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.     

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.     

A novel mutant of mouse lymphoma cells sensitive to alkylating agents and caffeine

Two methyl-methanesulfonate-sensitive strains have been isolated, one of which, M10, was cross-sensitive to X-rays as reported before. Sensitivities of parental L5178Y, M10, and newly isolated MS-1 cells to various mutagens were examined. Mutgans tested were UV, X-rays, 4-nitroquinoline 1-oxide (4NQO), caffeine and alkylating agents; methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS) and mitomycin C (MMC).In terms of D₃₇ values, M10 cells were 2.5–7 times more sensitive to EMS, MMC and 4NQO as well as to MMS and X-rays than were parental L5178Y cells, while the new mutant MS-1 was about 3 times more sensitive to MMS, EMS, MMC and caffeine than were parental cells. The characteristics in sensitivities of M10 cells to X-rays, alkylating agents and 4NQO resemble some ataxia telangiectasia cells; and MS-1 cells to alkylating agents and caffeine are novel among mammalian cell mutants so far reported. Sensitivity of M10 cells to mutagens has so far been stable for one year, and that of MS-1 cells was stable for 6 months in continuous culture.     

Chromosomal hypersensitivity in mutant M10 and Q31 mouse cells exposed to ultraviolet radiation (UV) and 4-nitroquinoline-1-oxide (4NQO)

2 mutant mouse cells M10 and Q31 were examined for chromosomal aberrations induced by ultraviolet radiation (UV) and 4-nitroquinoline-1-oxide (4NQO), as compared with mouse lymphoma L5178Y cells. Q31 cells are UV- and 4NQO-sensitive cells isolated from L5178Y cells. M10 cells are similar but are sensitive to ionizing radiation and 4NQO. After treatment with UV or 4NQO, chromatid-type aberrations in these cell strains were induced more frequently in the first mitotic cells, at late fixation times. After UV exposure (2.4 J/m2), the maximal frequencies of chromatid-type breaks in Q31 cells were about 5 times higher than in L5178Y cells. In M10 cells such breaks were only as frequent as in L5178Y cells. After 4NQO treatment (50 ng/ml) the frequencies of chromatid-type breaks in M10 and Q31 cells were significantly higher than in L5178Y cells. From these results and those of previous studies (Takahashi et al., 1982), M10 cells may be considered hypersensitive to gamma-rays and 4NQO, but not to UV, and thus react similarly to L5178Y cells. The hypersensitivity of M10 cells to 4NQO may result from a defect in the ionizing-radiation repair mechanism as has been suggested to occur in ataxia telangiectasia (AT) cells. Q31 cells are hypersensitive to UV and 4NQO, but not to gamma-rays. Q31 cells may be considered to be deficient in a UV-like repair pathway. In conclusion, characteristics of murine M10 and Q31 cells are compared with those of human AT and xeroderma pigmentosum (XP) cells.     

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.     

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.     

Chromosomal instability in mutagen-sensitive mutants isolated from mouse lymphoma L5178Y cells. I. Five different genes participate in the formation of baseline sister-chromatid exchanges and spontaneous chromosomal aberrations

