The repair of methyl methanesulphonate-induced lesions in the DNA of a murine lymphoma cell

The introduction of single-strand breaks into the DNA of a murine lymphoma (L5178Y) cell treated in vivo with methyl methanesulphonate (MMS) and the behaviour of these breaks on post-treatment incubation were studied. A large proportion of single-strand breaks present after MMS treatment could be repaired as shown by sedimentation in alkaline sucrose. Two inhibitors of DNA synthesis, hydroxyurea and cytosine arabinoside affected the repair process differently-hydroxyurea had only a small effect while cytosine arabinoside blocked repair and at some doses allowed further degradation of the DNA. It was also found that the level of ‘repair replication’ in the presence of cytosine arabinoside was lower than that found in the presence of hydroxyurea.     

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.     

The repair of double-strand DNA breaks correlates with radiosensitivity of L5178Y-S and L5178Y-R cells

To better understand the basis for the difference in radiosensitivity between the variant murine leukemic lymphoblast cell lines L5178Y-R (resistant) and L5178Y-S (sensitive), the production and repair of DNA damage after X irradiation were measured by the DNA alkaline and neutral elution techniques. The initial yield of single-strand DNA breaks and the rates of their repair were found to be the same in both cell lines by the DNA alkaline elution technique. Using the technique of neutral DNA elution, L5178Y-S cells exhibited slightly increased double-strand breakage immediately after irradiation, most significantly at lower doses (i.e., less than 10 Gy). Nevertheless, even at doses that yielded equal initial double-strand breakage of both cell lines, the survival of L5178Y-S cells was significantly less than that of L5178Y-R cells. When the technique of neutral DNA elution was employed to measure the kinetics of DNA double-strand break repair, both cell lines exhibited biphasic fast and slow components of repair. However, the double-strand repair rate was much lower in the radiosensitive L5178Y-S cells than in the L5178Y-R cells (T1/2 of 60 vs 16 min). This difference was more pronounced in the fast-repair component. These results suggest that the repair of double-strand DNA breaks is an important factor determining the radiosensitivity of L5178Y 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.     

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.     

Radiation-Induced Breaks of DNA in Cultured Mammalian Cells

Mouse leukemic cells (L5178Y) in suspension culture were irradiated and the extent of single-strand breaks and double-strand cuts of DNA was estimated by sucrose gradient centrifugation. The radiation produced 3.0 single-strand breaks per cell (G(1) stage) per rad and approximately 0.3 double-strand breaks per cell (G(1) stage) per rad.     

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.     

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.     

The contribution of alkylation to the activity of quinone antitumor agents

Studies have shown that the quinone group can produce tumor cell kill by a mechanism involving active oxygen species. This cytotoxic activity can be correlated with the induction of DNA double strand breaks and is enhanced by the ability of the quinone compound to bind to DNA by alkylation. The cytotoxic activity and the production of DNA damage by model quinone antitumor agents were compared in L5178Y cells, sensitive and resistant to alkylating agents, to assess the contribution of alkylation to the activity of these agents. The resistant L5178Y/HN2 cells were found to be two fold and six fold more resistant to the alkylating quinones, benzoquinone mustard and benzoquinone dimustard, respectively, than parent L5178Y cells. In contrast, the L5178Y/HN2 cells showed no resistance to the nonalkylating quinones, hydrolyzed benzoquinone mustard and bis(dimethylamino)benzoquinone. The alkylating quinones produced approximately two fold less cross-linking in L5178Y/HN2 cells compared with L5178Y sensitive cells. DNA double strand break formation by hydrolyzed benzoquinone mustard and bis(dimethylamino)benzoquinone was not significantly different in sensitive and resistant cells. However, the induction of double strand breaks by the alkylating quinones benzoquinone mustard and benzoquinone dimustard was reduced by 5-fold and 15-fold, respectively, in L5178Y/HN2 cells. These results show that the alkylating activity of the alkylating quinones cannot directly explain all of the enhanced cytotoxic activity of these agents. Furthermore, they provide strong evidence that the enhanced formation of DNA double strand breaks by alkylating quinone agents is directly related to the ability of these agents to bind to DNA. This increased formation of strand breaks may account for the enhanced cytotoxic activity of the alkylating quinones.     

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.     

Effect of UVC and araC on L5178Y-R and L5178Y-S cells. Nucleoid sedimentation

The effects of UVC radiation (lambda = 254 nm, 85 J/m2) and/or 1-beta-D-arabino-furanosylcytosine (araC, 2 x 10(-3) M, 2 h) on two mouse lymphoma cell lines, UVC-sensitive and X-ray resistant L5178Y-R and UVC-resistant and X-ray sensitive L5178Y-S, were investigated. AraC treatment inhibited the semiconservative DNA replication to 1.4% and 3.8% in L5178Y-R and L5178Y-S cells, respectively, and decreased the sedimentation distance of nucleoids from the cells of both lines. The shortening of sedimentation distances induced by UVC and araC treatment was 8.1 mm for L5178Y-R cells and 11.8 mm for L5178Y-S, and indicated a higher number of DNA breaks in L5178Y-S cells. Assuming that such breaks are the result of the inhibition of DNA repair replication by araC, we conclude that L5178Y-S cells have a greater number of repaired sites than L5178Y-R cells.     

