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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Survival and mutagenic responses of mitomycin C-sensitive mouse lymphoma cell mutants to other DNA cross-linking agents

Mitomycin C-sensitive mutants MCN 151 (complementation group I) and MCE 50 (complementation group II) derived from mouse lymphoma L5178Y cells were found to be also highly sensitive to the lethal effects of other DNA cross-linking agents, such as photoaddition of 8-methoxypsoralen (8-MOP) and cis-diamminedichloroplatinum II (cis-DDP). They were less sensitive to the monofunctional derivative 3-carbethoxypsoralen (3-CPs) and to trans-DDP to trans-DDP than their bifunctional counterparts. Incorporation levels of labeled 8-MOP or 3-CPs in wild-type cells and 2 mutants were almost the same, indicating that the sensitivity is not caused by differential incorporation of the agents. The rates of photoinduced mutations to 6-thioguanine resistance in the mutants, per unit dose of 8-MOP, were about 4 times higher for MCN 151 and 3 times higher for MCE 50 than that in L5178Y cells. However, the rates of induced mutations per viable cells in the mutants were nearly equal to those in wild-type cells. Cross-link repair was compared between mutants and wild-type cells by using the alkaline sucrose-gradient sedimentation technique. The results show that normal cells and both mutants are able to incise the cross-linked DNA, which is the first step of cross-link repair.     

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.     

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.     

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.     

X-ray-sensitive mutants of Chinese hamster ovary cell line. Isolation and cross-sensitivity to other DNA-damaging agents

A standard technique of microbial genetics, which involves the transfer of cells from single colonies by means of sterile toothpicks, has been adapted to somatic cell genetics. Its use has been demonstrated in the isolation of X-ray-sensitive mutants of CHO cells. 9000 colonies have been tested and 6 appreciably X-ray-sensitive mutants were isolated. (D10 values 5-10-fold of wild-type D10 value.) A further 6 mutants were obtained which showed a slight level of sensitivity (D10 values less than 2-fold of wild-type D10 value). The 6 more sensitive mutants were also sensitive to bleomycin, a chemotherapeutic agent inducing X-ray-like damage. Cross-sensitivity to UV-irradiation and treatment with the alkylating agents, MMS, EMS and MNNG, was investigated for these mutants. Some sensitivity to these other agents was observed, but in all cases it was less severe than the level of sensitivity to X-irradiation. Each mutant showed a different overall response to the spectrum of agents examined and these appear to represent new mutant phenotypes derived from cultured mammalian cell lines. One mutant strain, xrs-7, was cross-sensitive to all the DNA-damaging agents, but was proficient in the repair of single-strand breaks.     

A fourth complementation group among ionizing radiation-sensitive Chinese hamster cell mutants defective in DNA double-strand break repair.

The XR-V9B mutant of Chinese hamster V79 cells which exhibits hypersensitivity to ionizing radiation was isolated by the replica plating technique. The increased sensitivity of XR-V9B cells to X rays (approximately 4-fold, as judged by the D10) was accompanied by increased sensitivity to other DNA-damaging agents such as bleomycin (approximately 17-fold), VP16 (approximately 6-fold), and adriamycin (approximately 5-fold). Only a slightly increased sensitivity was observed after exposure to UV radiation, MMS, or mitomycin C (1.4-, 1.7-, and 2-fold, respectively). As measured by neutral elution after exposure to X rays, XR-V9B cells showed a defect in the rejoining of double-strand breaks (DSBs); after 4 h of repair more than 50% of DSBs remained in comparison to 5% in wild-type cells. No difference was observed in the kinetics of single-strand break rejoining between XR-V9B and wild-type cells, as measured by alkaline elution. To determine whether XR-V9B represents a new complementation group among ionizing radiation-sensitive Chinese hamster cell mutants defective in DSB repair, XR-V9B cells were fused with XR-V15B, XR-1, and V-3 cells, which have impaired DSB rejoining and belong to three different complementation groups. In all cases, the derived hybrids regained the sensitivity of wild-type cells when exposed to X rays, indicating that the XR-V9B mutant represents a new fourth complementation group among X-ray-sensitive Chinese hamster cell mutants defective in DSB repair.     