In a search for cell mutants that show an increase or a decrease in the frequency of baseline sister-chromatid exchanges (SCEs) or spontaneous chromosomal aberrations (CAs), large numbers of mutagen-sensitive clones previously isolated from mouse lymphoma L5178Y cells were analyzed. In addition to two SCE mutants (ES 4 and AC 12) previously reported, three other mutants were identified as an SCE mutant. An ethyl methanesulfonate-sensitive mutant ES 2 and an alkylating agent-sensitive mutant MS 1 exhibited, respectively, 1.4-fold and 1.8-fold higher baseline SCE frequencies than did the parental L5178Y. In contrast, M10, which is sensitive to X-ray and 4-nitroquinoline 1-oxide, showed a reduced frequency of baseline SCEs (0.65-fold). These 5 mutants including ES 4 and AC 12 had 3--9-fold increases in spontaneous CA frequencies. Measurement of baseline SCE formation in inter-mutant hybrids revealed that M10 mutation is dominant, MS 1 and ES 4 mutations are semidominant, and ES 2 and AC 12 mutations are recessive. Because SCE frequencies in hybrids formed between pairs of 4 mutants (ES 2, MS 1, ES 4 and AC 12) were significantly lower than those in the tetraploid mutant cells, these 4 mutants probably belong to different complementation groups. Since M10 behaved dominantly with respect to SCE phenotype, it was not possible to determine by complementation test whether it belongs to a different group from the other mutants. However, the finding that M10 is complemented by other mutants for EMS sensitivity indicates that the M10 mutation is different from the other mutations. From these results, it is concluded that at least 4 different genes participate in the formation of high levels of baseline SCEs. The defects in ES 2, MS 1, ES 4, and AC 12 produce common lesions responsible for the formation of both SCEs and CAs. In contrast, the defect in M10 is associated with a high increase in spontaneous CA frequency, but conversely associated with a decrease in baseline SCE frequency. This suggests that M10 is defective in the process involved in the formation of baseline SCEs.     

Mutagen-sensitive cell lines are obtained with a high frequency in V79 Chinese hamster cells

A replica-plating technique has been adopted for the isolation of mutagen-sensitive mutants of Chinese hamster V79 and CHO cell lines. After the mutagenic treatment (ENU) clones derived from these cell lines were replica plated into micro wells and replicas were treated with UV (254 nm), X-ray, MMC, EMC or MMS. Clonal cell lines which demonstrated mutagen sensitivity were retested by the determination of survival. Only one UV-sensitive line was obtained in 1500 clonal lines derived from CHO cells. This mutant appeared also sensitive to 4NQO and MMC. The sensitivity to UV and MMC was 2-3-fold enhanced, while the increase in sensitivity to 4NQO was 4-5-fold. In V79 cells 9 mutagen-sensitive lines were found after screening of 500 clonal lines; six of them showed increased sensitivity towards UV, two towards MMC, and one cell line was found to be X-ray sensitive. A considerable cross-sensitivity for the various agents was found among the isolated mutants. When a 2-fold increase is taken as a minimum to indicate mutagen sensitivity 6 mutants were sensitive to UV, 8 mutants were sensitive to MMC, 6 mutants were sensitive to 4NQO and 4 mutants were sensitive to X-rays. The difference in sensitivity to UV versus 4NQO makes it unlikely that 4NQO can be considered as a UV-mimetic agent. The sensitivity to MMC appears to fall into 2 classes: a class with moderate sensitivity (2-8-fold) and a class with high sensitivity (30-100-fold). The presence of similar classes is indicated for UV. Except for the two lines V-E5, V-B7 and the two lines V-H11, V-H4 all obtained mutants have a different spectrum of mutagen sensitivities which suggests that different genetic alterations underly these effects. The observed high frequency of mutagen-sensitive mutants in V79 cells, although unexpected and substantially higher than those published for CHO cells and L5178Y cells, can still be explained by the presence of functionally hemizygous loci.     

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.     

Sensitivity to bleomycin and hydrogen peroxide of DNA repair-defective mutants in Neurospora crassa

Mutations were induced in Neurospora which cause increased sensitivity to MMS (methyl methane-sulfonate) and other mutagens. Genetic analysis of such mus demonstrated that some of them defined new DNA repair genes (mus-21, and mus-27 to mus-30), while others represented new alleles in previously known genes. To characterize them further, and especially to identify rec- types which have not yet been found in this species, many MMS-sensitive strains were tested for cross-sensitivities to bleomycin (BLM) and to hydrogen peroxide (H2O2) to which some rec- of other species are hypersensitive. In Neurospora, many of the MMS-sensitive mutants were found to be cross-sensitive to BLM and frequently these were also hypersensitive to ionizing radiation. Bleomycin sensitivity was demonstrated for all alleles of 10 different genes, 4 of them new ones, with mus-27 being the most sensitive of the latter (resembling uvs-6; Koga and Schroeder, 1987, Mutation Res., 183, 139). In contrast, very few of the MMS-sensitive mutants were hypersensitive to H2O2 and, in general, results of H2O2 tests were variable and differences between strains small. However, consistent deviations from wild type were observed in a few cases (most clearly for mus-9 and mus-11) when results from treatments of germinating conidia were compared with those of non-growing ones.     

