H2O2.     

Increase in the frequency of gamma-ray induced chromosomal aberrations in mammalian cells by post-treatment with novobiocin

The effect of novobiocin (an inhibitor of DNA topoisomerase and polymerase) on the frequency of chromosomal aberrations was examined in Chinese hamster V79 cells irradiated with gamma-rays in the plateau phase of growth and subcultured in the presence of novobiocin until the first mitosis after irradiation. Novobiocin alone affected cell survival, DNA synthesis and the mitotic frequency of unirradiated cells in a dose-dependent manner, without causing any significant increase in the frequency of chromosome- or chromatid-type aberrations. The frequency of chromosome-type aberrations induced by gamma-radiation was not influenced by novobiocin at 200 microM, but the frequency of chromosome deletions (but not rings and dicentrics) showed a two-fold increase when 300 microM novobiocin was present. Irradiation produced a low level of chromatid-type aberrations and post-treatment with novobiocin at concentrations greater than 100 microM significantly increased the frequency of chromatid gaps and breaks. The results support the idea that different radiation-induced lesions lead to chromosome- as opposed to chromatid-type aberrations.     

Quantitative analyses of the induction of chromosome aberrations and sister-chromatid exchanges in human lymphocytes exposed to gamma-rays and mitomycin-C in combination

Most chemicals are S-dependent and are potent inducers of SCE, but do not produce chromosome-type aberrations in the first metaphases after exposure. Ionizing radiation, which is an S-independent agent, produces chromosome-type aberrations, especially dicentrics and rings, but inefficiently produces chromatid-type aberrations. A series of experiments has been performed to investigate whether cytogenetic damage induced by ionizing radiation (gamma-rays) might be assessed separately from that induced by the alkylating chemical, mitomycin C (MMC), when human lymphocytes were exposed to these 2 agents in combination. Whole-blood cultures of human lymphocytes in G0 phase were exposed to gamma-rays and MMC in combination or separately. Cytogenetic analyses were done for both chromosome aberrations (CA), analyzed in cultures incubated for 56 h without BrdUrd, and sister-chromatid exchanges (SCEs) in cultures incubated for 72 h with BrdUrd. The frequency of chromosome-type aberrations (dicentrics and rings) increased with increasing doses of gamma-rays from 0.5 to 4.0 Gy. The dose-response relationships were the same with or without concomitant treatment with MMC (10(-6) M). Although the SCE frequency increased with increasing doses of MMC, the increase was nearly the same as when cells were treated with both MMC and gamma-rays (2 Gy). There was no interaction between MMC and gamma-rays concerning these 2 endpoints.