Interaction between radiation and drug damage in mammalian cells. V. DNA damage and repair induced in LZ cells by adriamycin and/or radiation

A multi-drug-resistant cell line selected in increasing concentrations of Adriamycin and designated LZ (J. A. Belli, Radiat. Res. 119, 88-100, 1989) is shown to exhibit a survival response characterized by radiation sensitivity and Adriamycin resistance. To determine if this response is due to alterations in either the initial levels of damage induced or the repair of DNA damage, LZ cells and the parental V79 cells were exposed to either radiation or Adriamycin and the damage and repair were measured with alkaline or nondenaturing filter elution. After exposure to radiation, induction and repair of both single-strand and double-strand breaks were equivalent. LZ cells exposed to 100 micrograms/ml Adriamycin for 1 h contained no measurable damage while the same treatment induced breaks and crosslinks in V79 cells. Pretreatment of LZ cells for 1 h with Adriamycin before irradiation did not alter either the initial levels of induced damage or the repair of strand breakage. These results suggest that (1) mechanisms other than differential induction and repair of strand breaks are responsible for the increased radiation sensitivity in LZ, and (2) the lack of Adriamycin-induced DNA damage in LZ is at least partially responsible for the increased cell survival after treatment.     

Quantitative morphological analysis of adriamycin-resistant human K562 leukemic cells

The morphological changes associated with Adriamycin resistance in a human leukemic cell line have been investigated by image analysis. An Adriamycin-resistant subline of the human erythroleukemic K562 cell line has been established. Three sets of cells have been analysed: sensitive cells, resistant cells cultured in the continuous presence of Adriamycin, and resistant cells cultured without the drug. Image analysis shows that Adriamycin-resistant K562 cells display significant morphological changes as compared with sensitive cells, at both the nuclear and cytoplasmic levels. These changes make it possible to separate sensitive and resistant cells automatically and with a classification accuracy of 76% and only four cytological parameters. Image analysis may therefore offer an interesting tool for studying drug resistance in leukemic cells, from both an experimental and a clinical point of view.     

Effect of liposomes on P-glycoprotein function in multidrug resistant cells.

Presentation of doxorubicin in liposomes has shown to enhance the sensitivity of multidrug resistant CH LZ cells to the drug (Thierry et al. Cancer Commun. 1:311-316, 1989). We confirmed that liposomally encapsulated doxorubicin may partially overcome multidrug resistance in the human ovarian carcinoma SKVLB cell line and that this effect is, at least in part, due to an increase of cellular drug accumulation. When used at high concentration, empty liposomes appear to be specifically cytotoxic in the MDR SKVLB and CH LZ cells. As observed with certain multidrug resistance modulators, empty liposomes inhibited the specific [3H]-vincristine binding to P-glycoprotein-enriched membranes isolated from CH LZ cells (60% at 0.2 mg lipid/ml). Our data suggest that liposomes may alter the P-glycoprotein function by direct interaction.     

Thioltransferase overexpression increases resistance of MCF-7 cells to adriamycin

Thioltransferase, a small redox protein with thiol-disulfide oxidoreductase and dehydroascorbate reductase activities, has been reported to be expressed at higher levels in Adriamycin-resistant MCF-7 human breast tumor cells (MCF-7 ADR(R)) when compared with Adriamycin sensitive MCF-7 WT (MCF-7 WT) cells. The present study examined the effects of stably transfecting MCF-7 WT cells with the cDNA for human thioltransferase and the effects of subsequent Adriamycin cytotoxicity in the MCF-7 WT transfected cells. All transfected cell lines overexpressing thioltransferase activity were more resistant to Adriamycin than untransfected MCF-7 WT cells, supporting the hypothesis that increases in thioltransferase expression are related to Adriamycin resistance. This resistance was independent of the ability of thioltransferase to catalyze reduction of dehydroascorbic acid to ascorbic acid, as the addition of an ascorbate generating derivative, L-ascorbic acid-2-phosphate, to the media did not additionally increase Adriamycin resistance.     

