Isolation and characterization of vitamin A-sensitive Chinese hamster lung cell lines

Retinyl acetate (RA)-sensitive variants (RAs-2 and RAs-3) of V79 cell line were isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. The variants were stable and showed a 3- to 4-fold increase in sensitivity to RA compared to parental V79 cells. The RAs-2 clone was also sensitive to retinol and retinol palmitate. The RA-sensitivity behaves as a recessive trait in all hybrids of RAs-2 and V79. A number of physiological parameters were indistinguishable in V79 and RAs-2 cells, including the extent of uptake of [3H]retinol, the release of K+ from the cells induced by RA, and the levels of retinol and retinoic acid binding proteins. However, one possible correlation with the RA-sensitive phenotype was observed: Gomori acid-phosphatase staining of RA-treated RAs-2 and V79 cells indicated that lysosomal membrane of RAs-2 cells was more labile than those of the parental V79 cells.     

Isolation of chloroquine-resistant Chinese hamster V79 cell variants that are also resistant to ammonium chloride

Chloroquine-resistant (CQr) clones (CQ-21 and CQ-22) have been isolated from mutagenized hamster lung V79 cells by exposing the cells to a high dose of chloroquine. CQ-21 and CQ-22 showed about 3-fold higher resistance to chloroquine than the parental V79 cells, and they showed specific cross-resistance to another amine, NH4Cl, which is also concentrated in lysosomes. CQr clone showed no cross-resistance to other unrelated agents. Chloroquine-induced inhibition of [125I]ricin internalization was observed in both cell lines at neutral pH, but the inhibition of uptake was less in the variant. Also, the degradation of endogenous protein was slowed in the mutant; further, treatment of cells with 30 micrograms/ml of chloroquine inhibited the degradation of endogenous proteins in the parental V79, but not in CQ-22 cells. Similar levels of acid phosphatase, beta-glucuronidase and cathepsin D were observed in V79 and CQ-22 cells, but the level of cathepsin B was lower in the mutant. Electron microscopy showed an increased number of electron-dense bodies, possibly autophagosomes/lysosomes, in the mutant cells grown for 4 days with 5 micrograms/ml of chloroquine. Similar aberrant structures were observed in the parental V79 cells treated for only 3 h with 5 micrograms/ml of chloroquine.     

Induction of DNA-protein crosslinks by dichloromethane in a V79 cell line transfected with the murine glutathione-S-transferase theta 1 gene

Dichloromethane (DCM) is considered a probable human carcinogen. Laboratory studies have shown an increased incidence of lung and liver cancer in mice but not in rats or hamsters. Despite the correlation between metabolism of DCM by the glutathione-S-transferase (GST) pathway and the occurrence of tumors in different species, the mechanism of tumor induction by DCM metabolites produced through the GST pathway remains unclear. In this study a V79 cell line stably transfected with the murine GST theta 1 gene (mGSTT1) was compared to the parent cell line (MZ) to determine how the construct affects DCM metabolism and the sensitivity of the cell line to DNA damage and cytotoxicity. V79 cells were treated with DCM (2.5-10mM) or formaldehyde (150-600muM) for 2h. Also, formaldehyde produced by V79 cytosol metabolism of DCM was measured spectrophotometrically. DNA damage and DNA-protein crosslinks were measured by the standard and proteinase K-modified alkaline single cell gel electrophoresis (SCG) assays. Cytotoxicity was assessed by trypan blue stain exclusion, the Live/Dead((R)) cell viability/cytotoxicity kit for animal cells, and the neutral red assay. After DCM treatment a significant concentration-dependent increase in tail moment in the V79 MZ cells was observed compared to a significant concentration-dependent decrease in tail moment in the V79 mGSTT1 cells. Post-incubation with proteinase K significantly increased DNA migrations in DCM-treated V79 mGSTT1 cells. DCM formed significantly higher levels of formaldehyde in the cytosol of the V79 mGSTT1 cells than in the cytosol of the V79 MZ cells. Results using the cytotoxicity assays were comparable using the trypan blue and Live/Dead((R)) assays, neither showing a difference in response between the two cell lines when exposed to either formaldehyde or DCM. These results indicate that V79 mGSTT1 can metabolize DCM to a genotoxic and cytotoxic metabolite, which is likely formaldehyde. This is the first time that the magnitude of the GSTT1 effect can be observed in mammalian cells without confounding caused by using cells with different genetic backgrounds.     

