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N L Oleinick

Publications and source records attributed to N L Oleinick.

At least 73 records · Page 4Linked to original sources
PubMed1988

Repair of chromatin damage in glutathione-depleted V-79 cells: comparison of oxic and hypoxic conditions.

We have assessed the effects of two radiomodifying conditions, glutathione (GSH) depletion and hypoxia, on the formation and repair of radiation-induced chromatin damage, specifically DNA-protein cross-links (DPC). As measured by a nitrocellulose filter-binding assay, untreated V79 cells contain a low level of DPC (1-1.5% of the cellular DNA). The background level of DPC is elevated in cells treated with L-buthionine sulfoximine (BSO), in hypoxic cells, and in cells treated with BSO and made hypoxic (2.98%, 2.82%, and 7.71%, respectively). The dose response for production of radiation-induced DPC is approximately 6.0% DNA bound per 100 Gy for cells irradiated in air, and the dose response is not significantly different for BSO-treated cells but increases by a factor of about 1.4 for hypoxic cells and 1.7 for BSO-pretreated hypoxic cells. DPC were also assayed by alkaline elution with or without proteinase K treatment. By this analysis, the yield of DPC appears to be elevated in irradiated hypoxic and irradiated GSH-depleted cells. It is not possible to assay for background DPC alone in unirradiated cells by alkaline elution. Cells not exposed to BSO repair 70-80% of the radiation-induced DPC in 4 h. BSO-treated cells are considerably less efficient in repair of DPC. As analyzed by alkaline elution, GSH depletion had little or no effect on the yield of radiation-induced single-strand breaks (SSB) but slowed their repair. The data suggest that depletion of GSH impairs an enzyme system(s) responsible for the turnover of both background and radiation-induced DPC and that hypoxia elevates both the background level of DPC and the ratio of radiation-induced DPC to SSB.

Animals↗
PubMed1988

Inhibition of radiation-induced DNA-protein cross-link repair by glutathione depletion with L-buthionine sulfoximine.

Cells depleted of their glutathione (GSH) by treatment with L-buthionine sulfoximine (BSO) are more sensitive to ionizing radiation and chemotherapeutic agents. To assess the effects of GSH depletion on repair of radiation-induced DNA damage, we have determined DNA-protein cross-links (DPC) in A549 cells by a nitrocellulose filter binding assay. Untreated A549 cells have a low level of DPC (0.7%), whereas cells incubated with BSO for 92 hours have an elevated level of DPC (1.5%). The dose response for production of radiation-induced DPC is approximately 0.4% DNA bound per 10 Gy for both BSO-treated and non-pretreated cells. Cells not exposed to BSO repair 85% of the radiation-induced DPC in 4 hours. Less repair (55%) is observed for BSO-treated cells incubated for 4 hours postirradiation in conditioned medium, and DPC repair is nearly completely blocked if GSH-depleted cells are given fresh medium during the 4-hour repair interval. The DPC repair is not influenced by the pH of the media between 6.6 and 7.4. Data indicate that intracellular GSH regulates the level of a sulfhydryl-containing repair enzyme, and that other, as yet undefined, nutrients may further influence the steady-state level of DPC.

Buthionine Sulfoximine↗
PubMed1987

Induction of DNA strand breaks in transcriptionally active DNA sequences of mouse cells by low doses of ionizing radiation.

The efficiency of DNA single-strand break induction was measured in transcriptionally active DNA, transcriptionally inert satellite DNA, and bulk DNA sequences of mouse L929 cells using the alkaline filter elution assay. The cells were exposed to increasing doses of X-radiation up to 1000 rad. DNA which either eluted from or was retained on polycarbonate filters during the assays was collected onto nitrocellulose filters and hybridized against radiolabeled poly(A+)RNA (to probe transcribing DNA sequences) or mouse satellite DNA. The increasing rate and extent of elution of bulk DNA or specific DNA sequences after increasing radiation doses was taken as a measure of the increased frequency of radiation-induced DNA strand breaks. The results indicate that a significant fraction of transcriptionally active DNA contains endogenous strand breaks. With increasing dose, the efficiency of radiation-induced DNA strand breakage in bulk, transcriptionally active and satellite DNA sequences was observed to be the same when the sum of all eluted DNA was considered. However, the early eluting fractions contained DNA which was enriched in active sequences. Since DNA elutes as a function of size, the early fractions contain smaller DNA than later fractions. Therefore, our results indicate that the fraction of active sequences which elutes early resides on smaller fragments on the average than the later eluting DNA, and that even low doses of radiation preferentially cause breaks in regions of DNA containing active sequences.