Arsenic trioxide inhibits the growth of Adriamycin resistant osteosarcoma cells through inducing apoptosis

Given that arsenic trioxide (As₂O₃) has been successfully used as a chemotherapeutic agent for refractory malignant tumors, this study is aimed at investigating the effect of As₂O₃ on human Adriamycin resistant osteosarcoma cell line Saos-2. The mechanism underlying multi drug resistance (MDR) in osteosarcoma cells and the anti-tumor effect of As₂O₃ on Adriamycin resistant osteosarcoma cells were analyzed. In our experiment, we first selected Adriamycin resistant osteosarcoma cell line by growing the classic osteosarcoma cell line Saos-2 in the medium with increasing drug concentrations. Then, we compared the IC50s of the osteosarcoma cells treated with different anticancer drugs by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Subsequently, we assessed the expression of classic MDR related molecules, Pgp, multidrug resistance-associated protein (MRP) and glutathione (GSH) activity in the wild type and Adriamycin resistant Saos-2 cells. Furthermore, the apoptosis was assessed by concerning DNA fragment and flow cytometry with Annexin-V staining. To elucidate the underlying mechanism of the apoptosis, related proteins Bcl-2, Bcl-xL, Bax, Bak, cleaved Caspase-3 and cleaved Caspase-9 were analyzed by western blotting. The data showed that the resistance to Adriamycin affected the sensitivity of osteosarcoma cell to other chemotherapeutic agents. The IC50s of Saos-2/ADM cells for methotrexate (1.74-fold), Cisplatin (1.43-fold) and As₂O₃ (1.21-fold) were increased compared with Saos-2 control cells. The expression of Pgp was upregulated comparing with the control cells. No significant difference was detected about the MRP and the glutathione-S-transferase activity and intracellular GSH concentration among different treated osteosarcoma cells. Apoptosis was observed and proved. The western blotting showed that the expression of Bcl-2 and Bcl-xL was downregulated. Meanwhile, the level of Bax, Bak, cleaved Caspase-3 and cleaved Caspase-9 was upregulated after treated with As₂O₃. The study suggests that Adriamycin resistant osteosarcoma cells have good response to As₂O₃-based chemotherapy in vitro, probably via the pathway of inducing apoptosis. And As₂O₃ might serve as an excellent alternative candidate for adjuvant chemotherapeutic agent on this incurable pediatric sarcoma.     

Increased epidermal growth factor receptor in an estrogen-responsive,adriamycin-resistant MCF-7 cell line

We examined the expression of the estrogen and epidermal growth factor (EGF) receptors in a drug-resistant subline of MCF-7 cells in order to study potential alterations in hormone dependence or in the growth factor pathway that could be related to the development of drug resistance in human breast cancer. The drug-resistant subline was derived from MCF-7 cells by selection with Adriamycin in the presence of the P-giycoprotein antagonist, verapamil, to prevent acquisition of the classical multidrug resistance phenotype. The Adriamycin-resistant cells retain estrogen-binding, estrogen-responsive monolayer growth, and estrogen-dependent tumorigenesis. Estrogen-binding studies demonstrate 1.4 × 10⁶ sites per cell with unaltered affinity when compared to parental MCF-7 cells, which have 2.7 × 10⁵ sites per cell. An increase in expression of EGF receptor, eight to 12-fold, occurred early in the selection for drug resistance, and appears to be unrelated to verapamil exposure, since cells maintained in Adriamycin without verapamil also have increased EGF receptor expression. Partially drug-sensitive revertants carried a verapamil, but out of Adriamycin, demonstrate a decline in EGF receptor expression. We postulate that activation of growth factor pathways in drug-resistant cells may enhance mechanisms of drug resistance, or provide mitogenic stimuli for cells to recover after damage by drug exposure. © 1993 Wiley-Liss, Inc.     