A non-radioactive, PFGE-based assay for low levels of DNA double-strand breaks in mammalian cells

A PFGE method was adapted to measure DNA double-strand breaks (DSBs) in mammalian cells after low (0-25 Gy) doses of ionising radiation. Instead of radionuclide incorporation, DNA staining in the gel by SYBR-Gold was used, which lowered the background of DNA damage and could be applied to non-cycling cells. DSB level was defined as a product of a fraction of DNA released to the gel (FR) and a number of DNA fragments in the gel (DNA(fragm)) and expressed as a percentage above control value. The slope of the dose-response curve was two-fold higher compared to that with FR alone as DSB level indicator (31.4 versus 15.6% per Gy). Two alternative ways were proposed to determine the total amount of DNA, used for FR calculation: measurement of DNA content in a plug not subjected to electrophoresis, with the use of Pico-Green, or estimation of DNA released to the gel from a plug irradiated with 600 Gy of gamma-rays. The limit of DSB detection was 0.25 Gy for human G1-lymphocytes and 0.5-1 Gy for asynchronous cultures of human glioma M059 K and J or mouse lymphoma L5178Y-R and -S cells. Specificity of our PFGE assay to DSB was confirmed by the fact that no damage was detected after treatment of the cells with H(2)O(2), an inducer of single-strand DNA breaks (SSBs). On the contrary, the H(2)O(2) inflicted damage was detected by neutral comet assay, attaining 160% above control (equivalent to 2.5 Gy of X-radiation). DSB rejoining, measured in cells after X-irradiation with a dose of 10 Gy, generally proceeded faster than that measured previously after higher (30-50 Gy) doses of ionising radiation. Clearly seen were defects in DSB rejoining in radiosensitive M059 J and L5178Y-S cells compared to their radioresistant counterparts, M059 K and L5178Y-R. In some cell lines, a secondary post-irradiation increase in DSB levels was observed. The possibility is considered that these additional DSBs may accumulate during processing of non-DSB clustered DNA damage or/and represent early apoptotic events.     

Ligase-deficient yeast cells exhibit defective DNA rejoining and enhanced gamma ray sensitivity.

Yeast cells deficient in DNA ligase were also deficient in their capacity to rejoin single-strand scissions in prelabeled nuclear DNA. After high-dose-rate gamma irradiation (10 and 25 krads), cdc9-9 mutant cells failed to rejoin single-strand scissions at the restrictive temperature of 37 degrees C. In contrast, parental (CDC9) cells (incubated with mutant cells both during and after irradiation) exhibited rapid medium-independent DNA rejoining after 10 min of post-irradiation incubation and slower rates of rejoining after longer incubation. Parental cells were also more resistant than mutant cells to killing by gamma irradiation. Approximately 2.5 +/- 0.07 and 5.7 +/- 0.6 single-strand breaks per 10(8) daltons were detected in DNAs from either CDC9 or cdc9-9 cells converted to spheroplasts immediately after 10 and 25 krads of irradiation, respectively. At the permissive temperature of 23 degrees C, the cdc9-9 cells contained 2 to 3 times the number of DNA single-strand breaks as parental cells after 10 min to 4 h of incubation after 10 krads of irradiation, and two- to eightfold more breaks after 10 min to 2.5 h of incubation after 25 krads of irradiation. Rejoining of single-strand scissions was faster in medium. After only 10 min in buffered growth medium and after 10 krads of irradiation, the number of DNA single-strand breaks was reduced to 0.32 +/- 0.3 (at 23 degrees C) or 0.21 +/- 0.05 (at 37 degrees C) per 10(8) daltons in parental cells, but remained at 2.1 +/- 0.06 (at 23 degrees C) or 2.3 +/- 0.07 (at 37 degrees C) per 10(8) daltons in mutant cells. After 10 or 25 krads of irradiation plus 1 h of incubation in medium at 37 degrees C, only DNA from CDC9 cells was rejoined to the size of DNA from unirradiated cells, whereas at 23 degrees C, DNAs in both strains were completely rejoined.     

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.     

