Plateau-phase Chinese Hamster Ovary Cells Research Articles

Base substitutions, targeted single-base deletions, and chromosomal translocations induced by bleomycin in plateau-phase mammary epithelial cells.

Previous work showed that treatment of plateau-phase Chinese hamster ovary cells with the radiomimetic double-strand cleaving agent bleomycin induced very small deletions as well as interchromosomal reciprocal translocations, both of which could be ascribed to errors in end joining of DNA double-strand breaks. In an attempt to assess the possible role of TP53 in suppressing such repair errors, bleomycin-induced mutagenesis at the HPRT locus was examined in immortalized 184B5 human mammary epithelial cells (TP53(+)), and in a TP53-defective derivative, 184B5-E6tfxc6. For both cell lines, the most frequent bleomycin-induced mutations were base substitutions, with no apparent targeting to major bleomycin lesions. However, both lines also sustained single-base deletions that were targeted to expected sites of double-strand breaks, suggesting that they arose by end-joining repair of the breaks. Surprisingly, only a few large deletions or rearrangements, and no interchromosomal events involving the HPRT locus were detected among the mutants. The results suggest that in both cell lines, errors in double-strand break repair resulting in heritable large deletions and rearrangements are rare. Spectral karyotyping of bleomycin-treated 184B5 cells showed that a significant number of translocations were present shortly after bleomycin exposure, but their frequency decreased upon continued culture of the cells. Thus, for these cells, the lack of induced interchromosomal rearrangements can be explained in part by selection against such events as the cells proliferate.

Mechanism of radiosensitization by halogenated pyrimidines: the contribution of excess DNA and chromosome damage in BrdU radiosensitization may be minimal in plateau-phase cells.

We measured the contribution of increased DNA double-strand break (dsb) and interphase chromosome break induction in BrdU-mediated radiosensitization in exponentially growing (DNA dsb measurements only) and plateau-phase Chinese hamster ovary cells using an approach developed by Webb et al. (1993). The approach is based on the scavenging capacity of acetone for hydrated electrons, which are thought to react with bromine and form excess DNA and chromosome damage in BrdU-containing cells. In irradiated exponentially growing cells, acetone (1 M) removes the majority of excess DNA dsb induced in the presence of 4 microM BrdU (approximately 40% replacement of thymidine by BrdU), but does not restore cell radiosensitivity to the levels observed in BrdU-free cells. Although BrdU radiosensitizes cells by decreasing both D0 and Dq of the survival curve, acetone only restores D0 to levels measured in BrdU-free cells, but leaves Dq at levels measured in BrdU-containing cells. In plateau-phase cells, acetone removes the majority of excess DNA dsb and interphase chromosome breaks induced in the presence of 4 microM BrdU (approximately 50% replacement of thymidine by BrdU) but has only a small effect on BrdU-mediated radiosensitization to killing. These observations suggest that increased DNA damage production has a variable contribution in BrdU radiosensitization: it constitutes a major, albeit not the sole, component in the radiosensitization of exponentially growing cells, but only a minor component in the radiosensitization of plateau-phase cells. The results suggest that BrdU radiosensitization does not derive exclusively from increased DNA damage induction and support our previous hypothesis invoking repair inhibition/damage fixation as a component in the mechanism of radiosensitization. The results further suggest that repair inhibition is a major component in BrdU radiosensitization in exponentially growing cells, but the main cause of radiosensitization in plateau-phase cells.