Increased radiosensitivity in cells of two human cell lines treated with bystander medium from irradiated repair-deficient cells

Radiation-induced bystander factors have been shown to be more toxic if they are from medium harvested from irradiated repair-deficient cells. The aim of this study was to test the hypothesis that the radiosensitivity of repair-proficient cells can be increased by exposing them to medium-borne factors harvested from sensitive cells and vice versa. Cells from a mismatch repair (MMR)-deficient cell line (Raji 10) with a sensitive response to radiation or the wild-type parent cell line were irradiated to 0.5 Gy gamma rays and then monitored for growth rate in their own medium or in the alternative conditioned medium. In other experiments, cells or conditioned medium were added to reporter cells (HPV-G, which are relatively sensitive keratinocytes, or highly radioresistant HT29 cells). The subsequent responses of the two cell lines to a 0.5-Gy dose of (60)Co gamma rays were measured. The results show that prior exposure of resistant cells to medium from irradiated sensitive cells reduced the clonogenic survival of the subsequently irradiated resistant cells. The reverse is also true. Measurement of the apoptosis index and BCL2 expression confirmed that the harvested medium was capable of modulating apoptosis after irradiation. This may have important applications in tumor therapy and also in the understanding of mechanisms involved in induction of adaptive responses.     

The clonogenic response of bovine aortic endothelial cells in culture to radiation

The effect of ionizing radiation on the survival of bovine aortic endothelial (BAE) cells was determined by the in vitro colony formation method. The BAE cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum, antibiotics, and growth factors obtained from the culture of mouse S-180 cells. The cultured BAE cells were positive to the staining of antibodies against human factor VIII and formed clones in plastic culture flasks with a plating efficiency of about 11%. The survival curve of the BAE cells following an exposure to a single dose of X rays was characterized by D0 = 101 rad, Dq = 65 rad, and an extrapolation number (n) of 1.9. These parameters were not modified by the absence of growth factors at the time of irradiation. The response of BAE cells to radiation was dose-rate dependent. The split-dose studies demonstrated that the BAE cells were able to repair sublethal radiation damage within 1 h after irradiation.     

Control of pigment production in mouse melanoma cells in vitro. Evocation and maintenance

A clonally derived amelanotic melanoma cell line repeatedly has been forced to produce pigment by the inhibitor of DNA synthesis, I-β-D-arabinofuranosylcytosine (ara-C) at sublethal levels. One ara-C-derived melanotic line has been cloned, and has continued to produce pigment for 2 years on normal medium. The inhibitor is most effective when administered to synchronized cells in four pulses on successive days at 1.8 x 10^-5 M during the S phase of the cell cycle. Colcemid at a sublethal concentration, and growth on medium solidified with agar also evoked pigment production in this line, but a large number of other inhibitors of biosynthetic processes did not, under the conditions tested. The melanotic lines are active producers of tyrosinase (DOPA oxidase), whereas the amelanotic line produces an inhibitor of tyrosinase activity. Both enzyme and inhibitor are labile at 4° C and -20° C, and decay of the inhibitor in homogenates of amelanotic cells reveals a low level of residual DOPA oxidase activity. The mean population doubling time of a cloned melanotic line is 23 hr, and that of a cloned amelanotic line 16.5 hr. A similar decrease in rate of growth is found in other melanotic lines and is believed to be a significant factor in maintaining this differentiated function. Rapid growth may be related to the production of an inhibitor by the amelanotic cells.     

Differential sensitivity of murine myeloid FDC-P1 cells and apoptosis resistant mutant(s) to anticancer drugs