Lack of synchrony among multiple nuclei induces partial DNA fragmentation in V79 cells polyploidized by demecolcine

Abstract. The nuclear morphology of polyploidized cells was examined in V79 Chinese hamster cells polyploidized by demecolcine or K-252a, inhibitors of spindle fibre formation and protein kinases, respectively. A variety of nuclear morphologies, including multinuclei, were observed in V79 cells polyploidized by demecolcine but not by K-252a, which produced mononuclear cells. A lack of synchrony in the nuclear cycle was observed among nuclei in multinuclear polyploidized cells. Partial DNA fragmentation, defined as DNA fragmentation of a nucleus in a multinuclear cell, was detected using the TUNEL method in V79 cells polyploidized by demecolcine but not by K-252a. Apoptosis occurred earlier in cell populations treated with demecolcine than in these treated with K-252a once the drugs were removed from the medium, suggesting that polyploidized cells with separate nuclei tend to apoptose earlier than those with mononuclei.     

Differences in phorbol-ester-induced down-regulation of protein kinase C between cell lines.

Down-regulation of protein kinase C induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) was examined in Swiss 3T3, V79, MDBK and C6 cells by Western blotting. Variations in the rate of down-regulation caused by treatment with 100 nM-TPA were observed; TPA treatment for 5 h caused maximal down-regulation in V79 cells, whereas TPA treatment for 10 h or 30 h was needed for maximal down-regulation of protein kinase C in MDBK or Swiss 3T3 cells respectively. The decrease in amount of immunologically detectable protein kinase C was 30% in MDBK cells and 100% in V79 and Swiss 3T3 cells. MDBK and C6 cells could be completely depleted of protein kinase C by treatment with 250 nM-TPA. In C6 cells, after treatment with 500 nM-TPA, an 80% loss of protein kinase C was seen over 10 h. Measurement of the numbers of phorbol-ester-binding sites remaining in each cell line when protein kinase C was maximally down-regulated indicated that in MDBK and Swiss 3T3 cells loss of phorbol-ester-binding sites paralleled loss of protein kinase C, whereas in V79 and C6 cells no such correlation was observed.     

gamma-Glutamyltranspeptidase-dependent metabolism of 4-hydroxynonenal-glutathione conjugate.

A major pathway for detoxification of the highly reactive lipid peroxidation product, 4-hydroxy-2,3-trans-nonenal (HNE) is through the conjugation with glutathione (GSH). We have studied the metabolism of GS-HNE conjugate by the enzyme gamma-glutamyltranspeptidase (GGT) using its purified form, as well as a GGT-overexpressing fibroblast cell line (V79 GGT). Using mass spectrometry analysis we identified for the first time cysteinylglycine-HNE (CysGly-HNE) as the GGT metabolite of GS-HNE. Furthermore, the GGT-dependent metabolism of GS-HNE in the V79 GGT cell line was associated with a considerable increase of cytotoxicity as compared to a control cell line which does not express GGT (V79 Cl). The cytotoxic effect was dose- and time-dependent (100% cellular death at 200 microM GS-HNE after 24 h incubation) in V79 GGT cells, whereas no decrease of viability was observed in V79 Cl cells. A similar cytotoxic effect was obtained when cells were incubated directly with CysGly-HNE, demonstrating that this GGT-dependent metabolite unlike GS-HNE, exhibits cytotoxic properties.     

An investigation of the genotoxic effects of N-nitrosomorpholine in mammalian cells

N-nitrosomorpholine (NMOR) is a well-known hepatocarcinogen. Since this compound is representative of the group of indirect-acting N-nitrosamines, its metabolic activation should be essential. However, the mechanism of NMOR-induced carcinogenesis is still not completely clear. In this paper we tried to further our understanding of the genotoxic effects of NMOR. The central aim of this study was to elucidate to what extent NMOR requires metabolic activation. For evaluation of the mutagenicity of NMOR, V79 cells were used either in the presence or absence of the microsomal S9 fraction in the mutation assay and formation of reactive oxygen/nitrogen species (ROS/RNS) in Caco-2 cells treated with NMOR was measured by a fluorescent assay. A very weak rise of 6-thioguanine resistant mutations was observed in both NMOR-treated model cells, V79/-S9 and V79/+S9. A significant difference between the level of mutations in V79/-S9 and V79/+S9 cells was recorded on the 7th day of expression only. Data obtained by the fluorescent assay confirmed that NMOR caused generation of ROS/RNS. In summary, the presented results showed that NMOR might induce DNA damage not only indirectly by its activation by drug-metabolizing enzymes but also via direct formation of ROS/RNS.     

