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Biomedical subjects

R E Meyn

Publications and source records attributed to R E Meyn.

At least 91 records · Page 5Linked to original sources
PubMed1988

Cell survival and recovery processes in Chinese hamster AA8 cells and in two radiosensitive clones.

Cell survival and recovery after gamma irradiation were investigated in a Chinese hamster ovary cell line (AA8) and in two radiosensitive clones (EM9 and NM2) derived from it. When analyzed by the multitarget and linear-quadratic equations, the dose-response curves for survival of both EM9 and NM2 cells, compared with AA8 cells, were characterized by a decreased magnitude of the shoulder or single-hit region (as reflected by Dq or alpha, respectively) but no difference in the terminal slope or double-hit region (as reflected by DO or beta, respectively). Recovery from sublethal damage (SLD) and potentially lethal damage (PLD) was measured in the three cell lines to examine the relationship between the shoulder width of the survival curve and the magnitude of cellular recovery. NM2 cells exhibited a reduced shoulder on their survival curve and a reduced capacity for SLD recovery, compared with AA8 cells, after equitoxic doses of radiation. EM9 cells, which also had a reduced shoulder on their survival curve, displayed the same rate and extent of recovery as AA8 cells for both SLD and PLD. PLD recovery, as assayed in fed plateau-phase NM2 cells by delayed plating, occurred with slower initial kinetics but to the same final extent as that in AA8 cells, resulting in modification of both the shoulder and the slope of the survival curve. However, PLD recovery, as assayed in log-phase NM2 cells by postirradiation treatment with hypertonic salt, was normal and affected predominantly the slope of the survival curve. These data demonstrate that although both SLD and PLD recovery play a role in determining cell survival, cell-survival curve parameters may not always be useful in predicting cellular recovery capacity.

Animals↗
PubMed1987

DNA cross-linking following exposure to cis-platinum in primary and serially passaged cultured cells derived from two murine fibrosarcomas.

We compared the kinetics of the repair of total (ISC plus DPC) cross-links and of proteinase-resistant (ISC) cross-links in cultured cells derived from two murine fibrosarcoma tumors, FSA and NFSA, after treatment with cis-platinum (cis-DDP), using a modification of the alkaline elution technique. The two tumors had previously been characterized for their response to cis-DDP in vivo; FSA cells gradually removed cross-links from their genome, whereas the NFSA cells showed no capacity to repair these lesions. The aim of the present study was to establish whether treatment of cells from these same two tumors grown under controlled culture conditions would affect either the nature of the lesions induced by cis-DDP or the kinetics of repair of these lesions when compared with tumors treated with cis-DDP in vivo. The culture conditions represent two situations: in the first, the cells in culture approximated the proportion of tumor and normal host cells present in vivo, and in the second, the normal host cells had been eliminated by subculturing to produce cultures composed entirely of tumor cells. All cells were exposed to cis-DDP (either 10 or 20 micrograms/ml) for 1 h. The relative amounts of total cis-DDP-induced DNA crosslinks and of ISCs were then determined at various times after treatment. The results show that there was little difference in the behavior of these cultured cells compared to the in vivo response of the tumor from which they were derived. For FSA, each cell culture exhibited a capacity to repair DNA cross-links comparable to that of the tumor in vivo. For NFSA, the passaged cells again paralleled the behavior of that tumor in vivo, although in this case by showing no measurable capacity to repair cross-links. The absence of a significant repair response in the NFSA tumor therefore appears to be an intrinsic characteristic of these tumor cells.

Animals↗
PubMed1987

Applicability of the alkaline elution procedure as modified for the measurement of DNA damage and its repair in nonradioactively labeled cells.

We have critically evaluated various modifications of the alkaline elution methodology that were required to adapt the method for measuring DNA damage in cells from animal tissues treated in vivo. These modifications involved the use of a fluorometric assay for the eluted DNA using the dye Hoechst 33258, which in turn required the use of a different combination of filter and lysis conditions than those used in conventional assays. This protocol was compared with the conventional protocols by examining the DNA damage produced in cultured Chinese hamster ovary cells after treatment with three agents (gamma-rays, cis-dichlorodiammineplatinum (DDP) and trans-DDP) that differ widely in the type and repairability of the DNA lesions that they induce. For both gamma-rays and trans-DDP, the results obtained by the various protocols were equivalent with respect to the amount, type, and rate of repair of the DNA damage produced. On the other hand, for cis-DDP, where the repair time for DNA crosslinks was significantly long relative to the cell-cycle time, DNA replication appeared to be a potentially complicating factor in the measurement of crosslink repair. However, even after treatment of rapidly dividing cultured cells, where any discrepancy between the radioactivity and Hoechst assays due to DNA replication should be maximal, the resulting difference in the amount of repair measured using the two assays was relatively small. Finally, in experiments using cis-DDP and trans-DDP, the data suggested that when polycarbonate and polyvinyl chloride filters were compared using the same cell lysis conditions, their relative sensitivity to detect DNA-protein versus DNA-interstrand crosslinking were comparable. The modified alkaline elution protocol for the measurement of DNA damage in vivo therefore appears, in most cases, to produce results comparable with those obtained by the conventional protocols.