Identification of a new seventh complementation group of UV-sensitive mutants in Chinese hamster cells

The UV-sensitive mutant V-B11, isolated from the V79 Chinese hamster cell line (Zdzienicka and Simons, 1987) was further characterized. V-B11 has a slightly increased cross-sensitivity to 3me4NQO, whereas no increased sensitivity towards 4NQO was observed. A slightly increased sensitivity towards EMS and MMS was also found. The mutant shows a defect in the ability to perform the incision step of nucleotide-excision repair after UV irradiation: 2 h after UV exposure, the accumulation of incision breaks in V-B11, in the presence of HU and araC, was about 30% of that found in wild-type V79 cells. V-B11 was crossed to a panel of 6 UV-sensitive Chinese hamster ovary (CHO) cells, which represents all the previously identified 6 complementation groups of UV-sensitive Chinese hamster mutants. Since in all crosses complementation has been observed, V-B11 appears to be the first mutant of a new, 7th, complementation group.     

Mutagen sensitivities and mutator effects of MMS-sensitive mutants in Neurospora

7 mus (mutagen-sensitive) mutants of Neurospora crassa, which are more sensitive to the toxic effects of MMS (methyl methanesulfonate) than wild-type, were investigated for cross-sensitivities to other mutagens and inhibitors. These mutants have recently been mapped in 5 new genes, mus-7 to mus-11, and mutant alleles from each gene were checked for their effects on mutation frequencies. It was found that mutants in 3 of these 5 genes showed radiation-induced mutation frequencies similar to wild-type. These included 2 alleles of the gene mus-10, which were cross-sensitive only to UV and were the only mutants that produced some viable ascospores in homozygous crosses. The mutant of the second gene, mus-8, was especially sensitive to UV and mitomycin C and produced slightly reduced frequencies of spontaneous mutation. In contrast, the mutant of the third gene, mus-7, was not UV-sensitive but showed some cross-sensitivity to X-rays; mus-7 was highly sensitive to MMS and also to histidine, which inhibits various repair-defective mutants at concentrations well below those that reduce wild-type growth. None of these mus resemble mutants previously found in Neurospora, nor do they conform clearly to mutant types identified in E. coli or yeast. On the other hand mutants in 2 further genes, mus-11, and especially 2 alleles of mus-9, are very similar to uvs-3 of Neurospora and generally resemble mutants that are considered to be defective in "error-prone" repair. They were UV- as well as X-ray-sensitive, and showed strong spontaneous mutator effects but almost no increase in recessive lethal frequencies in heterokaryons after UV-treatments.     

Response of two strains of L5178Y cells to cis-dichlorobis(cyclopentylamine)platinum(II). II. Differential effects of caffeine.

Two strains of L5178Y murine lymphoma, inversely cross-sensitive to X-rays and UV light, were shown previously to respond to treatment with an antitumour platinum complex, cis-dichlorobis(cyclopentylamine)-platinum(II) (cis-PAD), in a similar manner as to UV. Enhancement of chromosomal damage and potentiation of lethal effect of cis-PAD by 0.75 mM caffeine were found in cis-PAD and UV light-resistant L5178Y-S strain but not in cis-PAD and UV light-sensitive L5178Y-R strain. These results suggest that the extreme sensitivity of L5178Y-R strain to cis-PAD and UV light is caused to some extent by deficiency in a caffeine-sensitive post-replication repair system.     

