Synergistic effect of inhibitors of topoisomerase I and II on chromosome damage and cell killing in cultured Chinese hamster ovary cells

Simultaneous treatment of cultured Chinese hamster ovary cells with the topoisomerase I inhibitor camptothecin and the topoisomerase II inhibitor 4-(9-acridinylamino)-methanesulfon-m-anisidide results in a clear synergistic effect on both chromosome damage detected at metaphase and loss of colony-forming ability. In contrast, the effect of combined treatment with these topoisomerase inhibitors on sister chromatid exchanges was not significantly different from that expected if the effects were additive. Taken as a whole, these results seem to support the hypothesis that topoisomerase inhibitors can lead to cell death, presumably when DNA replication forks collide with drug-stabilized cleavable complexes. Nevertheless, no evidence of apoptosis was obtained from DNA fragmentation analysis. The possible clinical implications of our findings are discussed.     

The effect of hyperthermia on the cytotoxicity of melphalan in Chinese hamster ovary cells

The heat stability of melphalan during incubation at temperatures from 37 degrees C to 45 degrees C was determined by spectrophotometric and HPLC analyses and by direct measurement of melphalan cytotoxicity using a colonogenic assay Although OD 250 changed little during exposure to temperatures up to 45 degrees C for periods of up to 1 h, the melphalan HPLC peak decreased as function of incubation time and temperature. Loss of cytotoxicity following heating paralleled the decay of the melphalan HPLC peak. Despite the inactivation of melphalan by heat, the cytotoxic effects of melphalan were enhanced at elevated temperatures from 38 degrees C to 42 degrees C and synergism was observed at lethal temperatures above 42 degrees C.     

Effect of hyperthermia on activity of three glycosyltransferases in Chinese hamster ovary cells

We measured activities of three glycosyltransferases at various times during heat-induced thermotolerance development. Glycosyltransferases are normally located in the Golgi apparatus and catalyze cellular glycosylation reactions. UDP-Gal:N-acetylglucosamine beta 1,4-galactosyltransferase (beta 1,4-GalT) is known to participate in the formation of N-linked glycoproteins; when compared to cell survival, beta 1,4-GalT activity was significantly more heat resistant (50% loss of activity: 80 min, 45 degrees C) and showed little elevation at a time when thermotolerance was fully expressed. However, beta 1,4-GalT activity increased twofold by 24-h postheating when thermotolerance had begun to decay. Activity of beta 1,4-GalT was compared with glycosyltransferase activities that are considered to be specific for O-linked glycoproteins: UDP-Gal:N-acetylgalactosamine-beta 1,3-galactosyltransferase (beta 1,3-GalT), and UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (Gal-NAcT). Heat-inactivation experiments with heating times up to 60 min at 45 degrees C failed to reduce either activity below that of unheated control cells. Instead both beta 1,3-GalT and GalNAcT activity increased approximately twofold immediately after 10 min at 45 degrees C. Activity of beta 1,3-GalT rapidly decreased with time after heating and returned to control levels by 6-h postheating. In contrast, GalNAcT activity continued to increase with time after 10 min at 45 degrees C, and was 4.5-fold above unheated controls by 6-h postheating. GalNAcT activity returned to control levels 24- to 48-h postheating. A comparison with the cellular survival response showed that GalNAcT activity preceded thermotolerance expression by 2-4 h and also decayed more rapidly than heat resistance in thermotolerant cells. These data, together with other published results, suggest that expression of thermotolerance may be associated with enhanced glycosylation of intracellular proteins.     

The effect of hyperthermia on intracellular K+ in Chinese hamster ovary cells

Dietary retinyl palmitate was administered for 22-30 weeks in CD-1 mice which had been initiated with 0.15 mumol of 7,12-dimethylbenz[a]anthracene (DMBA) and promoted with 8 nmol of 12-O-tetradecanoylphorbol-13-acetate (TPA) twice weekly thereafter. This treatment resulted in a dose response in both the tumoricidal capacity of a selected number of isolated peritoneal macrophages (PM) and in skin tumor prevention. At 350 I.U./g of diet, retinyl palmitate (RP) also resulted in a 3-fold increase in the number of DM. RP significantly increased the total capacity of macrophage host defenses by increasing the number and individual capacity for cytotoxicity. Selenium (Se), at 2 parts/million in the drinking water, did not enhance PM tumoricidal capacity, although it did result in 60% reduction of mouse tumor burden.     