Abrogation of the effects of aphidicolin on NIH3T3 and V79 cells by caffeine

In this communication I show that caffeine (1,3,7-trimethylxanthine) stimulates [³H]thymidine incorporation in aphidicolin-treated V79 and NIH3T3 cells. Flow microfluorometric analysis showed that caffeine, partially or fully, abrogates the cell cycle progression block produced by aphidicolin. Increased cell growth is also observed in cultures treated with both aphidicolin and caffeine compared to cultures treated with aphidicolin only. Microscopic examination of V79 cultures treated with aphidicolin for 8 h showed a marked reduction in the freqeuncy of round mitotic cells, as is expected from a drug which inhibits progression through the cell cycle by inhibiting DNA replication; this effect of aphidicolin was also reduced by caffeine. Biochemical analysis showed that caffeine did not directly interfere with the inhibition of DNA polymerase-α by aphidicolin. Analysis of dNTP pools indicated that caffeine increased the level of dCTP in V79 cells. In aphidicolin-treated V79 cells, the increase in the dCTP level due to exogenous cytidine was almost completely blocked; caffeine also substantially overcame this effect of aphidicolin. These results indicate that caffeine produces its effects on aphidicolin-treated cells by altering the dCTP metabolism.     

RNA dependence in the cell cycle of V79 cells

The cell cycle of V79 Chinese hamster lung cells synchronized by hydroxyurea was investigated by flow cytometry. The metachromatic fluorochrome acridine orange was used to differentially stain DNA and RNA of V79 cells. Green and red fluorescence from individual cells, representing cellular DNA and RNA, respectively, was measured by flow cytometry. Periodic changes of cellular DNA and RNA contents were observed over nine cell cycles. The duration of G1, S, and G2 + M phases of synchronized V79 cells whose RNA content was close to that of the cells in balanced growth was 3, 4.5, and 1.5 hours, respectively. The duration of G1 and S phases of cells containing RNA above a certain threshold was inversely proportional to the RNA content. The RNA content of cells containing RNA above the normal level regressed to normal after a few generations. Coefficients of variation for RNA content were significantly larger than those for DNA. An explanation for the decay of synchrony in a synchronized cell population is proposed.     

(6-4) photoproducts and not cyclobutane pyrimidine dimers are the main UV-induced mutagenic lesions in Chinese hamster cells.

A partial revertant (RH1-26) of the UV-sensitive Chinese hamster V79 cell mutant V-H1 (complementation group 2) was isolated and characterized. It was used to analyze the mutagenic potency of the 2 major UV-induced lesions, cyclobutane pyrimidine dimers and (6-4) photoproducts. Both V-H1 and RH1-26 did not repair pyrimidine dimers measured in the genome overall as well as in the active hprt gene. Repair of (6-4) photoproducts from the genome overall was slower in V-H1 than in wild-type V79 cells, but was restored to normal in RH1-26. Although V-H1 cells have a 7-fold enhanced mutagenicity, RH1-26 cells, despite the absence of pyrimidine dimer repair, have a slightly lower level of UV-induced mutagenesis than observed in wild-type V79 cells. The molecular nature of hprt mutations and the DNA-strand specificity were similar in V79 and RH1-26 cells but different from that of V-H1 cells. Since in RH1-26 as well as in V79 cells most hprt mutations were induced by lesions in the non-transcribed DNA strand, in contrast to the transcribed DNA strand in V-H1, the observed mutation-strand bias suggests that normally (6-4) photoproducts are preferentially repaired in the transcribed DNA strand. The dramatic influence of the impaired (6-4) photoproduct repair in V-H1 on UV-induced mutability and the molecular nature of hprt mutations indicate that the (6-4) photoproduct is the main UV-induced mutagenic lesion.     