Induction by H 2 O 2 of DNA and Interphase Chromosome Damage in Plateau-Phase Chinese Hamster Ovary Cells

The induction by H2O2 of DNA breaks, DNA double-strand breaks (DSBs), and interphase chromatin damage and their relationship to cytotoxicity were studied in plateau-phase Chinese hamster ovary (CHO) cells. Damage in interphase chromatin was assayed by means of premature chromosome condensation (PCC); DNA DSBs were assayed by nondenaturing filter elution (pH 9.6), and DNA breaks by hydroxyapatite chromatography. Cells were treated with H2O2 in suspension at 0 degrees C for 30 min and treatment was terminated by the addition of catalase. Concentrations of H2O2 lower than 1 mM were not cytotoxic, whereas concentrations of 40 and 60 mM reduced cell survival to 0.1 and 0.004, respectively. An induction of DNA breaks that was dependent on H2O2 concentration was observed at low H2O2 concentrations that reached a maximum at approximately 1 mM; at higher H2O2 concentrations induction of DNA breaks either remained unchanged or decreased. Damage at the chromosome level was not evenly distributed among the cells, when compared to that expected based on a Poisson distribution. Three categories of cells were identified after exposure to H2O2: cells with intact, control-like chromosomes, cells showing chromosome fragmentation similar to that observed in cells exposed to ionizing radiation, and cells showing a loss in the ability of their chromatin to condense into chromosomes under the PCC reaction. The fraction of cells with fragmented chromosomes, as well as the number of excess chromosomes per cell, showed a dose response similar to that of DNA DSBs, reaching a maximum at 1 mM and decreasing at higher concentrations. The results indicate that induction of DNA and chromosome damage by H2O2 follows a complex dependence probably resulting from a depletion of reducing equivalents in the vicinity of the DNA. Reducing equivalents are required to recycle the transition metal ions that are needed to maintain a Fenton-type reaction. The absence of cell killing at H2O2 concentrations that yielded the maximum amount of DNA and chromosome damage suggests that this damage is nonlethal and repairable. It is suggested that lethal DNA and chromosome damage is induced at higher concentrations of H2O2 where cell killing is observed by an unidentified mechanism.

Effects of 5′-iododeoxyuridine on the repair of radiation induced potentially lethal damage interphase chromatin breaks and DNA double strand breaks in chinese hamster ovary cells

The effect of 5'-iododeoxyuridine incorporation into DNA on radiation sensitivity, cellular repair capability, induction and repair of interphase chromatin breaks, as well as induction and repair of DNA double strand breaks was investigated in plateau-phase Chinese hamster ovary cells exposed to X rays. Repair of potentially lethal damage, as measured by delayed plating plateau-phase cells, was used to assay cellular repair capacity. Induction and repair of interphase chromatin breaks were assayed by means of premature chromosome condensation, whereas induction and repair of DNA double strand breaks were assayed by pulsed field gel electrophoresis. Incorporation of 5′-iododeoxyuridine iodo DNA sensitized cells to radiation. Radiosensitization increased with increasing percent thymidine replacement and was accompanied by an increase in the number of chromatin breaks scored per Gy and a small increase in the number of DNA double strand breaks produced. Cells grown in the presence of 5′-iododeoxyuridine were able to repair potentially lethal damage. When the comparison was made at equal doses, the extent of this repair was higher but its rate slower in 5′-iododeoxyuridine containing cells. At equal survival levels, cells that had incorporated IdU also repaired PLD to a slightly higher extent than control cells grown in IdU free medium. The magnitude of potentially lethal damage repair increased as cells “aged” in the plateau-phase, particularly for high 5′-iododeoxyuridine concentrations (8 μM). Incorporation of 5′-iododeoxyuridine reduced the rate of repair of interphase chromatin breaks and the rate of repair of DNA double strand breaks (both the fast and the slow component). The results suggest that reduction in the efficiency of repair of DNA double strand breaks and chromatin breaks, produced by radiation in 5′-iododeoxyuridine containing cells, is one determinant of the radiosensitization observed.