Characterization of an X-ray-hypersensitive mutant of V79 Chinese hamster cells

A V79 Chinese hamster cell line XR-V15B exhibiting hypersensitivity to X-ray has been isolated and characterized. Additionally to increased X-ray-sensitivity (approximately 8-fold, as judged by D10 values), cross-sensitivity to bleomycin (3-fold increase), 4NQO (3-fold), H2O2, EMS, MMS (2-fold) were observed also. No increased sensitivity to UV and MMC was found. Genetic complementation analysis indicates that XR-V15B belongs to the same complementation group as the X-ray-sensitive (xrs) mutants of Chinese hamster ovary (CHO) cells described by Jeggo (1985). Biochemical analysis of XR-V15B confirms this finding: the mutant showed a decreased ability to rejoin double-strand breaks induced by X-ray as measured by neutral elution. After 4 h of repair more than 50% of the double-strand breaks remain in comparison to 3% in V79 cells. No difference was observed between wild-type and XR-V15B cells in the initial number of single-strand breaks induced, in the kinetics of their rejoining and in the final level of unrejoined single-strand breaks. Treatment with 5-azacytidine did not have an effect on the reversion frequency of XR-V15B, contrary to the results obtained with the xrs mutants. XR-V15B has been grown in continuous culture for more than 3 months without evidence of reversion. The mutation induction by X-ray irradiation at the HPRT locus is not significantly increased in the mutant, but at doses giving the same degree of cell killing, XR-V15B cells are hypomutable.     

SCH28080, a K+-competitive inhibitor of the gastric H,K-ATPase,binds near the M5-6 luminal loop,preventing K+ access to the ion binding domain

Inhibition of the gastric H,K-ATPase by the imidazo[1,2-alpha]pyridine, SCH28080, is strictly competitive with respect to K+ or its surrogate, NH4+. The inhibitory kinetics [V(max), K(m,app)(NH4+), K(i)(SCH28080), and competitive, mixed, or noncompetitive] of mutants can define the inhibitor binding domain and the route to the ion binding region within M4-6. While mutations Y799F, Y802F, I803L, S806N, V807I (M5), L811V (M5-6), Y928H (M8), and Q905N (M7-8) had no effect on inhibitor kinetics, mutations P798C, Y802L, P810A, P810G, C813A or -S, I814V or -F, F818C, T823V (M5, M5-6, and M6), E914Q, F917Y, G918E, T929L, and F932L (M7-8 and M8) reduced the affinity for SCH28080 up to 10-fold without affecting the nature of the kinetics. In contrast, the L809F substitution in the loop between M5 and M6 resulted in an approximately 100-fold decrease in inhibitor affinity, and substitutions L809V, I816L, Y925F, and M937V (M5-6, M6, and M8) reduced the inhibitor affinity by 10-fold, all resulting in noncompetitive kinetics. The mutants L811F, Y922I, and I940A also reduced the inhibitor affinity up to 10-fold but resulted in mixed inhibition. The mutations I819L, Q923V, and Y925A also gave mixed inhibition but without a change in inhibitor affinity. These data, and the 9-fold loss of SCH28080 affinity in the C813T mutant, suggest that the binding domain for SCH28080 contains the surface between L809 in the M5-6 loop and C813 at the luminal end of M6, approximately two helical turns down from the ion binding region, where it blocks the normal ion access pathway. On the basis of a model of the Ca-ATPase in the E2 conformation (PDB entry 1kju), the mutants that change the nature of the kinetics are arranged on one side of M8 and on the adjacent side of the M5-6 loop and M6 itself. This suggests that mutations in this region modify the enzyme structure so that K+ can access the ion binding domain even with SCH28080 bound.     

Genetic analysis of X-ray-sensitive mutants of the CHO cell line

The genetic diversity of 6 X-ray-sensitive (xrs) mutants of the CHO cell line has been investigated. Hybrids were constructed by fusing ouabain- and 6-thioguanine-resistant cells to ouabain- and 6-thioguanine-sensitive cells and selecting in HAT and ouabain medium. Hybrids were examined for ploidy and X-ray sensitivity. Crosses between xrs mutants and wild-type showed that each mutant was recessive. Crosses between different xrs mutants showed that all were in the same complementation group. Although all the mutants are primarily sensitive to ionizing radiation and bleomycin, and all have a defect in double-strand break rejoining, their cross-sensitivity to other DNA-damaging agents differed to some degree. One explanation is that this repair gene is involved in a pleiotropic response to DNA damage.     

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.