Activity of the plant flavanol quercetin in the mouse lymphoma L5178Y TK+/− mutation,DNA single-strand break,and Balb/c 3T3 chemical transformation assays

The activity of quercetin was investigated in (a) the L5178Y TK^+/− mutation assay system, using trifluorothymidine (TFT) as the selection agent; (b) the DNA single-strand break assay in L5178Y cells after the same treatment used for the mutation assay; and (c) the Balb/c 3T3 chemical transformation assay (foci method). Quercetin was active in the TK^+/− mutation assay, increasing the frequency of TFT-resistant colonies from a control value of 37 per 10⁶ viable cells to 355 per 10⁶ viable cells at 20 μg/ml. When S9 was present, the activity was decreased at each concentration tested. As the S9 concentration employed (mg/ml protein) was decreased, the induced mutant frequency increased. DNA single-strand breakage was observed without S9 at 10 μg/ml, using the alkaline elution technique; a maximal rate of elution was reached at 20 μg/ml. In the chemical transformation experiments, transformation just at the level of 0.05% significance (if both intermediate and typical transformed colonies were combined) was observed. The evidence is sufficiently strong that additional attention should be given to its role as a dietary caused of human cancer.     

The mutagenicity of 5-azacytidine and other inhibitors of replicative DNA synthesis in the L5178Y mouse lymphoma cell

The mutagenic potential of the cytidine analog, 5-azacytidine (Aza Cyd), was tested at the thymidine kinase (TK) gene locus of L5178Y mouse lymphoma cells. 3-h exposure to as little as 20 ng/ml Aza Cyd yielded a substantial increase in TK-deficient L5178Y cells as measured by drug-induced resistance to trifluorothymidine (TFTres) 48 h later. This mutagenic effect was diminished up to 75% when Aza Cyd was tested in the presence of either enzymatically active or heat-denatured 9000 X g supernatant prepared from rat liver homogenate. The mutagenicity of Aza Cyd was also decreased in the presence of 1-5 X 10(-3) M thymidine and eliminated in the presence of greater than 1 X 10(-5) M cytidine. Two L5178Y TK-deficient cell lines had no selective survival advantage compared to TK-competent L5178Y cell stock when plated in soft-agar medium that contained Aza Cyd. Four other specific inhibitors of scheduled DNA synthesis in mammalian cells, deoxyadenosine, aphidicolin, 1-beta-D-arabinofuranosylcytosine, and hydroxyurea were also L5178Y/TK mutagens. These data along with other published results suggest that chemicals known to disrupt nucleotide biosynthesis, alter deoxyribonucleotide pools, or directly inhibit DNA polymerase can cause stable, heritable increases in TFT resistance through mechanisms dependent upon altered replicative DNA synthesis, yet not necessarily dependent upon DNA incorporation or the binding of these mutagenic agents to nuclear DNA.     

Discrepancy between the initial DNA damage and cell survival after camptothecin treatment in two murine lymphoma L5178Y sublines

Two L5178Y (LY) murine lymphoma cell sublines, LY-R, resistant, and LY-S, sensitive, to X-irradiation display inverse cross-sensitivity to camptothecin (CPT): LY-R cells were more susceptible to this specific topoisomerase I inhibitor than LY-S cells. After 1 h incubation with CPT, the doses that inhibited growth by 50 per cent (ID₅₀) after 48 h of incubation were 0·54μM for LY-R cells and 1·25 μM for LY-S cells. Initial numbers of DNA–protein crosslinks (DPCs) measured at this level of growth inhibition were two-fold higher in LY-R (5·6 Gray-equivalents) than in LY-S cells (3·1 Gray-equivalents), which corresponds well with the greater in vitro sensitivity of Topo I from LY-R cells to CPT.^1,2 Conversely, the initial levels of single-strand DNA breaks (SSBs) and double-strand DNA breaks (DSBs) were lower in LY-R cells (4·2 Gray-equivalent SSBs and 5·8 Gray equivalent DSBs) than in LY-S cells (8·0 Gray-equivalent SSBs and 12·0 Gray-equivalent DSBs). Dissimilarity in the replication-dependent DNA damage observed after 1 h of treatment with CPT was not due to a difference in the rate of DNA synthesis between the two cell lines, but may have arisen from a substantially slower repair of DNA breaks in LY-S cells.³ Release from G₂ block by caffeine co-treatment significantly increased cell killing in the LY-S subline, and only slightly inhibited growth of LY-R cells. These results show that after CPT treatment cells arrest in G₂, allowing them time to repair the long-lived DSBs. As LY-S cells are slower in repairing the DSBs, they were more susceptible to CPT in the presence of caffeine.     

Cytogenetic analysis of an immunogenic mutant of the L5178Y lymphoma

A mutant of the uniformly lethal L5178Y lymphoma, called the L5178Y/Manitoba (L5178Y/M), was rejected after subcutaneous challenge in syngeneic DBA/2 mice. Karyotypic analysis revealed that the parent L5178Y lymphoma had four chromosome markers, with the mutant L5178Y/M sharing one of them as well as possessing two distinguishing markers. One diploid and two hypotetraploid clones were isolated from the L5178Y/M; they contained all the marker chromosomes and were also rejected by the syngeneic host. In addition to the shared chromosome markers, the L5178Y/M possessed antigens in common with the parent L5278Y. DBA/2 mice made immune to the mutant by subcutaneous immunization were able to slow the growth of the parent tumor but not the unrelated P-815-X2 mastocytoma.     

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.