Animals↗
PubMed1987

The formation, identification, and significance of DNA-protein cross-links in mammalian cells.

DNA-protein cross-links (DPC) are formed by a variety of radiations and chemicals which act via free radical formation. Covalency is inferred from the resistance of the cross-links to harsh treatments. In mammalian cells, a background of DPC (6000 per V79 cell) may result from normal associations of chromosomal loops with the nuclear protein matrix. After ionizing radiation, the elevated level of DPC (150 per Gy per V79 cell) are enriched in actively transcribing DNA and in a subset of proteins of the nuclear matrix. DPC formation is reduced by hydroxyl radical scavengers, by oxygen, and by hypertonic medium and is enhanced by hypotonic medium and by removal of intracellular glutathione. DPC are repaired more slowly than single-strand breaks and not at all when formed during metaphase. During the postirradiation period, changes in the sequence composition of the DNA of residual DPC are consistent with the preferential repair of DPC in actively expressed genes. Excision repair mechanisms have been proposed. Unrepaired DPC may block normal functions of the nuclear matrix, such as replication and transcription.

Animals↗
PubMed1986

Modification of DNA damage in transcriptionally active vs. bulk chromatin.

Our previous experiments have demonstrated that regions of nuclear chromatin, containing transcriptionally active DNA sequences and associated with the nuclear matrix, are hypersensitive to the production of both single-strand breaks and DNA-protein cross-links upon gamma-irradiation of exponentially growing mammalian cells. In this study, we have irradiated Chinese hamster V79 cells in buffered saline with or without DMSO to scavenge hydroxyl radicals and in buffered salines of various tonicities to expand or condense chromatin. The yield of DNA-protein cross-links was assayed by a nitrocellulose filter binding technique and the DNA recovered from the cross-links hybridized to 125I-poly(A+)RNA to determine the relative frequency of transcriptionally active sequences in the cross-links compared to the bulk DNA. In all cases, the data show that active DNA is affected to a greater extent than bulk, primarily inactive DNA. The more extensive alteration of the level of ionizing radiation-induced damage in active DNA by the diffusible agents tested suggests that other agents, such as chemical sensitizers and protectors, which need to diffuse to the nuclear DNA, may also be acting primarily on active, matrix-associated DNA.

Animals↗
PubMed1986

The effect of L-buthionine sulfoximine on the aerobic radiation response of A549 human lung carcinoma cells.

Our data show that A549 cells are increasingly radiosensitive with prolonged exposure to L-BSO. The resulting glutathione and protein thiol depleted cells show both loss of shoulder and slope modification. Furthermore, there is an increase in single strand DNA breaks and irrepairable cross-linking. The aerobic radiation damage in the thiol depleted state appears to be different from that obtained with hypoxic cells. Any postulated role for GSH in reducing or preventing peroxidative radiation damage must also include protection against single strand DNA breaks as well as involvement in repairing DNA-protein cross-links. The latter effect may be related to decreased protein thiol content as reflected in a decreased enzyme capacity to repair DNA damage.

Buthionine Sulfoximine↗
PubMed1986

Nuclear matrix proteins are crosslinked to transcriptionally active gene sequences by ionizing radiation.