Rapid loss of stress fibers in Chinese hamster ovary cells after hyperthermia

This study was initiated to characterize the effect of hyperthermia (45 degrees) on the distribution of actin stress fibers in Chinese hamster ovary cells using rhodamine-conjugated phalloidin, a probe specific for F-actin. Fluorescent microscopy revealed a rapid loss of stress fibers after immersion in a 45 degrees water bath. After 5-min immersion at 45 degrees, approximately 90% of the cells analyzed did not contain observable stress fibers. Stress fibers were visible after incubation of cells at 37 degrees after heating. The recovery of the appearance of the stress fibers occurred as protein synthesis resumed, and addition of protein synthesis inhibitors following heat treatment blocked the reappearance of these structures. These results support the hypothesis that cytoskeletal components may be a target of hyperthermia, explaining the pleotropic biological effects of heat and, in particular, heat radiosensitization.     

Effects of hyperthermia on the intracellular pH and membrane potential of Chinese hamster ovary cells

The effects of hyperthermia (exposure to 41-47 degrees C) on the intracellular pH and membrane potential have been studied using Chinese hamster ovary HA-1 cells. Our goal was to determine whether intracellular pH changes or changes in membrane potential correlated with cell killing. The intracellular pH (pHi) was measured using the DMO partitioning technique. A rapid acidification of the intracellular environment was observed at all the elevated temperatures studied. The pHi reached a plateau value of approximately 6.9, and started reversing towards normal values upon prolonged exposure to heat. Similar patterns were seen for delta pH (pHi-pHo). The membrane potential difference (delta psi) was measured using the fluorescence quenching of 3,3-dipropylthio-carbocyanine, and calibrated using a 86Rb+ diffusion potential. We found that delta psi falls to zero only upon prolonged exposure to temperatures above 43 degrees C. When the external pH was changed from normal values the drop in delta psi occurred more readily. Development of thermotolerance resulted in an increase in the time required to make delta psi change by half. The changes in delta psi were shown to be irreversible. When the proton electrochemical gradient (delta mu H+) was calculated using the measured values of delta psi and delta pH, the trends observed were the same as those seen for delta psi. The changes observed for pHi can be accounted for by the changes in the pK values of the components involved in the intracellular buffering. The changes in delta psi and delta mu H+ may reflect the physical breakdown of the transmembrane H+ gradients, which may be the actual mechanical process of cell death. No correlation of cell survival with the measured parameters was observed.     

The effect of hyperthermia on the uptake and cytotoxicity of melphalan in Chinese hamster ovary cells

The effect of temperature on the cytotoxicity of melphalan in CHO cells was studied in an in vitro clonogenic assay. The cytotoxicity of melphalan was significantly increased at elevated temperatures with a 4 fold increase in cytotoxicity at 42 degrees C compared to 37 degrees C. The effect of temperature on membrane permeability to melphalan was studied to determine whether the increase in cytotoxicity was due to increased intracellular drug levels. Melphalan influx and efflux rates both increase with increasing temperature. There is, however, a small net increase in steady state intracellular drug levels with increasing temperature with a 20% increase in intracellular drug levels at 42 degrees C compared to 37 degrees C. The increase in drug uptake observed in insufficient to explain the much greater increase in cytotoxicity with increasing temperature.     

The effect of hyperthermia on glucose transport in normal and thermal-tolerant Chinese hamster ovary cells

The effect of hyperthermia on glucose transport was studied in CHO cells to test the hypothesis that interference with membrane transport might be related to cell death at elevated temperatures. It was shown that passive diffusion of 2-deoxyglucose increases steadily over the temperature range 4-50 degrees C. Facilitated diffusion increases from 4 degrees C to 35 degrees C then exhibits a broad optimum before decreasing rapidly above 45 degrees C. The temperature dependence of glucose transport in thermally resistant cells was not however different from that of normal cells suggesting that this membrane transport process is not a critical target in cell killing by heat.     