Increased resistance of the radiosensitive M10 mutant cells of the L5178Y mouse lymphoma cell line to heat-induced apoptosis.

M10 cells, which are deficient in the repair of DNA DSBs and are therefore radiosensitive, are about twofold more thermoresistant than their parental L5178Y cells. We found that, after heat shock at 43 degrees C under conditions resulting in 10% survival (D(10)), M10 cells did not undergo apoptosis, whereas L5178Y cells did undergo apoptosis. M10 cells, but not L5178Y cells, constitutively expressed Hsp72 protein. Moreover, the M10 cells accumulated higher amounts of the heat-inducible form of Hsp72. The patterns of activation of the DNA-binding activity of HSF (heat-shock factor) differed in M10 and L5178Y cells. In response to heat shock, M10 cells accumulated greater amounts of Trp53 protein (formerly known as p53) than the parental cells. Cdkn1a (formerly known as p21, Waf1) was constitutively expressed and further accumulated after heat shock only in M10 cells. We suggest that heat-inducible Hsp72 to a larger extent, and constitutive Hsp72 to a lesser extent, together with Cdkn1a may be involved in the protection of M10 cells against heat-induced apoptosis. Apoptosis in these cells is likely to occur in Trp53-dependent manner.     

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.     

Genetic complementation between UV-sensitive CHO mutants and xeroderma pigmentosum fibroblasts

The purpose of this study was to determine the feasibility of doing complementation analysis between DNA-repair mutants of CHO cells and human fibroblasts based on the recovery of hybrid cells resistant to DNA damage. Two UV-sensitive CHO mutant lines, UV20 and UV41, which belong to different genetic complementation groups, were fused with fibroblasts of xeroderma pigmentosum in various complementation groups. Selection for complementing hybrids was performed using a combination of ouabain to kill the XP cells and mitomycin C to kill the CHO mutants. Because the frequency of viable hybrid clones was generally < 10⁻⁶ and the frequency of revertants of each CHO mutant was 2×10⁻⁷, putative hybrids required verification. The hybrid character of clones was established by testing for the presence of human DNA in a dot-blot procedure.

Hybrid clones were obtained from 9 of the 10 different crosses involving 5 complementation groups of XP cells. The 4 attempted crosses with 2 other XP groups yielded no hybrid colonies. Thus, a definitive complementation analysis was not possible. Hybrids were evaluated for their UV resistance using a rapid assay that measures differential cytotoxicity (DC). All 9 hybrids were more resistant than the parental mutant CHO and XP cells, indicating that in each case complementation of the CHO repair defect by a human gene had occurred. 3 hybrids were analyzed for their UV-radiation survival curves and shown to be much more resistant that the CHO mutants but less resistant than normal CHO cells. With 2 of these hybrids, sensitive subclones, which had presumably lost the complementing gene, were found to have similar sensitivity to the parental CHO mutants. We conclude that the extremely low frequency of viable hybrids in this system limits the usefulness of the approach. The possibility remains that each of the nonhybridizing XP strains could be altered in the same locus as one of the CHO mutants.     

Isolation and characterization of mitomycin-C-sensitive mouse lymphoma cell mutants

26 mutants with increased sensitivity to the lethal effects of mitomycin C (MMC) were isolated from mouse lymphoma L5178Y cells by a replica-plating technique. Most of them were about 5-10 times more sensitive in terms of D37 values to MMC than were parental cells. 5 of the MMC-sensitive mutants isolated from independently mutagenized cell populations were further analyzed. They were highly sensitive to the killing by decarbamoyl (DC) MMC, a monofunctional derivative of MMC, but were not sensitive to ultraviolet radiation, X-rays, 4-nitroquinoline-1-oxide or methyl methanesulfonate. These 5 mutants were classified into at least 2 genetic complementation groups. The implication of these mutations in cross-link and mono-adduct repair of DNA damage induced by MMC and DCMMC is discussed.     