There is growing evidence which suggests that dysregulation of apoptosis may lead to several disease states including cancer. To investigate the mechanism controlling the induction of cell death, apoptosis defective/resistant (Apt-) mutants were isolated and characterized in this study. FDC-P1, a mouse myeloid cell line that depends upon IL-3 for survival and growth but undergoes apoptosis when deprived of growth factor, was mutagenized by treatment with ethyl methane sulfonate. We selected cells that survived the growth factor deprivation but did not grow without the factor. Surviving cells were cloned by limiting dilution and four clones that showed the least morphological characteristics and biochemical changes of apoptosis were chosen. Unlike the parent FDC-P1, these mutants were cross resistant to apoptosis induced by a variety of antitumor drugs such as Adriamycin, Dexamethasone, VP-16, as well as reactive oxygen species (ROS) generated by xanthine/xanthine oxidase (X/XO). We used one of these Apt- mutant to test candidate death genes. Our findings suggest that the preferential increase in Bax/Bcl-2 ratio, p53, c-Myc, Caspase-3 and decrease in AP-1 on treatment with various anticancer drugs may contribute to the preferential apoptotic response in FDC-P1 cells but to varying degrees. Whereas, the higher constitutive level of antioxidant enzymes superoxide dismutase and catalase in the Apt- mutant may contribute at least in part to its resistance.     

Adriamycin tolerance in human mesothelioma lines and cell surface NADH oxidase

Adriamycin tolerant human mesothelioma cell lines derived from a single tumor prior to either chemotherapy or radiation therapy and a susceptible cell line were investigated. Not only was growth resistant to low doses of adriamycin but an unusual pattern of resistance was encountered in which cells seemed to better tolerate high adriamycin doses than intermediate doses. The differential growth susceptibility of the tolerant lines compared to A549 lung carcinoma and the bimodal dose response correlated with differences in the specific activity of a plasma membrane-associated NADH oxidase (NOX). Plasma membrane fractions of high purity were isolated by aqueous two-phase partition and assayed directly. The NADH oxidase activity of the plasma membranes for the susceptible cell line was maximally inhibited by 1 microM adriamycin whereas the NADH oxidase activity of the tolerant lines was less and was maximally inhibited by 0.1 microM adriamycin with 1 and 10 microM adriamycin being less inhibitory than 0.1 microM adriamycin. The findings suggest a relationship between the growth response to adriamycin of the adriamycin tolerant mesothelioma lines and the activity of the plasma membrane-associated NADH oxidase activity of the cell surface in these cell lines.     

Mutation to ouabain-resistance in Chinese hamster cells: induction by ethyl methanesulphonate and lack of induction by ionising radiation

The spontaneous frequency of mutants resistant to growth inhibition by ouabain (OUAR mutants) was found to be about 5:10(-5) per viable cell in uncloned cultures of Chinese hamster V79-4 cells. In freshly-isolated clones or cultures started from a few cells this frequency was initially reduced to about 1.10(-6) in 1 mM ouabain. No increase in the frequency of OUAR mutants was found in cultures treated with gamma-rays despite exploration of such variables as radiation dose, ouabain concentration, post-treatment interval before selection, cell density in selective medium, and clonal state of the cells at the time of adding ouabain (in situ vs. respreading method). A similar negative result was found for accelerated helium ions, for which the mutagenic effectiveness per unit dose has been shown to be about 10 times higher than gamma-rays for the induction of thioguanine-resistant mutants in these cells. Some evidence was found for an interaction between cellular radiation damage and ouabain-resistance, which may lead to a reduction in the survival of OUAR mutants in irradiated populations, but this damage seemed insufficient to account for inability to detect radiation-induced OUAR mutants. Reproducibly large increases in the frequency of OUAR mutants were found in cultures treated with various concentrations of ethyl methanesulphonate (EMS) by respreading cells in 1 mM ouabain for up to 8 days after EMS treatment. The concentration-OUAR mutant induction curve was approximately linear with low EMS concentrations. Recent evidence is reviewed in support of the suggestion, made in earlier studies, that ionising radiation is unable to induce OUAR mutants because of the severity of the genetic damage it causes.     

Distinguishable transformation-defective phenotypes among temperature-sensitive mutants of Rous sarcoma virus.