Expression of rat liver glutathione-S-transferase GSTA5 in cell lines provides increased resistance to alkylating agents and toxic aldehydes

The glutathione-S-transferases (GST) are a major contributor to the eukaryotic cell's defences against chemical and oxidative stress. However, the role of individual GST isoenzymes in conferring resistance to xenobiotics has not been fully determined. We have examined the effect of the rat GSTA5 isoenzyme in the detoxication of alkylating agents and aldehydes by constructing a cell line in which it is stably expressed. The hamster fibroblast cell line V79 was transfected with a construct expressing GSTA5 from the CMV promoter. A stable clone (V79-GSTA5) was isolated after selecting for the neomycin phosphotransferase gene present on the introduced DNA. The cell line showed significantly increased levels of resistance towards the alkylating agents chorambucil and melphalan. Levels of resistance were 4-6-fold greater in V79-GSTA5 cells than in control cells. Increased levels of resistance were also observed towards the lipid peroxidation product acrolein (IC(50)=80 microM compared with 17 microM in control cells). The V79-GSTA5 cells also showed a 4-fold increase in resistance to trans, trans muconaldehyde (IC(50)=4 micro compared with l microM for control cells). GSTA5 did not protect against 4-hydroxynonenal, but it did provide greater levels of protection to hydrogen peroxide, with an IC(50) of 380 microM in V79-GSTA5 compared with 180 microM in control cells. In contrast, V79-GSTA5 cells were more sensitive to methyl glyoxal, suggesting that a methyl glyoxal-glutathione conjugate is more toxic that the parental compound. These data contribute towards the evaluation of the role of GSTA5 in the detoxication of these compounds.     

Cis-platinum(II) diamine dichloride causes mutation, transformation, and sister-chromatid exchanges in cultured mammalian cells.

The anti-tumor agent cis-platinum(II) diamine dichloride caused dose-dependent toxicity in V79 Chinese hamster cells and in secondary Syrian hamster embryo cells. Chromosome aberrations were induced and positive dose--response relationships were observed for induction of sister-chromatid exchanges and 6-thioguanine-resistant mutations in V79 cells and morphologic transformation of secondary Syrian hamster embryo cells. The findings suggest that this chemical is a potential human carcinogen.     

Genotoxic effects of paracetamol in V79 Chinese hamster cells

Paracetamol was studied for possible genotoxic effects in V79 Chinese hamster cells. Paracetamol (0.5 mM for 30 min) reduced the rate of DNA synthesis in exponentially growing V79 cells to about 50% of control. A further decrease in the DNA synthesis was seen during the first 30 min after termination of paracetamol exposure. Paracetamol (3 and 10 mM for 2 h) caused a small increase in DNA single-strand breaks, as measured by the alkaline elution technique. After 16 h elution, the amount of DNA retained on the filters was 79 and 70% of controls in cells treated with 3 and 10 mM paracetamol respectively. No indication of DNA damage was seen in measuring the effect of paracetamol (0.25-10 mM for 2 h) on unscheduled DNA synthesis in growth-arrested cultures of V79 cells. At the highest concentrations (3 and 10 mM paracetamol), decreased unscheduled DNA synthesis was observed. Also UV-induced DNA-repair synthesis was inhibited by 3 and 10 mM paracetamol. DNA-repair synthesis was, however, inhibited at a much higher concentration than that inhibiting replicative DNA synthesis. The number of sister-chromatid exchanges (SCE) increased in a dose-dependent manner on 2 h exposure to paracetamol from 1 mM to 10 mM. At the highest dose tested (10 mM), the number of SCE increased to 3 times the control value. Co-culturing the V79 cells with freshly isolated mouse hepatocytes had no further effect on the paracetamol induced sister-chromatid exchanges. The present study indicates that paracetamol may cause DNA damage in V79 cells without any external metabolic activation system added.     

Induced resistance in cells exposed to repeated low doses of H2O2 involves enhanced activity of antioxidant enzymes

We have derived cells from the Chinese hamster V79 cell line by conditioning them with repeated low doses of hydrogen peroxide (H(2)O(2)). This mimics the physiological condition where cells are repeatedly exposed to low levels of oxidants. In an attempt to characterize such cells, we have exposed both conditioned cells (V79(C)) and the parental V79 cells (V79(P)) to different types of cytotoxic agents and compared their sensitivity to cell killing. The V79(C) cells were found to be stably resistant to killing by agents that produced toxicity through oxidative stress, e.g. H(2)O(2) and cisplatin. It was also found that the lipid peroxidation produced by these agents were considerably lower in the V79(C) cells. Thus, the difference in sensitivity could be due to lesser extent of damage to these cells. V79(C) cells had greater antioxidant defense through higher GSH content and greater activity of enzymes such as Cu-Zn superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), which provided protection from damage. Enzyme activities were also assayed at different times after treatment with various cytotoxic agents; there was a relatively large increase in SOD activity which perhaps plays a key role in determining the resistance of the V79(C) cells to killing.     