Effect of the metal chelating agent o-phenanthroline on the DNA and chromosome damage induced by bleomycin in Chinese hamster ovary cells

A 30-min pulse treatment with bleomycin to Chinese hamster ovary cells in culture produces DNA degradation and chromosomal aberrations in a dose-dependent manner. Bleomycin also induces a long-lasting effect on the cell cycle producing a lengthening of two or more cycles after the treatment. The presence of o-phenanthroline, which chelates metal ions, totally inhibits DNA cleavage and the appearance of chromosome aberrations while partially correcting the lengthening of the cell cycle. These findings suggest that an important cellular target for bleomycin is the DNA. Chromosomal aberrations are a secondary effect resulting from DNA cleavage. On the other hand, the increase in the duration of the cell cycle is probably induced by DNA degradation and, perhaps, by damage to other cellular structures.     

Interaction of multidrug-resistant Chinese hamster ovary cells with amphiphiles.

The interaction of membrane-active amphiphiles with a series of MDR Chinese hamster ovary (CHO) cell lines was investigated. Cross-resistance to cationic amphiphiles was observed, which was effectively sensitised by verapamil. MDR cells showed collateral sensitivity to polyoxyethylene amphiphiles (Triton X-100/Nonidet P-40), which reached a maximum at 9-10 ethylene oxide units. Resistant lines were also highly collaterally sensitive (17-fold) to dibutylphthalate. mdrl transfectants showed cross-resistance to cationic amphiphiles, but no collateral sensitivity to nonionic species. Triton X-100/Nonidet P-40 inhibited 3H-azidopine photoaffinity labelling at low concentrations, perhaps reflecting a specific interaction with P-glycoprotein. Further investigation of the molecular basis of collateral sensitivity revealed that association of 3H-Triton X-100 with MDR cells reached steady state levels rapidly, and occurred by a non-mediated mechanism. The equilibrium level of X-100 uptake was inversely related to drug resistance. Collateral sensitivity is thus not a result of decreased Triton X-100 association with the cell. The fluorescent probe merocyanine 540 was used to examine the MDR plasma membrane microenvironment for physicochemical changes. Increasing levels of drug resistance correlated with a progressive shift in the mean cell fluorescence to lower levels, which suggests that the packing density in the outer leaflet of MDR cells is increased relative to that of the drug-sensitive parent.     

Induction of major chromosome aberrations in Chinese hamster ovary cells by alpha-difluoromethylornithine

DL-alpha-Difluoromethylornithine (DFMO) is a specific irreversible inhibitor of ornithine decarboxylase (EC 4.1.1.17) and has antitumor effects. In this paper, we show that DFMO inhibits the growth of and causes severe chromosomal damage in Chinese hamster ovary cell strain A7 which grows without serum but has deficient arginase activity and therefore requires ornithine or polyamines for continuous replication. In ornithine-containing medium, the A7 cells had very few chromosome aberrations, but incubation of these cells with 0.5 mM DFMO for 7 days induced chromosome aberrations in 12 to 46% of the mitoses. Depletion of polyamines by omitting ornithine from the medium also caused chromosome aberrations. The chromosomal damage found after DFMO treatment alone and ornithine deprivation alone were of similar nature. In addition to chromosome breaks, there were chromosome fragmentation and structurally changed chromosomes including rings, chromatid exchange configurations, and chromosome elongations. A phenomenon resembling premature chromosome condensation was also seen. Double-minute chromosomes were visible in some mitoses, and the chromosome elongations sometimes gave an impression of homogeneously staining regions.     

Neocarzinostatin-mediated DNA damage and repair in wild-type and repair-deficient Chinese hamster ovary cells

