DNA strand breaks and repair synthesis in Yoshida sarcoma cells with differential sensitivities to bifunctional alkylating agents and UV light

The induction of DNA-strand breaks and repair synthesis has been examined in cultured Yoshida sarcoma cell lines sensitive (YS) and resistant (YR) to methylene dimethanesulphonate (MDMS). Using an alkaline DNA unwinding-hydroxylapatite technique, we were able to detect breaks in DNA immediately after MDMS treatment and at similar levels in both YS and YR cells. MDMS treatment and post-treatment incubation in the presence of 1-β-D-arabino-furanosylcytosine (araC) lead to a large increase in the numbers of breaks when compared with MDMS treatment alone which indicated that many of the DNA-strand breaks seen after MDMS treatment were intermediates in excision repair. The magnitude of break incidence with the araC treatment was again equal in YS and YR cells indicating that these 2 lines made enzymic incision next to MDMS-induced lesions with equal capacities.During incubation following MDMS treatment, the levels of DNA-strand breaks in YR cells were found to decrease more rapidly than in YS cells. Parallel DNA-repair synthesis estimations, using BND-cellulose chromatography, revealed that the increased rate of decline in breaks in YR cells was accompanied by an increase in repair-synthesis activity compared to YS cells. This was interpreted as indicating that an intermediate step in an excision-repair pathway for MDMS-induced lesions was relatively deficient in YS compared to YR cells.A similar difference in the rates of decline of DNA-strand breaks between YS and YR cells was also observed following treatment with UV light to which MDMS-resistant YR cells also display cross-resistance. However, no such difference was detected following treatment with the monofunctional alkylating agent, methyl methanesulphonate, to which YS and YR cells are equally sensitive. These results suggest that resistance to MDMS in the YR cell line is achieved by an increased efficiency in the gap-sealing component of the excision-repair process.     

DNA strand breaks in resistant and sensitive murine lymphoma cells detected by the hydroxyapatite chromatographic technique.

Strand breaks were determined in L5178YS and L5178YR cell lines with DOS of 0.75 and 1.75 Gy, respectively, for 7 MeV electrons. The hydroxyapatite chromatographic technique was used to measure the breaks produced by 7 MeV electrons or 7 MeV neutrons immediately after irradiation or after maximal repair. Oxygen enhancement values for survival as well as strand breaks were determined for both cell lines and both qualities of radiation. The results indicate that despite a differential response to the lethal effects of radiation the levels of DNA strand breaks induced in these two lines were identical. Furthermore the values obtained for oxygen enhancement ratios (o.e.r.) and relative biological effectiveness (r.b.e.) for DNA strand breaks were different from those for cell survival. These results show that the difference in radiation sensitivity for cell killing is not reflected by the extent of DNA strand breaks measured by this method.     

Involvement of poly (ADP-ribose) in the radiation response of mammalian cells

The evidence implicating poly (ADP-ribose) in the radiation response of mammalian cells is reviewed. It is concluded that the apparently conflicting results using inhibitors of ADP-ribosyl transferase (ADPRT) can be explained by a working hypothesis. This hypothesis maintains that poly (ADP-ribose) is required for repair of radiation damage (presumably to facilitate ligation). In most cells the synthesis of poly (ADP-ribose) is not rate limiting for repair and therefore, an almost complete inhibition of ADPRT activity is required to potentiate the radiation response. In radiation-sensitive cells (e.g. resting lymphocytes, L5178Y-S cells) with a deficient poly (ADP-ribose) metabolism, its synthesis can become rate limiting for repair. In such cells even a partial inhibition of ADPRT activity may enhance radiation-induced cell killing. It is suggested that if such differences exist between normal and cancer cells, they can be utilized to improve the therapeutic ratio of radiotherapy.     