Monoenoic fatty acid requirement in V79 cells

When lipids were removed from the culture medium, growth of V79 cells ceased. Supplementation with cis-octadecenoic acids satisfied the requirement for lipids by V79 cells. After starvation of the exogenous lipids by the shift-down of the medium to lipid-free medium, the content of octadecenoic acid in phospholipids increased more slowly in V79 cells than in V79-R cells, which can grow in the lipid-starved medium. The incorporation of [14C]acetic acid into monoenoic fatty acids and phospholipid molecular species containing monoenoic fatty acids in V79 cells was lower than that in V79-R cells. The reduced formation of monoenoic fatty acids was shown to be due to deficiency in the stimulation of activity of stearoyl-CoA desaturase which is a key enzyme to convert saturated fatty acids to monoenoic fatty acids.     

Impact of 864 MHz or 935 MHz radiofrequency microwave radiation on the basic growth parameters of V79 cell line

The aim of this study was to evaluate and compare the influence of 864 MHz and 935 MHz radiofrequency/microwave (RF/MW) fields on the growth, colony-forming ability, and viability of V79 cells (continuous line). Cell samples with 1 x 10(4) V79 cells each, were exposed to continuous wave frequencies of 864 MHz and 935 MHz for 1, 2 and 3 hours. Exposed samples were matched with unexposed control samples. Specific absorption rate (SAR) was 0.08 W/kg for the 864 MHz or 0.12 W/kg for the 935 MHz field. Cell growth and viability were determined by counting cells every day for five days after exposure. Colony-forming ability was assessed by counting colonies seven days after exposure. The growth of the 864 MHz-irradiated cells was significant after two- and three-hour exposure 72 hours after irradiation (p < 0.05). The similar was observed 72 hours after exposure for cells exposed to 935 MHz microwaves for three hours (p <0.05). Colony-forming ability and cell viability in V79 cells exposed to 864 MHz or 935 MHz microwaves did not significantly differ from control cells. The two applied RF/MW fields showed similar effects on the growth, colony-forming ability and viability of V79 cells. Cell growth impact was time-dependent for both fields.     

The Use of Macroporous Microcarriers for the Large-Scale Growth of V79 Cells Genetically Designed to Express Single Human Cytochrome P450 Isoenzymes and for the Characterization of the Expressed Cytochrome P450

In this study, macroporous microcarriers were used for the large-scale growth of parental V79 cells and V79 cells genetically engineered to express a single human cytochrome P4501A1 isoenzyme (V79h1A1). Starting from 2 × 10⁵cells/ml, approximately 1 × 10⁷cells/ml could easily be harvested after 6 days in the case of the parental V79 cells, or after 11 days in the case of the V79h1A1 cells, resulting in a total of 3.6 × 10¹⁰cells. For the first time, the presence of cytochrome P450 (CYP) in the expressed V79 cells could be demonstrated by CO difference spectra with a Soret maximum around 450 nm. CYP levels in microsomes derived from the V79h1A1 cells of 14 pmol/mg protein were achieved. Importantly, no CYP was detected in microsomal fractions of the parental V79 cells. Cytochrome b5 levels could also be measured by difference spectrophotometry. No significant differences were found between cytochrome b5 levels in microsomes derived from the large-scale growth of V79h1A1 cells and parental V79 cells, i.e., 16.7 ± 7.9 vs 14.5 ± 7.6 pmol/mg protein. The presence of human cytochrome P4501A1 (CYPh1A1) in microsomal fractions derived from the large-scale growth of V79h1A1 cells was further substantiated by measuring 7-ethoxyresorufin-O-deethylase (EROD), 7-ethoxycoumarin-O-dealkylase (ECOD), and testosterone-6β-hydroxylation activities. EROD, ECOD, and testosterone-6β-hydroxylation activities of the V79h1A1 microsomes were 40 pmol resorufin/min/pmol CYPh1A1, 13 pmol hydroxy-coumarin/min/pmol CYPh1A1, and 0.16 pmol 6β-hydroxytestosterone/min/pmol CYPh1A1, respectively, indicating the presence of a highly active human CYP1A1 enzyme system. Further confirmation that the CYP protein was correctly expressed was obtained by Western blotting. In conclusion, the use of macroporous microcarriers is suitable for large-scale growth of V79 cells expressing human CYP isoenzymes. The present method may provide an easy and rather inexpensive tool in obtaining large quantities of microsomes containing human CYP isoenzymes, which are involved in the bioactivation and bioinactivation of xenobiotics. High yields of microsomes containing human CYP isoenzymes may substantially facilitate the production of sufficient quantities of human metabolites to allow isolation and identification in an early stage of development of pharmacologically interesting drugs.     