Eight transformation-defective, temperature-sensitive (ts) mutants of the Prague strain of Rous sarcoma virus, subgroup A, have been isolated after mutagenesis with 5-bromodeoxyuridine followed by selection on the basis of focus tests. Five of these mutants, ts GI201, GI202, GI203, GI204, and GI205, exhibit properties like most previously reported isolates in that they show a temperature-sensitive response to each of a variety of transformation-specific parameters tested. Interestingly, GI201, in addition to the temperature-sensitive defect, carries a lesion that was observed as a nonconditional loss of expression of plasminogen activator protease. Three mutants, ts GI251, GI252, and GI253 have been disignated partial transformation-defective (PTD) mutants since they behave as ts mutants according to some tests for transformation and as wild type according to others. These three mutants fail to form foci at the nonpermissive temperature (41 degrees C) and art nontumorigenic in 3-week-old chickens (body temperature, 42 degrees C). The agglutinability by concanavalin A of cells infected with these mutants shows a definite temperature sensitivity, as do the rate of 2-deoxyglucose uptake and the disappearance of the 250, 000-dalton normal cell glycoprotein (large, external, transformation sensitive [LETS]). Although the PTD mutant-infected cells, unlike cells infected with other transformation mutants, exhibit a cell-bound plasminogen activator protease at the nonpermissive temperature, this activator is not detectable as a free protease in the medium, as it is with wild-type, virus-infected cells. The PTD mutants behave like the wild-type parent in their ability to induce transformed growth properties in the infected cells, i.e., growth beyond normal cell saturation density with or without serum-supplemented medium and growth leading to colony formation in soft-agar- or methyl cellulose-containing suspension media.     

Interaction between radiation and drug damage in mammalian cells. VI. Radiation and doxorubicin age-response function of doxorubicin-sensitive and -resistant Chinese hamster cells.

A comparative study of the radiation and/or doxorubicin (DOX) survival response for synchronous populations of Chinese hamster V79 cells and two DOX-resistant variants (77A and LZ-8) was performed. The greatest cellular radiation sensitivity was observed in mitosis, while the greatest resistance was observed during late S phase for the three cell lines. The variation in radiation response throughout the cell cycle was expressed as a change in the width of the shoulder of the survival curves (Dq) with little change in D0. This suggests that each phase of the cell cycle has a different capacity for accumulation of radiation injury. The radiation age-response function for the three cell lines revealed that 77A and LZ-8 cells were more radiosensitive than V79 cells throughout the cell cycle. Exposure of synchronous populations to DOX (1.84 microM for V79, 9.21 microM for 77A, and 921 microM for LZ-8) for 1 h as a function of cell cycle phase revealed that V79, 77A, and LZ-8 cells exhibited the greatest sensitivity to DOX in mitosis and the most resistance to DOX during S phase, as indicated by the differences in the slope of the initial component of the survival curve. Levels of P-glyco-protein (P-gp) are probably not a factor contributing to DOX age-response function since P-gp levels remain constant throughout the cell cycle in all three cell lines. Synchronous populations of V79, 77A, and LZ-8 cells sequentially treated with DOX and radiation at various cell cycle phases were also analyzed. The results showed that the interaction between radiation and DOX damage resulted in a reduced cellular capacity for the accumulation of radiation damage throughout the cell cycle, as indicated by a decrease in the width of the shoulder of the survival curve. Overall, both DOX-sensitive V79 cells and DOX-resistant 77A and LZ-8 cells exhibited (1) a similar age-response function for radiation or DOX, and (2) no differences in the effects of DOX on radiation-induced damage throughout the cell cycle. These results indicate that acquired resistance to DOX associated with increased levels of P-gp in the cell membrane did not appear to affect the age-response function for radiation or DOX, and the nature of the interaction between damage caused by radiation and DOX was also not affected.     

Amphibian cells in culture. II. Isolation of drug-resistant variants and an asparagine-independent variant.