Cell cycle effects on the basal and DNA-damaging-agent-stimulated ADPRT activity in cultured mammalian cells

ADP-ribosyl transferase (ADPRT) is a DNA-dependent chromatin-associated enzyme which covalently attaches ADP-ribose moieties derived from NAD+ to protein acceptors to form poly(ADP-ribose). ADPRT activity is strongly stimulated by breaks in DNA, and it is suggested that its activity is required for efficient DNA excision repair. In this paper, a cell-cycle-dependent fluctuation of basal ADPRT activity was demonstrated by measuring it in permeabilized FL cells. The cell used was subjected to arginine starvation for 48 h before being released from the block by replacement of deficient medium with complete medium and cells in different proliferating stages were traced by [3H]TdR pulse labelling and obtained at different intervals after block release. The peak basal ADPRT activity appeared 4-6 h after the appearance of the peak of DNA synthesis. After treating the cells with MNNG (10(-4) M), MMS (10(-3)-10(-4) M) and 4NQO (10(-5) M) for 90 min just after release of the block, the ADPRT activity was markedly stimulated. It was further demonstrated that the effects of MNNG/4NQO and cell cycle influence on the level of poly(ADP-ribose) synthesis appear to be additive. While concerning MMS, quite a different pattern of ADPRT stimulation in the cell cycle was demonstrated, i.e., the activity of ADPRT stimulation of 10(-3) M MMS was found to be completely dependent on the basal ADPRT activity. In the cells with the highest basal ADPRT activity 12 h after block release, the MMS-induced ADPRT stimulation could not be observed. It was suggested that more than one pathway might be present in ADPRT stimulation induced by DNA-damaging chemicals, and the cells synchronized in late G1 stage might be the most suitable for demonstrating poly(ADP-ribose) synthesis after DNA damage.     

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.     

The effect of post-treatment with caffeine on survival and UV-induced mutation frequencies in Chinese hamster and mouse lymphoma cells in vitro

The effect of post-treatment with caffeine on the survival of a number of cell lines after UV-irradiation has been studied. The mouse lymphoma cell lines P388 and L5178YS were sensitized by caffeine but only after UV doses of 50 erg/mm² and above. V79 cells also showed sensitization by caffeine but CHO cells and two cell lines YS and YR derived from Yoshida sarcoma of rats, sensitive and resistant to UV radiation, respectively, showed no effect.P388 and V79 cells were both mutable by UV, and caffeine, when studied at a single expression time (42–48 h) and at a single dose level (0.5 M and 0.75 M, respectively) suppressed the UV-induced mutation frequency in both cell lines. L51788YS cells although sensitized by caffeine showed no increase in frequency of thymidine-resistant (TdR^r) colonies when irradiated with UV.On more detaled examination, caffeine was found to delay the expression of UV-induced mutations inV79 cells, and the delay was dependent on the dose of caffine used. The effect on expression time was less when caffeine was present 0–48 h than when it was present throughout the post-irradiation incubation period. Similar results were obtained in P388 cells.The data are discussed in relation to those of other workers and to the concept that caffeine inhibits an error prone post-replication repair process in mammalian cells     

Unscheduled DNA synthesis induced by N-acetoxy-2-acetylaminofluorene is not sensitive to regulation by ADP-ribosyl transferase

We have directly compared in resting human mononuclear leukocytes the DNA repair effects caused by ADP-ribosyl transferase (ADPRT) activity following DNA damage induction by gamma radiation, UV radiation, ethylene oxide (EO) and N-acetoxy-2-acetylaminofluorene (NA-AAF). The presence of inhibitors of ADPRT during the quantitation of unscheduled DNA synthesis (UDS) resulted in about a 2-fold increase of UDS when induced by gamma radiation, UV radiation or EO. The stimulation of UDS by EO, UV- or gamma-radiation in the presence of an ADPRT inhibitor was equally strong whether 1 mM or 10 mM hydroxyurea was used to suppress scheduled DNA synthesis. The level of NA-AAF induced UDS was not affected by inhibitors of ADPRT. In addition, direct estimation of ADPRT activity revealed that at doses giving maximal UDS, NA-AAF damage did not induce a measurable enzymatic activity whereas gamma-radiation, UV radiation and EO all showed a significant dose response increase. We have interpreted our data to mean that NA-AAF induced UDS estimates DNA repair relating mainly to DNA lesions that are recognized with difficulty, and hence, the rate of endonuclease-induced DNA strand break accumulation is not sufficient to allow a stimulation of ADPRT and affect the quantitation of UDS.     

