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Nine mutants isolated from CHO · K1 cells with increased sensitivity to the lethal effect of plumbagin (PG), a powerful superoxide generator, were classified into five groups, A-E, according to their sensitivity to PG and methyl viologen (MV). Two mutants of group B (Pa13 and Pb4) were sensitive to both drugs, and two mutants of group C (Pa14 and Pa15) were moderately sensitive to PG and extremely sensitive to MV. To mitomycin C (MMC) these mutants showed cross-sensitivity; especially Pa13 and Pb4 (group B) were highly sensitive to MMC. Genetic complementation analyses of these four mutants were carried out using MV sensitivity. Sensitivity group B was divided into two complementation groups, I and II. Pa14 and Pa15 belonged to the same complementation group III. These four mutants were also classified into three complementation groups for MMC sensitivity. Because Pa13 and Pb4 were also sensitive to cis - diamminedichloroplatinum(II), they may have a defect in the repair of DNA crosslinks induced by these agents. A complementation group IV (Pa2 and Pa8) was also suggested based on the studies of MMC sensitivity.

The induction and repair of radiation-induced DNA damage was assessed in 3 mouse cell lines, including the parental L cell line, a radiation-sensitive, SL3-147 mutant cell line and the H5 revertant to radiation resistance. The yield of neither radiation-induced DNA single- nor double-strand breaks could explain the variable sensitivity of the 3 cell lines. Closure of DNA single-strand breaks proceeded at a similar rate in both the L and SL3-147 cell lines. Closure of DNA double-strand breaks however was significantly slower and less complete in the SL3-147 cell line than in either of the radiation-resistant cell lines. The results are consistent with the increased radiation sensitivity of the SL3-147 cell line resulting from a defect in their ability to repair radiation-induced DNA double-strand breaks.

A UV-sensitive mutant, Q31, isolated from mouse-lymphoma L5178Y cells, was studied for excision and post-replication repairs. 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 of detection, whereas Raji cells eliminated about 60% of the sites. These results during incubation for 24 h, and Q31 cells removed 3% of the sites, a fraction less than the limit 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 cell strains increased to comparable sizes after a 2-h chase.

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 D 10 values (dose required to reduced 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.

The mouse lymphoma L5178Y cell line and its radiosensitive variants M10 and LX830 were examined for DNA synthesis after X-irradiation. The dose-response curves show that the rates of DNA synthesis immediately after exposure are reduced in a dose-dependent fashion and that the extents of reduction in these 3 cell lines are similar to one another. But a difference was observed in the recovery of DNA synthesis with time of incubation. The recovered levels in M10 and LX830 cells were much higher than those in L5178Y cells at high doses of X-rays. These results are discussed in relation to radioresistant DNA synthesis in ataxia telangiectasia cells.

A mouse-cell mutant sensitive to methyl methanesulfonate (MMS), X-rays, ultraviolet light (UV), and crosslinking agents was selected using the replica plating and cell suspension spotting methods. This mutant (XUM1) is a mitomycin C-sensitive derivative of previously reported XU1, a mutant sensitive to MMS, X-rays and UV. Since XU1 is highly susceptible to the lethal effect of 4-nitroquinoline-1-oxide (4NQO), XUM1 is also hypersensitive to 4NQO. Growth inhibition area tests showed that low concentrations of mutagens were detected with the multiple mutagen-sensitive mutant XUM1. Hence XUM1 cells will be useful in detecting with high sensitivity a wide range of mutagens and carcinogens which mimic X-rays, UV and crosslinking agents.

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.

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.

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 D 37 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.

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 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 MNC 151 and 3 times higher for MCE 506 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 gradient sedimentation technique. The results show that normal cells and both mutants are able to incise the cross-linked DNA, which is the fisrst step of cross-link repair.

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.