The formation and repair of neocarzinostatin (NCS)-mediated DNA damage were examined in two strains of Chinese hamster ovary cells. The response in strain EM9, a mutant line selected for its sensitivity to ethyl methanesulfonate and shown to have a defect in the repair of X-ray-induced DNA breaks, was compared with that observed in the parental strain (AA8). The DNA strand breaks and their subsequent rejoining were measured using the method of elution of DNA from filters under either alkaline (for single-strand breaks), or nondenaturing conditions (for double-strand breaks). Colony survival assays showed that the mutant was more sensitive to the action of NCS than was the parental strain by a factor of approximately 1.5. Elution analyses showed that the DNA from both strains was damaged by NCS; the mutant displayed more damage than the parent under the same treatment conditions. Single-strand breaks were produced with a frequency of about 10 to 15 times the frequency of double-strand breaks. Both strains were able to rejoin both single-strand breaks and double-strand breaks induced by NCS treatment. The strand break data suggest that the difference in NCS-mediated cytotoxicity between EM9 and AA8 cells may be directly related to the enhanced production of DNA strand breaks in EM9. However, the fact that much higher doses of NCS were required in the DNA studies compared to the colony survival assays implies that either a small number of DNA breaks occur in a critical region of the genome, or that lesions other than DNA strand breaks are partly responsible for the observed cytotoxicity.     

Effects of hyperthermia on the cytoskeleton and cell survival in G1 and S phase Chinese hamster ovary cells

The effects of acute hyperthermia on three cytoskeletal systems (microtubules (MT), microfilaments (MF), and vimentin intermediate filaments (VIMF] were observed in G1 and S phase Chinese hamster ovary (CHO) 10B cells using immunofluorescence microscopy and compared to cell survival. A scoring system was devised to express the degree of cytoskeletal collapse induced by heat and the degree of recovery 20 h following heat treatments. A positive correlation was found between recovery from heat-induced cytoskeletal disruption and surviving fractions (SF) of cells heated in G1 but not with SF of cells heated in S phase. Recovery of MT arrays, for example, averaged 96.5%, 71.6% and 20.3% for heat doses of 5 min, 15 min and 25 min, 45 degrees C, respectively. The corresponding SF (means) were 0.92, 0.68 and 0.23, respectively. However, in S phase cells, where restoration of MT and VIMF patterns averaged 94.2%, 83.8% and 33.0% for heat doses of 5 min, 15 min and 25 min, 45 degrees C respectively, SF were 0.70, 0.09 and 0.02. These results suggest that heat-induced cytoskeletal alterations may play a role in the death of cells heated in G1, and that these alterations do not significantly influence death of cells heated in S phase. This work is in agreement with previous studies showing that cells heated in G1 or S phase appear to die by different mechanisms, and further emphasizes the need to use synchronous populations of cells in order to understand the mechanisms whereby cells die following hyperthermia.     

Urine of tobaccolareca nut chewers causes genomic damage in Chinese hamster ovary cells

The chromosome-damaging effects of urine concentrates (UCs)from tobacco plus areca nut (T/AN) chewers (a highly popularhabit and a major risk factor for oral cancer in India) wereevaluated on Chinese hamster ovary (CHO) cells employing twocytogenetic end-points, namely chromosome aberration (CA) andsister chromatid exchange (SCE) frequencies. Urine creatininelevels were comparable between controls and T/AN chewers. CAand SCE frequencies in CHO cells were found to be elevated significantly(P < 0.001) following treatment with UCs prepared from T/ANchewers (UC-T/AN chewers) as well as with UCs of non-chewercontrols (UC-control subjects). Moreover, elevation of thesetwo parameters by UC-T/AN chewers was significantly higher incomparison to that of UC-controls. The results of the presentstudy indicated that besides the oral cavity, which is a targetorgan for T/AN chewers, mutagen/scarcinogens in tobacco andareca nut might be playing a causative role in cancer of theurinary bladder as well.     

Superoxide dismutase levels in Chinese hamster ovary cells and ovarian carcinoma cells after hyperthermia or exposure to cycloheximide