Radiosensitization of hypoxic cells by a nitrofuran; dose-modifying and shoulder effects.

The radiosensitizer nifurpipone dihydrochloride (5-nitro-2-furaldehyde N-methyl piperazino acetyl hydrazone dihydrochloride) sensitizes hypoxic V79 mammalian cells by at least two mechanisms. Sensitization is by a reduction of ? in addition to an increase in slope. Both these affects are absent under oxygenated conditions. When hypoxic V79 cells are irradiated in the presence of nifurpipone dihydrochloride combined with Ro-07-0582, sensitization greater than that due to air alone is observed; this effect is due to a reduction in ? and an increased slope. Again this effect is absent under oxygenated conditions. Rapid-mix studies using Serratia marcescens show that full senitization occurs with a pre-irradiation contact time of 4 msec; this contrasts with data for V79 cells where a pre-irradiation contact time of 40 msec is insufficient for any sensitization to occur. This sensitizer also exerts a differential toxic effect, being more toxic to hypoxic cells than to oxygenated ones. It is concluded from these results that nifurpipone dihydrochloride sensitizes by at least two mechanisms, one of which resembles that of the electron-affinic type.     

Interaction of leukotriene C4 and Chinese hamster lung fibroblasts (V79A03 cells). 1. Characterization of binding

A novel, specific, and potent biological action of leukotriene C4 (LTC4) was demonstrated in the Chinese hamster lung fibroblast cell line V79A03 (V79 cells), namely the confirment of protection against subsequent gamma-irradiation. Consequently, studies were conducted to determine whether LTC4-conferred radioprotection could be attributed to a receptor-mediated phenomenon. Specific binding sites for leukotriene C4 (LTC4) were identified and characterized using intact V79 cells incubated at 4 degrees C in the presence of serine-borate, during which time conversion of LTC4 to LTD4 or LTE4 was undetectable. Binding was maximal in a broad region between pH 6.2 and 8.8. Ca2+, Mg2+, and Na+ were not required for binding, and binding was not altered by GTP, ATP, or cAMP, by leukotrienes B4, D4, or E4, or by the leukotriene end point antagonists LY 171883, FPL 55712, or Revlon 5901-5. Scatchard analyses and kinetic experiments indicated the presence of high-affinity [Kd = 2.5 +/- 0.63 nM, approximately 9.9 x 10(5) sites/cell] and low-affinity [Kd = 350 +/- 211 nM, approximately 2.7 x 10(6) sites/cell] binding sites. The observed binding characteristics of LTC4 to V79 cells are consistent with a receptor-mediated phenomenon. In a companion communication which follows this report, we report the subcellular distribution of LTC4 binding to V79 cells and demonstrate that this binding is unlikely to be attributed principally to interaction with glutathione-S-transferase.     

Evidence for similarities between radiation damage expressed by beta-araA and damage involved in the interaction effect observed after exposure of V79 cells to mixed neutrons and gamma radiation

Plateau-phase V79 cells were exposed sequentially to fast neutrons and gamma rays. A dose-dependent reduction in the shoulder width of the gamma-ray survival curve was observed after preexposure of cells to neutrons. A similar effect was demonstrated on the neutron survival curve when cells were preirradiated with gamma rays. Treatment of cells with 150 microM beta-araA after either gamma or neutron irradiation reduced primarily the shoulder of the survival curve. When beta-araA was given to the cells after exposure to mixed radiation modalities, survival curves similar to those observed after exposure to a single radiation modality and treatment with beta-araA were obtained. The kinetics of loss of the interaction observed after exposure of cells to gamma rays following neutron irradiation was similar to the kinetics of loss of sensitivity to beta-araA (T1/2 = 1 h) measured by delaying drug administration after exposure to gamma rays. The results suggest that the PLD expressed by beta-araA is at least partly involved in the interactive effect observed after combined exposure of plateau-phase V79 cells to neutrons and gamma rays.