Mechanism of Radiosensitization by Halogenated Pyrimidines: Bromodeoxyuridine and β-Arabinofuranosyladenine Affect Similar Subsets of Radiation-Induced Potentially Lethal Lesions in Plateau-Phase Chinese Hamster Ovary Cells

Chinese hamster ovary (CHO) cells were grown to plateau phase in the presence of various amounts of bromodeoxyuridine (BrdU) and treated after irradiation with beta-arabinofuranosyladenine (ara-A), an inhibitor of DNA and potentially lethal damage (PLD) repair, in order to investigate the importance of repair reactions in general and of PLD repair, in particular, on the mechanism of radiosensitization by halogenated pyrimidines. The degree of BrdU-mediated radiosensitization observed in ara-A-treated cells was compared to that of cells incubated after irradiation in the absence of ara-A. A substantial reduction in BrdU-mediated radiosensitization was observed in cells treated with ara-A at concentrations that, when given alone, produced maximum potentiation in cell killing (500-1500 microM). The residual BrdU-mediated radiosensitization observed at high levels of thymidine replacement could be explained by a BrdU-mediated increase in DNA and chromosome damage induction per gray. These findings are similar to those reported previously for a repair-deficient mutant of CHO cells, the xrs-5 cell line, and consistent with the hypothesis that BrdU-mediated radiosensitization has two distinct components, one that derives from an increase in damage induction per gray, and a second one that derives from an effect of BrdU on the repair of radiation-induced damage. It is proposed that the reduction in BrdU-mediated radiosensitization observed in ara-A-treated cells is the result of ara-A-mediated expression of radiation damage, the repair of which would have been otherwise modulated by BrdU. Since ara-A is known to act by fixing a form of radiation-induced PLD (alpha-PLD), we further propose that BrdU acts by fixing alpha-PLD. A synergistic effect in the potentiation of cell killing was observed between ara-A and BrdU when ara-A was given at concentrations below 100 microM. This result suggests that a benefit may be expected in the clinic from the combined application of halogenated pyrimidines with repair inhibitors, if administered at a carefully screened range of concentrations.

Molecular characterization of X-ray-induced mutations at the HPRT locus in plateau-phase Chinese hamster ovary cells

CHO-K1 cells were irradiated in plateau phase to determine the effects of dose, dose fractionation, and delayed replating on the type, location and frequency of mutations induced by 250 kVp X-rays at the hypoxanthine-guinine phosphoribosyl transferase (HPRT) locus. Independent HPRT-deficient cell lines were isolated from each group for Southern blot analysis using a hamster HPRT cDNA probe. When compared with irradiation with 4 Gy and immediate replating, dose fractionation (2 Gy + 24 h + 2 Gy) and delayed plating (4 Gy + 24 h) appeared to have no effect on the frequency of deletions encompassing the entire gene. Since an increase in survival was noted under these conditions, these data suggest that repair of sublethal and potentially lethal damage acts equally on all premutagenic lesions, regradless of type or location. Differences in the mutation spectrum were noted when cells were irradiated at 2 Gy and replated immediately. The location of the deletion breakpoints was determined in 15 mutants showing partial loss of the HPRT locus. In 12 of these cell lines one or both of the breakpoints appearead to be located near the center of the gene, indicating a nonranrandom distribution of mutaions. These results indicate that damage induced by ionizing radiation results in a nonrandom distribution of genetic damage, suggesting that certain regions of the genome may be acutely sensitive to the mutagenic effect of ionizing radiation.

Multiple Components of Split-Dose Repair in Plateau-Phase Mammalian Cells: A New Challenge for Phenomenological Modelers

Split-dose experiments using starved plateau-phase Chinese hamster ovary cells have been used to investigate the kinetics of repair, expressed in terms of enhancement of reproductive survival. The results show two distinct components of repair, one having a characteristic time of just over 1 h for the removal of a lesion, the other, about 18 h. The rate at which each component removes damage and the fraction of the total damage that each removes appear to be independent of the initial amount of damage produced, i.e., dose. This lack of dose dependence is not consistent with some simple models of ionizing radiation damage and repair, such as those which assume that saturation of a repair process, depletion of enzyme pools, or the interaction of pairs of sublesions is responsible for the curvature in the dose-response relationship. However, the relationship between the amounts of each type of damage and dose appears to be consistent with models that assume that only a portion of the initial damage is directly accessible to the repair systems or that the initial damage consists of a mixture of potentially lethal and sublethal lesions.