Animals↗
PubMed1987

Induction and repair of DNA single-strand breaks in EM9 mutant CHO cells treated with hydrogen peroxide.

In this study we investigated the induction and rejoining of DNA single-strand breaks (SSBs) produced by H2O2 in the repair-deficient EM9 mutant Chinese hamster ovary (CHO) cell line. The effect of the poly(ADP-ribose)-transferase inhibitor 3-aminobenzamide (3-ABA) on SSB-rejoining and on cell killing was also evaluated. Results were compared with those obtained previously with the parent cell line (AA8). Cells were treated with H2O2 on ice for 1 h, after which they were either harvested or allowed to repair their damage at 37 degrees C either in the presence or absence of 3-ABA (5 mM). The cells were then assayed either for survival using a colony-forming assay or for their level of DNA SSBs using alkaline elution. EM9 cells were somewhat more sensitive than AA8 cells to the cytotoxic effects of H2O2. However, because the repair mutant showed slightly lower levels of DNA SSBs than did its parental cell line, this sensitivity could not be explained on the basis of alterations in initial damage. The rejoining of the H2O2-induced DNA SSBs followed exponential kinetics in both cell lines; however, EM9 cells rejoined these breaks at a slower rate (t1/2 of 10 min) than did AA8 cells (t1/2 of 5 min). The increased sensitivity of the EM9 cells therefore appears to correlate with a reduced ability to remove these lesions from their DNA. As previously demonstrated for the AA8 cells, 3-ABA treatment resulted in both a retardation of the removal of H2O2-induced DNA SSBs and potentiation of cytotoxicity in the EM9 cells. However, the degree of these effects were similar for both AA8 and EM9 cells. These data provide further evidence that the cytotoxic effects of low concentrations of H2O2 are mediated by damage to DNA, and suggest that the rate at which DNA SSBs are rejoined is important for cell survival.

Animals↗
PubMed1987

The induction of DNA-protein crosslinks in hypoxic cells and their possible contribution to cell lethality.

The induction of single-strand breaks (SSBs) in the DNA of Chinese hamster ovary cells by X rays under different irradiation conditions was measured by the alkaline elution technique. The oxygen enhancement ratio (OER) for SSB induction determined for cells irradiated in air versus irradiation of cells made hypoxic by metabolic depletion of O2 was 9.7. However, when proteinase K was included in the cell lysis solution the OER was reduced to 4.2. The proteinase affected the elution rate only of the cells irradiated under hypoxic conditions, suggesting that DNA-protein crosslinks (DPCs) are preferentially produced in hypoxic cells by radiation. The ability to repair these DPCs was compared in two cell lines: the wild-type AA8 line and an excision-repair-deficient mutant line, UV-41. The AA8 line removed about 80% of the DPCs induced by radiation under hypoxic conditions within a 24-h repair incubation. The UV-41 line, on the other hand, removed only about 20% of the DPCs in the same time. The OERs for cell survival of these two lines are 3.1 for AA8 but only 1.9 for UV-41, suggesting that the DPCs preferentially induced in the DNA of cells irradiated under hypoxic conditions may contribute to cell killing when the normal DNA-repair mechanisms are compromised.

Animals↗
PubMed1987

Enhancement of X-ray-induced sister chromatid exchanges in hypoxic cells.