Cytogenetical characterization of UV-sensitive repair-deficient CHO cell line 43-3B. II. Induction of cell killing, chromosomal aberrations and sister-chromatid exchanges by 4NQO, mono- and bi-functional alkylating agents

An established cell line of Chinese hamster ovary (CHO-9) cells and its UV-sensitive mutant 43-3B have been studied for the induction of cell killing, chromosomal aberrations and sister-chromatid exchanges (SCEs) after exposure to different types of DNA-damaging agents such as 4-nitroquinoline-1-oxide (4NQO), mitomycin C (MMC), diepoxybutane (DEB), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS) and ethyl nitrosourea (ENU). In comparison with the wild-type CHO cells, 43-3B cells showed very high sensitivity to the UV-mimetic agent 4NQO and the DNA cross-linking agents MMC and DEB. The 43-3B cells responded with higher sensitivity to the monofunctional alkylating agents (MMS, EMS and ENU). The increased cytotoxic effects of all these chemicals correlated well with the elevated increase in the frequency of chromosomal aberrations. In 43-3B cells exposed to 4NQO, MMC or DEB the increase in the frequency of chromosomal aberrations was much higher than the increase in the frequency of SCEs (4-10-fold) when compared to the wild-type CHO cells. This suggests that SCEs are results of fundamentally different cellular events. The responses of 43-3B cells to UV, 4NQO, MMC and DEB resemble those of 2 human syndromes, i.e., xeroderma pigmentosum and Fanconi's anemia. These data suggest that 43-3B cells are defective in excision repair as well as the other pathways involved in the repair of cross-links (MMC, DEB) and bulky DNA adducts (4NQO).     

Characterization of MMS-sensitive mutants of Neurospora crassa

Several MMS-sensitive mutants of Neurospora crassa were compared with the wild-type strain for their relative sensitivities to UV, X-ray, and histidine. They were also compared for the frequency of spontaneous mutation at the loci which confer resistance to p-fluorophenylalanine. The mutants were also examined for possible defects in meiotic behavior in homozygous crosses and for any change in the inducible DNA salvage pathways (as indicated by their ability to utilize DNA as the sole phosphate source in the growth medium). On the basis of these characterizations, the present MMS-sensitive mutants of Neurospora can be placed into three groups. The first group includes three mutants, mus-(SC3), mus-(SC13), and mus-(SC28). These are slow growers, insensitive to histidine with no apparent meiotic defects and may have reduced frequency of spontaneous mutation. In addition, their mycelial growth is sensitive to MMS but the conidial viability following MMS, UV or X-ray treatment appears normal or only slightly more sensitive than the wild-type. The second group includes only one mutant, mus-(SC15); its mycelial growth is very sensitive to MMS but the conidial survival following treatment with MMS or UV appears normal; however, the conidial survival following exposure to X-ray is significantly reduced. This mutant shows an increase (more than 10-fold) frequency of spontaneous mutation, but behaves normal like the wild-type with respect to fertility, growth rate and insensitivity to histidine. The third group includes mutants mus-(SC10), mus-(SC25), and mus-(SC29). These mutants are very sensitive to UV, X-rays and MMS and to histadine but have normal growth rates on minimal medium. Mutant mus-(SC10), but not mus-(SC25) and mus-(SC29), has an increased (11 ×) frequency of spontaneous mutation. On the basis of data presented, the MMS sensitivity of the first group of mutants cannot be ascertained to arise from a defect in the DNA repair pathways; instead, it may stem from altered cell permeability or other pleotropic effects of the mus mutations. However, it can be suggested that the second and third group of mus mutants may indeed result from a defect in the DNA repair pathways controlled by the mus genes; this conclusion is based on their cross-sensitivity to a number of DNA-damaging agents such as MMS, UV and/or X-ray, high frequencies of spontaneous mutation (mutator effects) and defects in meiotic behavior.