Superoxide dismutase (SOD) activity in Chinese hamster ovary (CHO) and ovarian carcinoma (OvCa) cells was measured after exposure to hyperthermia and correlated with the development of thermotolerance. The SOD activity of each cell type was largely copper- and zinc-containing SOD activity. Both cell types had similar but low levels of SOD activity when the cells were grown at 37 degrees C. After exposure for 2 h at 41.5 degrees C, SOD activity of OvCa cells, but not of CHO cells, was increased. After exposure to 45 degrees C for 15 min, SOD activity was also increased in the OvCa cells, but not in CHO cells. After 15 min at 45 degrees C followed by 1 h incubation at 37 degrees C, SOD activity was increased in OvCa and CHO cells; after an 8-h incubation at 37 degrees C, SOD activity doubled in each cell type. Thermotolerance is maximal after 2 to 3 h of exposure at 41.5 degrees C and after 8 to 10 h incubation at 37 degrees C following exposure to 15 min at 45 degrees C. The turnover of SOD activity in OvCa cells was estimated by the rate at which activity was lost following addition of cycloheximide (10 micrograms/ml). Twenty-four % of the activity was lost with a half-life of 10 min, and 76% was lost with a half-life of 4.5 h. Despite restriction of general protein synthesis 3 to 4 h after 45 degrees C hyperthermia, SOD activity was increased at 1 and 8 h after exposure, presumably coincidental with heat shock protein synthesis and development of thermotolerance. These data suggest that SOD activity may be important in protecting cells exposed to heat and that it may play a role in the development of thermotolerance.     

Phagocyte-induced mutation in Chinese hamster ovary cells

Methapyrilene, tested at both non-hepatocytotoxic and hepatocytotoxic concentrations, failed to induce somatic mutations in the hepatocyte-mediated Chinese hamster ovary/hypoxanthine-guanine phosphoribosyl transferase (HGPRT) mutational assay. These data support the conclusion that methapyrilene induces the carcinogenesis process through a non-DNA damaging mechanism, suggested when this antihistamine previously failed to induce a genotoxic response in several in vitro test systems developed to detect carcinogens and mutagens.     

Effects of hyperthermia and membrane-active compounds or low pH on the membrane fluidity of Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells heated in the presence of the membrane-active agents procaine, ethanol, butylated hydroxytoluene (BHT) and glycerol were analysed for changes in fluorescence polarization of the lipid probe (1-[4(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene, an indicator of plasma membrane fluidity). Cells were heated in normal and acid (pH 6.6) medium. Procaine, ethanol, BHT and low pH sensitized cells to heat-killing. Procaine, ethanol and BHT decreased fluorescence polarization (increased plasma membrane fluidity) significantly. Polarization distributions for cells heated with these sensitizers were broadened substantially and were skewed toward lower polarization values. Glycerol, a heat-protector, inhibited changes in fluorescence polarization due to heating at temperatures up to 45.0 degrees C. Heating cells at either 42 or 45 degrees C in pH 6.6 medium had no significant effect on the fluorescence polarization compared with controls, while survival was reduced substantially. Thus, heat-sensitization of low pH cannot be ascribed to changes in membrane fluidity. The changes in membrane fluidity caused by other sensitizers and protector indicate that membrane fluidity changes may be a contributing cause of cell killing by hyperthermia.     

Expression of human chromosome 2 ornithine decarboxylase gene in ornithine decarboxylase-deficient Chinese hamster ovary cells

Ornithine decarboxylase (ODC) belongs to a multigene family and some of these may very well be nonfunctional (pseudogenes). We isolated an ODC gene from a human chromosome 2-specific library and transfected the gene into ODC-deficient Chinese hamster ovary cells to directly demonstrate that this ODC gene is functional and ODC is essential for cell proliferation. After screening 2.5 X 10(5) plaques using a human ODC complementary DNA probe, a typical clone with a 5.4-kilobase insert was isolated and then cloned into the HindIII site of the pGem-1 vector. One (phODC 2B1) of these clones containing a 5.4-kilobase ODC gene insert was identified. Restriction enzyme analysis and partial sequencing data revealed that phODC 2B1 contained the full length protein-coding sequences but lacked first exon and 3'-polyadenylation sequences. Primer extension analysis indicated that human ODC mRNA has homologous sequences with the ODC gene from human chromosome 2. To determine that the chromosome 2 ODC gene is functional, ODC-deficient Chinese hamster ovary cells were transfected with the ODC expression vector (phSV2B1-neo) and several G418-resistant transfectants were isolated which expressed 70- to 400-fold more ODC activity than parental or wild-type Chinese hamster ovary cells. Furthermore, these stable transfectants exhibited a higher growth rate than wild-type cells. These results indicate that the ODC gene from human chromosome 2 encodes functional ODC protein, and ODC (and its product putrescine) is required for cell growth.