Analyzing the role of biochemical processes in determining response to ionizing radiations.

The interaction of biochemical processes and radiation damage appears to play a major role in determining long-term biological effects. It is responsible for both the removal of radiation-induced alterations in macromolecules and for the time-dependent changes in survival of irradiated cells. Restoration of macromolecules by such means as the rejoining of strand breaks in DNA suggests a variety of possible mechanisms which could lead to the observed enhancement of cell survival. However, even though a number of molecular repair mechanisms have been identified, specific links between any such mechanisms and a subsequent modification of cell survival have proved difficult, if not impossible, to demonstrate. Models of cellular response provide a means of attempting to establish this connection. Although details of radiation chemistry, chromatin structure, enzymatic repair, molecular genetics, and cell cycle kinetics are generally simplified, each individual model incorporates features based on a set of assumed mechanisms. For example, one group of models assumes that all damage is potentially lethal (capable of killing the cell unless it is repaired), while another assumes that part of the damage is sublethal (innocuous until it interacts with other damage). Using split-dose, dose-rate, and delayed-plating techniques, we have demonstrated two distinct components of repair in plateau-phase Chinese hamster ovary cells. One process has a characteristic time of about 1 h; the other, about 18 h. In both cases, the reaction rates and the fractions of damage repaired appear to be independent of the initial amounts of damage produced. These observations suggest that none of the simpler models adequately describes cell inactivation; i.e., reproductive death is inconsistent with all assumptions regarding any of them. Consequently, more-complex models involving combinations of sublethal and potentially lethal damage or multiple-step damage processes may be required. These findings help to define the effects of exposure at low doses and dose rates and to develop an understanding of the underlying biochemical mechanisms involved.

Two Distinct Modes of Hyperthermic Cell Death

Plateau-phase Chinese hamster ovary cells sterilized by heat died one of two distinct modes of death. A "rapid" mode, which predominated during the first few days postheating, was characterized by cell detachment and inhibited rates of protein, RNA, and DNA synthesis. A "slow" mode of death became evident after the cells had fully recovered from the heat-induced inhibition of macromolecular synthesis and cell detachment had ceased. These populations had reduced plating efficiencies relative to nonheated populations and contained a large fraction of cells with multiple nuclei. The multinucleated cells did not form colonies, but heated populations also contained increased numbers of uninucleated cells which were nonclonogenic. As the heat dose was increased and the surviving fraction decreased, the rapid mode of death predominated. These data show that heat damage is expressed in two distinct ways. This might result from the existence of two separate targets for heat killing, or a single target which manifests its effects in different ways as the damage it sustains increases.

Proliferation dependence of topoisomerase II mediated drug action.

Topoisomerase II mediated DNA scission induced by both a nonintercalating agent [4'-demethylepipodophyllotoxin 4-(4,6-O-ethylidene-beta-D-glucopyranoside) (VP-16)] and an intercalator [4'-(9-acridinylamino) methanesulfon-m-anisidide (m-AMSA)] was studied as a function of proliferation in Chinese hamster ovary (CHO), HeLa, and mouse leukemia L1210 cell lines. Log-phase CHO cells exhibited dose-dependent drug-induced DNA breaks, while plateau cells were found to be resistant to the effects of VP-16 and m-AMSA. Neither decreased viability nor altered drug uptake accounted for the drug resistance of these confluent cells. In contrast to CHO cells, plateau-phase HeLa and L1210 cells remained sensitive to VP-16 and m-AMSA. Recovery of drug sensitivity by plateau-phase CHO cells was found to reach a maximum approximately 18 h after these cells regained exponential growth and was independent of DNA synthesis. DNA strand break frequency correlated with cytotoxicity in CHO cells; log cells demonstrated an inverse log linear relationship between drug dose (or DNA damage) and colony survival, whereas plateau-derived colony survival was virtually unaffected by increasing drug dose. Topoisomerase II activity, whether determined by decatenation of kinetoplast DNA, by cleavage of pBR322 DNA, or by precipitation of the DNA-topoisomerase II complex, was uniformly severalfold greater in log-phase CHO cells compared to plateau-phase cells.