Unirradiated, exponentially growing Chinese hamster cells contain a low level (less than 5%) of their DNA firmly bound to protein, as measured by a filter-binding assay. That fraction of DNA is highly enriched in sequences which hybridize to poly(A+)RNA or ribosomal RNA. After 60 Gy gamma irradiation, the additional crosslinked DNA is also enriched in transcriptionally active sequences compared to bulk DNA, while DNA crosslinked by uv radiation has a frequency of active sequences which is no higher than the bulk DNA. DNA crosslinked to protein by gamma radiation but not by uv is largely released during a 4-h postirradiation incubation. The DNA which remains bound to protein during that period becomes depleted in active sequences; this is followed by an apparent restoration of the active gene-enriched protein complex found in unirradiated cells. When nuclear matrix-associated DNA was isolated free of the majority ("loop") DNA, an enrichment for active DNA sequences was found in the matrix-associated DNA, and the frequency of DNA-protein crosslinks was found to be 10- to 16-fold greater in the matrix fraction. Gel electrophoretic analysis of the crosslinking proteins identifies them as subset of proteins of the nuclear matrix. These data are consistent with known properties of the nuclear matrix and suggest that chromatin structure plays an important role in the formation and repair of gamma-radiation-induced DNA lesions.

Animals↗
PubMed1985

Poly(ADP-ribose) and the response of cells to ionizing radiation.

The activity of poly(ADP-ribose) polymerase is stimulated by DNA damage resulting from treatment of cells with ionizing radiation, as well as with DNA-damaging chemicals. The elevated polymerase activity can be observed at doses lower than those necessary for measurable reduction in cellular NAD concentration (less than 20 Gy). Several nuclear proteins, including the polymerase itself, are poly(ADP-ribosylated) at elevated levels in irradiated Chinese hamster cells. The addition of inhibitors of poly(ADP-ribose) polymerase to irradiated cells has been found to sensitize the cells to the lethal effects of the radiation, to inhibit the repair of potentially lethal damage, and to delay DNA strand break rejoining. Because of the nonspecificity of the inhibitors, however, it is as yet unknown whether their effects are directly related to the inhibition of poly(ADP-ribose) polymerase, to interference with the poly(ADP-ribosylation) of one or more chromosomal proteins, or to effects unrelated to the poly(ADP-ribosylation) process. The data are consistent with the involvement of poly(ADP-ribose) in the repair of radiation damage, but the nature of this involvement remains to be elucidated.

Animals↗
PubMed1984

The participation of elevated levels of cyclic GMP in the recovery from radiation-induced mitotic delay.

The levels of cyclic AMP and cyclic GMP have been measured in Physarum plasmodia before and after treatment with gamma-radiation, 2 mM caffeine, or combinations of the two agents and compared to the length of the radiation-induced mitotic delay. Caffeine alone produces a rapid transient elevation of cyclic AMP and a slower delayed elevation of cyclic GMP. Irradiation elicits an immediate transient increase in cyclic AMP and a later cyclic GMP increase which accompanies or precedes the delayed mitosis. A composite pattern is produced by combinations of radiation and caffeine, a distinctive feature of which is an elevated level of cyclic GMP near the time of the radiation-delayed and caffeine-promoted mitosis. With pretreatment by caffeine, the least radiation-induced mitotic delay occurs when plasmodia are irradiated during the caffeine-elicited increase in cyclic GMP. The plasmodium becomes refractory to the reduction of mitotic delay by caffeine at approximately the time it becomes refractory to the further elevation of cyclic GMP by caffeine. The data support a role for cyclic AMP in the onset of and for cyclic GMP in the recovery from mitotic delay induced by ionizing radiation.

Caffeine↗
PubMed1984

Differential processing of ultraviolet or ionizing radiation-induced DNA-protein cross-links in Chinese hamster cells.