With L-15 as the base medium, drug-resistant variants were isolated from two amphibian tissue culture strains: the Xenopus laevis A8 diploid cell line and the ICR 2A cell line of Rana pipiens. Four different classes of variants were obtained: (1) A8 cells resistant to chloramphenicol, an inhibitor of mitochondrial protein synthesis; (2) A8 cells resistant to ouabain, an inhibitor of the Na+/K+-activated ATPase of the plasma membrane;(3) ICR 2A cells resistant to low (20 microgram/ml) and high (300 microgram/ml) levels of bromodeoxyuridine (BUdR), a thymidine analog which interferes with the pyrimidine salvage pathway; and (4) ICR 2A cells resistant to 2,6-diaminopurine (DAP), an adenine analog which interferes with the purine salvage pathway. Unlike the other variants, isolation of BUdR resistant cells is a 2-step process. Resistance to low levels of BUdR is phenotypically expressed by a reduction in thymidine transport activities while resistance to high levels of this compound is evidenced by greatly reduced levels of thymidine kinase activity. DAP-resistant cells, which are characterized by reduced levels of adenine phosphoribosyl transferase (APRT) activity, do not die in AAT (adenine, aminopterin, thymidine) selection medium. This suggests that these cells utilize adenine efficiently. With MEM as the base medium, an asparagine independent clone was isolated from the ICR 2A cell line. When compared with the wild type, this variant exhibited a slightly reduced growth rate in the presence or absence of asparagine.     

Rescue of marker phenotypes mediated by somatic cell hybridization.

The effect of irradiation prior to virus-induced cell fusion on the frequency of hybrid production has been measured as a function of radiation dose. The Chinese hamster line wg3h (HGPRT-) was crossed with the TK- mutants; Chinese hamster A23 or mouse 3T34E, and hybrids were selected in HAT medium. Irradiation of one (marker rescue) or both (mutual rescue) partners before fusion yielded qualitatively different results. After X-irradiation marker rescue curves were of single-hit type, with D0 values about five-fold greater than the irradiated parent cell. Mutual rescue curves were of the multi-hit type, with zero-dose extrapolation value (n) greater than that of the more resistant partner, but no significant alteration in D0. Qualitatively similar results were obtained after U.V.-irradiation, but the probability of rescue per surviving parent cell was higher after U.V. than after X-rays. With both forms of radiation, reciprocal marker rescue curves were not significantly different. Control experiments showed that mutual rescue was not an artefact either of sensitization of parent cells due to TK- or HGPRT- mutations, or of the enhancement of recovery by feeder layers resulting from high-density mutant populations killed with graded radiation doses and HAT selection. Analysis of heterokaryon frequencies within 18 hours of fusion demonstrated that radiation doses up to four lethal hits, given to one or both parents of the cross wg3h x A23, did not increase heterokaryon formation.     

Structural differences between sensitive and resistant L1210 cells

The main structural differences between sensitive L1210 mouse leukaemic cells and their multidrug resistant counterpart, obtained by adaptation of the parental cell line to vincristine (VCR), concern the size and shape of the cells, their surface properties and changes in organelles involved in proteosynthesis and transport of substances. The resistant cells are larger with higher density of microvilli. In light and electron micrographs containing a group of cells, cells were found to be closer to each other in L1210/VCR cells than in L1210 cells. This difference in cell aggregation suggests different surface properties which could be visualised by decreased staining of L1210/VCR cell surface coat (glycocalyx) with a polycationic dye ruthenium red. A decrease in surface to volume ratio as a consequence of increased cell size in resistant cells is compensated by proliferation of villi and cytoplasmic protrusions of the cell surface. L1210/VCR cells were further distinguished by higher amount of euchromatin, increase in density of rough endoplasmic reticulum, more developed Golgi apparatus and aggregation of free ribosomes into tetrameric and pentameric polyribosomes. These structural changes may be interpreted as a sign of increase in proteosynthesis and transport of substances.     

Effect of low dose rate radiation on cell growth kinetics.

Experimental determinations were made of cell number as a function of time for two strains of L5178Y mammalian cells maintained continuously in various environments of radiation. One strain possessed a shoulder in its dose response curve whereas the other did not. Neither strain showed any significant difference in growth rate for interdivision doses on the order of the median lethal dose or less delivered continuously at a low dose rate or pulsed every 4 h at a high instantaneous dose rate. It was also shown that large numbers of dead cells have little effect on growth rate and that these dead cells last as discrete entities for many days. A simple theory of growth rate in the presence of radiation is presented, and the agreement with the observations implies that there is no effect of any sublethal low dose rate radiation received in one generation on the growth rate or radiation sensitivity of the succeeding generation. Further analysis of the data also showed that for the no-shoulder cells at 37 degrees C, tritiated water had a relative biological effect close to unity for cell sterilization.     