Effect of Hypoxia on Recovery from Damage Induced by Heat and Radiation in Plateau-Phase CHO Cells

The effect of hypoxia on the induction of and recovery from damage by radiation alone and in combination with heat has been investigated using plateau-phase Chinese hamster ovary (CHO) cells. Postirradiation hypoxia reduced the potentially lethal damage recovery (PLDR) in cells irradiated under an euoxic state and completely eliminated PLDR in cells irradiated under hypoxia. Cells which were maintained under hypoxia during both irradiation and a 4-hr recovery period and then incubated for a further period of 4 hr under euoxic conditions showed PLDR, suggesting that the inhibition of PLDR by hypoxia is reversible. Oligomycin, an inhibitor of energy metabolism, completely eliminated PLDR when present at a concentration of 1 microM during the postirradiation period. Pre- or postirradiation heat treatment at 42.5 degrees C for 30 min appreciably sensitized the cells to the induction of lethality. Thermal enhancement ratio (TER) was 1.7 for cells irradiated and heat treated under hypoxic conditions. The same heat treatment reduced the oxygen enhancement ratio (OER) associated with gamma radiation from 3.1 to 2.5. Cells subjected to this postirradiation heat treatment showed a small extent of PLDR, whereas the pre-heat-treated cells showed as much recovery as non-heat-treated cells. When hypoxic conditions prevailed during the post-treatment incubation period, PLDR was reduced in preheated cells and completely eliminated in postheated cells. The kinetics of interaction between heat and radiation damage were studied by introducing a time gap of 4 hr between the treatments. Cells maintained under euoxic conditions between the treatments showed an appreciable decrease in interaction, suggesting recovery from damage induced by the first treatment. Hypoxic conditions intervening the two treatments largely inhibited the loss of sensitization. Analysis of the results suggests that cells fail to recover from sublethal heat damage when held for 4 hr under hypoxic conditions. Cells held under hypoxic conditions partly recover from the radiation damage which subsequently interacts with sublethal heat damage, resulting in cell lethality.

Modification of the Thermal Response by D 2 O: II. Thermotolerance and the Specific Inhibition of Development

In a companion paper it was shown that D/sub 2/O protects cells against the lethal effects of heat and that D/sub 2/O interfers with the induction of thermotolerance. In this paper we investigate the latter phenomenon with particular reference to an operational model of thermotolerance. Plateau-phase Chinese hamster ovary cells (HA-1) were treated at elevated temperatures permissive for the development of thermotolerance (40-42.5/sup 0/C) in media prepared with H/sub 2/O or D/sub 2/O. Cells were given a more lethal dose of heat (45/sup 0/C/45 min with surviving fraction approx. =10/sup -4/) to assess the degree of thermotolerance. Cells pretreated in H/sub 2/O media expressed a two to three log increase in survival, while thermotolerance was effectively suppressed in cells pretreated in D/sub 2/O. However, cells treated in D/sub 2/O media for 4 hr at 42/sup 0/C, then permitted a 37/sup 0/C incubation period in H/sub 2/O media, rapidly recovered their thermotolerance. Interpreting results within the framework of a three-component model of thermotolerance (trigger, development, decay), we suggest the following: (1) D/sub 2/O inhibits the development component of tolerance; (2) the effect of D/sub 2/O on the trigger component is such that the cells behave as if they were treated atmore » a temperature lower by 2/sup 0/C in H/sub 2/O media; (3) the protection afforded by D/sub 2/O is mechanistically different from that of thermotolerance, at least in the sense that they augment each other.« less