In these studies we have used wild-type Chinese hamster ovary cells (AA8) and a mutant cell line (UV-41) deficient in excision repair to compare sister chromatid exchange (SCE) induction after X irradiation under oxic and hypoxic conditions. X irradiation of AA8 cells under oxic conditions induced only a slight increase in SCEs, whereas at each dose tested a significantly greater number of SCEs were induced in hypoxic cells. When AA8 cells were X-irradiated and the addition of bromodeoxyuridine (BrdU) was delayed for 20 h to allow DNA lesions to be repaired, the levels of SCEs detected in both oxic and hypoxic cells returned to background levels. X irradiation of UV-41 cells also induced only a slight increase of SCEs in oxic cells, whereas a significant number of SCEs were induced in hypoxic cells. However, in contrast to results with AA8 cells, when hypoxic UV-41 cells were X-irradiated and the addition of BrdU was delayed for 20 h, the number of SCEs remained significantly above background levels. In combination with previous alkaline elution data, these results are consistent with the possibility that DNA-protein crosslinks are responsible for the SCEs induced by X irradiation of hypoxic cells. Irrespective of the mechanism(s) involved, the data presented suggest that the SCE assay may potentially aid in the detection of hypoxic tumor cells.

Animals↗
PubMed1987

Differential repair of gamma-ray-induced DNA strand breaks by various cellular subpopulations of mouse jejunal epithelium and bone marrow in vivo.

We have examined the induction and repair of gamma-ray-induced DNA strand breaks in different subpopulations of cells in mouse jejunal epithelium and bone marrow using a modification of the alkaline elution methodology whereby different populations of cells are selectively labeled with radioactive DNA precursors. Mice were labeled by intraperitoneal injection with between 0.5 and 2.0 mu Ci/g of [3H]thymidine at various times prior to irradiation with 10 Gy of gamma rays. In the studies with jejunal epithelium, the timing of the injection of the radiolabel relative to the irradiation was varied between 6 and 72 h, depending on the cell population of interest. The DNA damage and repair characteristics representative of both the total cell population and the radiolabeled fraction of these cells were then measured. Little difference was noted in the amount of initial damage induced in these different populations of cells. However, for both the jejunum and bone marrow, cells that incorporated the radiolabel within 6 h after injection (i.e., rapidly proliferating cells) repaired their strand breaks more rapidly than did the remainder of the population. In the case of jejunum, the repair capacity of the radiolabeled cell population progressively diminished as the cells matured and differentiated so that cells that contained the radiolabel 72 h after injection (i.e., mature villus cells) actually repaired their strand breaks more slowly than did the bulk cells.

Animals↗
PubMed1986

Effect of 3-aminobenzamide on DNA strand-break rejoining and cytotoxicity in CHO cells treated with hydrogen peroxide.

The influence of the nuclear ADP-ribosyltransferase inhibitor 3-aminobenzamide on the DNA strand-break rejoining kinetics and cytotoxicity in Chinese hamster ovary cells following H2O2 treatment was investigated. For the DNA damage studies, cells were treated on ice with H2O2 (0-20 microM) for 1 h in serum-free medium, after which the H2O2 was removed and the cells were allowed to repair their damage in complete medium at 37 degrees C in the presence or absence of 3-aminobenzamide (5 mM) for periods up to 2 h. The DNA strand breaks remaining as a function of time were then estimated by alkaline elution. A linear relationship between the H2O2 concentration and the initial level of DNA single-strand breaks (zero time allowed for repair) was observed. No double-strand breaks or DNA-protein cross-links were detected at these doses. The rejoining of single-strand breaks after H2O2 (20 microM) alone was characterized by a single exponential process with a t1/2 of approx. 5 min. However, in the presence of 3-aminobenzamide, rejoining was much slower and biphasic, with t1/2 of approx. 10 and 36 min. The inhibitory action of 3-aminobenzamide was concentration-dependent and completely reversible in that, when the 3-aminobenzamide was removed from the treated cultures, the strand-break rejoining kinetics rapidly returned to the t1/2 of 5 min typical of H2O2 alone. Considerably higher concentrations of H2O2 (up to 600 microM) were required for cell killing compared to the DNA damage studies. Cell killing by H2O2 alone was characterized by a shoulderless, exponential survival curve (D0 = 880 microM). The cytotoxicity was potentiated when the cells were treated with 3-aminobenzamide (5 mM) for 1 h after the H2O2 treatment; the survival curve with 3-aminobenzamide also assumed a biphasic character (D0 of 212 microM and 520 microM). These results are consistent with the theory that OH.-induced single-strand breaks do not normally represent lethal lesions to the cell because of their rapid, efficient repair. However, interference with these repair processes (in this case by 3-aminobenzamide) can alter this relationship, possibly allowing lesion fixation.

Animals↗
PubMed1986

Survival of rat mammary tumor cell clones and DNA strand damage following adriamycin treatment.