The yield and repairability of DNA-protein cross-links have been compared after gamma- or U.V.-irradiation of Chinese hamster V79-379 lung fibroblasts. Using a filter-binding assay, cross-linked DNA can be specifically isolated after doses between 10 and 100 Gy of gamma-radiation and fluences between 20 and 300 J/m2 of U.V.-radiation. After ionizing radiation, the majority of DNA cross-linked to protein is released with biphasic kinetics, requiring 1 h for removal of 50 per cent of the cross-linked DNA and 24 h for 90 per cent release. In these cells, U.V.-induced cross-linked DNA is not removed; on the contrary, the yield of apparent DNA-protein complexes increases during postirradiation incubation. Prior gamma-irradiation, to initiate the associated repair system, does not stimulate release of U.V.-induced cross-linked DNA. Inhibition of protein synthesis by cycloheximide affects neither the removal of gamma-ray-induced cross-linked DNA nor the increase in U.V.-induced cross-linked DNA. 3-Aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase, slows the second phase of release after gamma-irradiation as well as the increase in apparent cross-links after U.V.-irradiation. Thus, even though both types of DNA-protein cross-links can be detected by the same assay, their structures or other factors must be substantially different, since the repair system for one type does not recognize the other.

Animals↗
PubMed1984

Cyclic nucleotide responses and radiation-induced mitotic delay in Physarum polycephalum.

The response of the plasmodial levels of cyclic AMP and cyclic GMP in Physarum polycephalum to several putative phosphodiesterase inhibitors and to ionizing radiation has been measured. Isobutylmethylxanthine (2 mM) induces a rapid transient threefold elevation of cyclic AMP alone, with maximum response in about 10 min and return to the base line in about 30 min. Theophylline (2 mM) induces a rapid, sustained twofold elevation of cyclic GMP only. Caffeine (2 mM) and Ro-20-1724 (18 microM) both elicit a rapid transient rise in cyclic AMP, resembling the isobutylmethylxanthine response, and a slow transient elevation of the cyclic GMP level. Of particular interest is the rapid threefold transient elevation of the cyclic AMP, but not of the cyclic GMP, level by gamma radiation. These results (1) emphasize the organism and agent specificity of cyclic nucleotide responses, (2) indicate that the kinetics and apparent refractory response are compatible with the function of previously identified membrane receptor systems, i.e., a blue light receptor and a choleragen receptor mediating cyclic AMP responses, and (3) suggest a relationship between the agent-specific cyclic nucleotide responses and the modification of gamma-radiation-induced delay of mitosis by the same agents, i.e., a decrease in the delay by those which elevate cyclic GMP levels and an increase in the delay by those which elevate only cyclic AMP levels.

1-Methyl-3-isobutylxanthine↗
PubMed1984

Gamma radiation as a probe of chromatin structure: damage to and repair of active chromatin in the metaphase chromosome.

Cobalt-60 gamma radiation has been employed as a means of preferentially damaging actively transcribing chromatin within interphase and metaphase Chinese hamster V79-379 lung fibroblasts. The single-strand size distribution and break frequency of bulk 3H-labeled DNA have been compared to those same parameters for active sequences, i.e., sequences complementary to 125I-labeled poly(A+)RNA. The results show that (a) sequences active during interphase are more sensitive than inactive sequences to single-strand break formation by gamma radiation even when the chromatin is condensed in metaphase, (b) repair of strand breaks in the bulk DNA is slower in metaphase than in interphase cells, but (c) during metaphase, repair is faster in active sequences than in the bulk DNA. Furthermore, this study demonstrates that chromatin structure can be probed within intact cells by a method which circumvents isolation of nuclei or chromatin and the use of exogenous nucleases.

Animals↗
PubMed1982

Hypersensitivity of DNA in transcriptionally active chromatin to ionizing radiation.

We have examined the size distribution of single-strand fragments of total 3H-labeled DNA and of DNA sequences complementary to specific probes in gamma-irradiated and unirradiated mouse L929 cells. Those DNA sequences which hybridize to rDNA or to poly(A+)RNA have lower number average molecular weights and sustain 5--6-times the number of single-strand breaks as do satellite DNA sequences or the bulk DNA. We therefore conclude that transcriptionally active DNA sequences are more susceptible to ionizing radiation-induced damage than are inactive sequences, and suggest that these differential susceptibilities are a likely consequence of differences in their chromatin organization.

Animals↗