The effects of benzamide ADP-ribosyl transferase inhibitors on cell survival and DNA strand-break repair in irradiated mammalian cells

We have recently shown that 3-acetamidobenzamide (3-AAB), a highly effective inhibitor of ADP-ribosyl transferase (ADPRT), can act as a post-irradiation (electrons) sensitizer on the mouse lymphoma cell lines L5178Y R and S. We have now shown that this compound sensitizes human derived skin fibroblasts but to a lesser extent. Fibroblasts derived from normal, Friedreich's ataxia, and ataxia-telangiectasia individuals were equally sensitized by 3-AAB to electron radiation. 3-AAB was also effective in sensitizing the mouse lymphoma lines to fast neutron irradiation. In addition DNA strand break repair was retarded as had been found after electron irradiation. 3-Nitrobenzamide is structurally a potentially dual action radiation sensitizer with electron affinic and ADPRT inhibitory properties. It is a weaker inhibitor of ADPRT compared to 3-AAB, and results in a smaller sensitization of mouse lymphoma cells in air. However, a much greater sensitization is achieved in anoxia. This greater sensitization appears to be a synergistic rather than an additive combination of its electron affinic and ADPRT inhibitory properties.     

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.     

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

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

The 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.     

Evidence for caffeine-sensitive damage in methylazoxymethanol acetate-treated L5178Y cells.

The effects of methylazoxymethanol (MAM) acetate on colony survival, cell proliferation and DNA synthesis of murine lymphoma L5178Y cells are studied. Decreased sensitivity and immediate depression of cell proliferation and DNA synthesis were found in L5178Y cells in contrast to the reports on HeLa cells. Pre-labelling with 5-bromodeoxyuridine (BUdR) did not enhance significantly the carcinogen-induced cell lethality. Post-treatment with caffeine greatly enhanced cell lethality and depression of cell proliferation. These effects of caffeine were diminished when the cells had passed through two generations following the MAM acetate treatment. Experiments with synchronized cells showed that the action of caffeine was located primarily in S phase following the MAM acetate-treatment. These results strongly suggest that in L5178Y cells, MAM acetate induces damage, which is repaired by a mechanism analogous to post-replication repair of UV light-induced damage.     

Correlation of dihydrofolate reductase elevation with gene amplification in a homogeneously staining chromosomal region in L5178Y cells

A methotrexate (MTX)-resistant murine lymphoblastoid cell line has been obtained by serial passage in increasing concentrations of MTX which is greater than 100,000-fold resistant to MTX (L5178YR) and has dihydrofolate reductase (DHFR) levels 300-fold higher than the parental line. The L5178YR cell line synthesizes approximately 10-11% of its total soluble cell protein as DHFR regardless of growth phase, as measured by direct immunoprecipitation with a monospecific antiserum. Molecular hybridization of a purified [3H]DNA probe complimentary to DHFR specific mRNA with cellular DNA and RNA indicates that DHFR coding sequences are elevated several hundred fold in both nucleic acid species in the mutant cell line. Giemsa-banding studies of the diploid mutant line indicate the presence of a large homogeneously staining region on chromosome No. 2. In situ molecular hybridization studies indicate that the DHFR genes are localized in this homogeneously staining region. The homogeneously staining region probably consists of tandom repeats of a basic segment approximately 800 kilo base pairs long.     

细胞周期及DNA损伤剂引起的细胞NAD含量变化及其意义

本文观察了FL细胞中ADP-核糖基转移酶(ADPRT)底物NAD含量的细胞周期性变化及其与DNA复制之间的关系。FL细胞NAD含最在G_1期最高,而在S期DNA合成高峰后0—3小时(S/G_2期)达到最低点。ADPRT抑制剂3 AB能够抑制NAD含量的细胞周期性变化,而且S期DNA合成亦受到抑制,并呈现S期延长,提示ADP-核糖基化作用可能参与DNA复制过程。本文还观察了三种DNA损伤剂MNNG、MMS及4NQO对处于细胞周期不同时相的FL细胞NAD含量的影响,以及ADPRT抑制剂3 AB及尼克酰胺对此影响的作用。证明ADPRT抑制剂可以特异地抑制DNA损伤性NAD含量下降而对正常FL细胞NAD含量及代谢抑制剂2,4-DNP所致的NAD含量下降没有影响。从而有可能建立一个以测量细胞内NAD含量为指标的简便、快速、特异的检测DNA损伤因子的方法。     

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.     