Tumor cell subpopulations have been shown to be heterogeneous in a number of phenotypic characteristics, including responses to cytotoxic drugs. This phenotypic heterogeneity has been used here to study mechanisms associated with Adriamycin (doxorubicin HCl)-induced cytotoxicity. Clonogenic survival and alkaline elution methods were employed to examine the response of two tumor cell subpopulations to Adriamycin. The cells were derived from a primary 13762NF rat mammary adenocarcinoma (clone MTC) and a lung metastasis in the same animal (clone MTLn3). The MTC cells were significantly more resistant to Adriamycin than were the MTLn3 cells; the dose effective in reducing cell survival by 50% was 10-fold higher. Protein-associated DNA strand breakage assayed by alkaline elution was dose-dependent in both clones, and MTC cells were again more resistant to break induction than were MTLn3. These results showed that clonal tumor subpopulations isolated from a primary tumor and its metastases possessed different intrinsic survival responses to Adriamycin treatment in vitro and that this survival response correlated with Adriamycin-induced production of protein-associated DNA single-strand breaks.

Animals↗
PubMed1986

Potential methods for predicting tumor radiocurability.

Several predictors of tumor radiocurability are already integrated into clinical practice, for example, tumor size, gross morphology (that is, infiltrative or exophytic), histologic type and grade. These are nonspecific and relatively imprecise. The aim of research into predictive assays is not only to refine the discrimination of existing predictors, but also to suggest specific experimental approaches for overcoming tumor radioresistance in individual patients. Two broad categories of predictive assays can be defined: direct and indirect measurement of tumor cell survival and/or repair capability following irradiation, and measurement of cellular and extracellular parameters affecting radiosensitivity. The ongoing research at The University of Texas M. D. Anderson Hospital is overviewed to illustrate potential methods for predicting radiocurability.

Animals↗
PubMed1986

Cell cycle-dependent cytotoxicity of alkylating agents: determination of nitrogen mustard-induced DNA cross-links and their repair in Chinese hamster ovary cells synchronized by centrifugal elutriation.

Chinese hamster ovary cells were synchronized into the different phases of the cell cycle by centrifugal elutriation and treated with nitrogen mustard (HN2) in order to investigate the role of DNA damage and repair processes in the cell cycle-dependent cytotoxicity of this alkylating antitumor agent. In agreement with previous studies, cell populations enriched in G1 were the most sensitive to HN2, and those enriched in late S phase-G2 were more resistant, as determined by clonogenic assay. Although the variation in surviving fraction through the cell cycle in response to a single dose (3 micrograms/ml; 1.0 h) of HN2 was as great as a factor of 10, complete dose-response curves generated for the most sensitive and most resistant elutriator fractions indicated that such changes could be accounted for by a ratio of D0 values of only 1.4 Cells synchronized by this same method were also analyzed for their relative levels of HN2-induced DNA cross-linking using the sensitive technique of alkaline elution. There was no significant difference in the levels of either DNA interstrand or DNA-protein cross-links induced in the two elutriator fractions described above immediately after the HN2 treatment. When the amount of DNA cross-linking in the two fractions was measured 6 h after treatment, considerable repair had occurred; however, there was no measurable difference in the rate of repair of either type of cross-link (i.e., DNA interstrand and DNA-protein) in the different phases. Differences in DNA damage and repair processes could not therefore be resolved within the confidence limits of available assays and probably cannot account for the differential cytotoxicity of HN2 towards cells in the different cell cycle phases.

Alkylating Agents↗
PubMed1985

DNA damage in normal and neoplastic mouse tissues after treatment with misonidazole in vivo.

Alkaline elution has been used to examine the integrity of DNA isolated from various tissues from mice treated with misonidazole (MISO). High doses (1-3 mg/g) of MISO caused extensive DNA strand breakage in cells isolated from two fibrosarcoma tumors that were known to contain hypoxic cells, and also in cells from certain normal tissues (liver and kidney in particular). The incidence of strand breaks gives further support to the suggestion that MISO can be metabolically nitroreduced beyond the singly reduced nitro radical-anion in some normal tissues as well as in hypoxic tumor cells, generating DNA-reactive species. Nitroreductases must therefore be able to compete successfully with molecular oxygen for the MISO nitro radical-anion in such tissues.

Animals↗
PubMed1985

Kinetics of DNA cross-linking in normal and neoplastic mouse tissues following treatment with cis-diamminedichloroplatinum(II) in vivo.