Effects of v-src oncogene activation on radiation sensitivity in drug-sensitive and in multidrug-resistant rat fibroblasts.

Recent work has implicated the activated ras oncogene, whose gene product is a G-protein located in the plasma membrane, as well as the activated raf oncogene, whose gene product is a membrane-associated protein kinase, in contributing to radioresistance. Another transforming oncogene whose gene product is localized to the plasma membrane is v-src. We have examined a rat fibroblast line (RAT-1) infected with an avian sarcoma virus carrying a temperature-sensitive mutation in the v-src tyrosine kinase domain (LA-24). At 40 degrees C, LA-24 cells have a flat morphology and grow as a contact-inhibited monolayer, while at 35 degrees C, LA-24 cells have a transformed morphology, lose contact inhibition, grow in soft agar, and exhibit 3.5-fold higher tyrosine kinase activity. The parental RAT-1 line, not infected by the virus, grows at both temperatures as a contact-inhibited monolayer. This well-characterized system represents a good model for examining the effect of v-src transformation on radiosensitivity. RAT-1 and LA-24 cells grown at 35 and 40 degrees C were irradiated with graded doses of radiation, and clonogenic survival was assayed. For LA-24 cells grown at 35 and 40 degrees C, and for RAT-1 cells grown at 35 and 40 degrees C, calculated D0, n, alpha, and beta values did not differ significantly. To determine whether there might be differences in radiation damage repair capacity too subtle to detect by comparing radiation survival curves, sublethal damage repair capacity was assessed. There was no difference in sublethal damage repair capacity for LA-24 cells grown at 35 or 40 degrees C. Other studies have associated multidrug resistance with radioresistance. We have examined the radiation sensitivity of two colchicine-resistant LA-24 clones with four- to fivefold amplification of the P-glycoprotein gene, which are four-to fivefold more resistant to colchicine than the parental LA-24 line. In these multidrug-resistant clones, v-src activation does appear to increase radiation resistance. This did not appear to be due to alteration in cell cycle kinetics. We conclude that oncogene activation, or even protein kinase activity per se, does not necessarily lead to radiation resistance. Rather, radiation resistance following oncogene activation depends upon the oncogene and cell line studied, and perhaps upon specific protein phosphorylation.     

Dominance of a repair function for X-ray-, U.V.- and alkylating agent-induced damage in hybrids between mouse lymphoma cells of different sensitivity.

Cell lines resistant to IUdR and 6-thioguanine were isolated from a radiosensitive mouse lymphoma line LS and its radioresistant derivative AII respectively. Their biological and biochemical characteristics, as measured by plating in HAT medium IUdR and TG and by (14C) hypoxanthine and (3H) thymidine uptake were measured and found to be consistent with those expected for drug resistant cell lines. Two hybrid cell lines were isolated from crosses between the LS IUdRr line and AII TGr line and their radiosensitivity measured relative to that of the parental lines. Radioresistance was found to be dominant in both the hybrid lines as was resistance to EMS and U.V. to which the parental lines also show differential sensitivity. The ability to recover from sublethal damage after X-irradiation was found to be temperature sensitive in radiosensitive cells, an inverse effect being seen when LS cells were irradiated at 20 degrees C and held at 20 degrees C between dose fractions. Hybrid cells showed a normal amount of recovery from sub-lethal damage when compared with that in AII cells but this occurred at t slightly slower rate at 20 degrees "c than at 37 degrees C. Recovery from sub-lethal damage in AII cells was unaffected by lowering the temperature. These findings suggest that the radiosensitive cells may be temperature sensitive, in some step or steps in the ligation process.