The relationship of DNA and chromosome damage to survival of synchronized X-irradiated L5178Y cells. II. Repair

The purpose of this study was to investigate the role of DNA and chromosome repair in determining the difference in radiosensitivity between a radiosensitive murine leukemic lymphoblastoid cell line, L5178Y-S, and its radioresistant counterpart, L5178Y-R. Populations of cells in the G1 or G2 phase of the cell cycle were obtained by centrifugal elutriation and irradiated with X-ray doses up to 10 Gy and allowed to repair at 37 degrees C for various periods. The kinetics of DNA double-strand break repair was estimated using the DNA neutral filter elution method, and the kinetics of chromosome repair was measured by premature chromosome condensation. L5178Y-S cells exhibited decreased repair rates and limited repair capacity at both the DNA and chromosome level in both G1 and G2 phases when compared to L5178Y-R cells. For the repair-competent L5178Y-R cells, the rate of DNA repair was similar in G1 and G2 cells and exhibited both fast and slow components. While the kinetics of chromosome break repair in G1 cells was similar to that of DNA repair, chromosome repair in G2 cells had a diminished fast component and lagged behind DNA repair in terms of fraction of damage repaired. Interestingly, concomitant with a diminished repair capacity in L5178Y-S cells, the number of chromatid exchanges in G2 cells increased with time, whereas it remained constant with repair time in L5178Y-R cells. These results suggest that the basis for the exceptional radiosensitivity of L5178Y-S cells is a defect in the repair of both DNA double-strand breaks and chromosome damage.     

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.     

Poly(ADP-ribose) and the recovery from damage in Chinese hamster cells due to 5-bromodeoxyuridine photolysis

Exposure of Chinese hamster cells to near-u.v. light, following the uniform incorporation of 5-bromodeoxyuridine (BrdUrd) into their DNA, resulted in cell killing that was close to exponential. An inhibitor of poly(ADP-ribose) synthesis, 3-aminobenzamide (3-ABA), enhanced the cytotoxic effect of this treatment when present for 2 h at 20 mM after light exposure. The dose modifying factor was 1.4. Under conditions that resulted in a sigmoidal survival curve (a 30 min BrdUrd pulse in S phase, followed 90 min later by light exposure) the effect of 3-ABA was to remove the shoulder of the survival curve with very little change in its final slope. Using various inhibitors of ADP-ribosyl transferase (ADPRT) the enhanced cell killing was found to correlate with the inhibitors' relative potency. Cellular NAD+, the substrate for poly(ADP-ribose) synthesis, was rapidly depleted after exposure. This depletion was largely prevented by 3-ABA; the activity of ADPRT increased with the fluence of near-u.v. light; and the concentration of cellular NAD+ decreased with exposure. ADPRT activity was maximal immediately after exposure to near u.v. light and then decayed to pre-exposure levels within 30 min (37 degrees C). The enhanced cytotoxicity of BrdUrd + near-u.v. light, when followed by 3-ABA treatment, disappeared at a rate similar to that of the decay in ADPRT activity. We conclude from these results that poly(ADP-ribose) synthesis is important for the recovery from BrdUrd photolysis damage in DNA. Because this damage and its repair are relatively specific (e.g. compared to ionizing radiation) and relatively easy to manipulate, it could serve as a model system for the study of the role of poly(ADP-ribose) in the repair of DNA damage.     

The relationship of DNA and chromosome damage to survival of synchronized X-irradiated L5178Y cells. I. Initial damage

We investigated the role of initial DNA and chromosome damage in determining the radiosensitivity difference between the variant murine leukemic lymphoblast cell lines L5178Y-S (sensitive) and L5178Y-R (resistant) and the difference in cell cycle-dependent variations in radiosensitivity of L5178Y-S cells. We measured initial DNA damage (by the neutral filter elution method) and chromosome damage (by the premature chromosome condensation method) and compared them with survival (measured by cloning) for both cell lines synchronized in G1 or G2 phase of the cell cycle (by centrifugal elutriation) and irradiated with low doses of X rays (up to 10 Gy). The initial yield of DNA and chromosome damage in G2 L5178Y-S cells was almost twice that in G1 L5178Y-S cells and G1 or G2 L5178Y-R cells. In all cases DNA damage expressed as relative elution corresponded with chromosome damage (breaks in G1 chromosomes, breaks and gaps in G2 chromosomes). Also we found that the initial DNA and chromosome damage did not determine cell age-dependent radiosensitivity variations in L5178Y-S cells, as there was less initial damage in the more sensitive G1 phase than in the G2 phase. L5178Y-R cells showed only small changes in survival or initial yield of DNA and chromosome damage throughout the cell cycle. Because survival and initial damage in sensitive and resistant cells irradiated in G2 phase correlated, the difference in radiosensitivity between L5178Y-S and L5178Y-R cells might be determined by initial damage in G2 phase only.