The formation and repair of cis-diamminedichloroplatinum(II) (cis-DDP)-induced DNA cross-links in cells from a number of different mouse tissues, both normal and neoplastic, were compared in three different populations of animals, tumor-free mice and mice bearing a transplanted fibrosarcoma (either FSa or NFSa) in their thighs. Groups of mice were given i.v. injections of 4-12-mg/kg doses of cis-DDP, and the amount of cis-DDP-induced DNA cross-linking was determined at different times after injection using an adaptation of the alkaline elution technique. The degree of cross-linking in each tissue was linearly related to the dose of cis-DDP at either 6 or 24 h after injection and varied significantly among the different tissues, with FSa, NFSa, kidney, and liver showing the highest level of cross-linking of the tissues studied. The relative contributions of DNA-interstrand and DNA-protein cross-links to the elution profiles were estimated by proteinase K (PK) digestion. At either 6 or 24 h after injection with cis-DDP, the rate of elution of the DNA was substantially increased by PK, indicating a large contribution of DNA-protein cross-links. This effect was observed in all tissues studied, although the proportion of PK-resistant lesions appeared to vary from tissue to tissue, liver and spleen showing a significantly lower proportion of DNA-interstrand to total cross-links than either of the tumors. For liver, virtually no interstrand cross-links could be detected after PK treatment. The kinetics of the repair of cis-DDP-induced DNA cross-linking in these tissues were also compared. In cells from tumor-free animals, the amount of total (DNA-interstrand plus DNA-protein) cross-linking increased gradually, reaching a maximum after about 6 h; however, little evidence of repair of these lesions was observed in any of these normal tissues. In fact, the degree of cross-linking tended to increase somewhat between 6 and 24 h after injection. The kinetics of cross-linking in cells isolated from the FSa tumor were very different; while there was an initial increase in cross-linking up to 6 h, these lesions were subsequently repaired, although at a somewhat slower rate than has been reported for cultured mammalian cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

Use of mammalian DNA repair-deficient mutants to assess the effects of toxic metal compounds on DNA.

Wild-type and repair-deficient cell lines ( EM9 ) of Chinese Hamster Ovary cells were utilized to assess cytotoxic responses towards metals that produce lesions in DNA. Alkaline elution studies indicated that both CaCrO4 and HgCl2 induced single-strand breaks in the DNA. CaCrO4 and HgCl2 treatments of intact Chinese hamster ovary cells also caused the induction of DNA cross links. The mutant cells, which are thought to have a defect in the repair polymerase enzyme and therefore exhibit greater sensitivity towards a variety of agents that produce lesions in the DNA such as X-rays and ultraviolet-light, also displayed a greater sensitivity, compared to wild-type cells, towards the cytotoxic response of HgCl2 and CaCrO4 . For example, the IC50 (concentration producing a 50% growth inhibition) following exposure for 6-hr to CaCrO4 or 1 hr to HgCl2 was 3.4-fold or 1.8- to 3.9-fold greater in wild-type cells compared to repair-deficient cells respectively. Mutant cells compared to wild-type cells were not more sensitive to growth inhibition by agents whose primary site of action was not at the DNA level (i.e. amphotericin B, trifluoroperazine and cycloheximide). The DNA crosslinks induced by exposure to 10 microM CaCrO4 for 6 hr were almost completely repaired in wild-type cells within 24 hr, whereas in similarly exposed mutant cells this lesion was initially more pronounced and was only partially repaired following a 24-hr recovery period in the absence of CaCrO4 . The repair of single-strand breaks induced by CaCrO4 was more rapid and similar in both wild-type and mutant cells. Since Hg(II) inhibits repair of single-strand breaks, we could not study repair of this lesion induced by this agent; however, at very low concentrations (1 microM) binding of 203Hg(II) to DNA was greater in the mutant cells compared to the wild-type cells. Following removal of 203Hg(II) from the media, mutant cells generally retained more 203Hg bound to DNA relative to the total 203Hg(II) present in the cell. These results demonstrate that an important toxic action of CaCrO4 and HgCl2 involves injury to DNA since the concentrations of these metals causing measurable DNA damage were consistent with their respective cytotoxic concentrations and DNA repair-deficient mutants displayed both enhanced cytotoxicity and decreased repair of metal-induced lesions.

Amphotericin B↗
PubMed1984

DNA damage produced by combined hyperglycemia and hyperthermia in two mouse fibrosarcoma tumors in vivo.

In this study we used alkaline elution to examine DNA damage produced in two murine fibrosarcomas after hyperthermia (42 degrees C), with or without preinduced hyperglycemia. The work was stimulated by a recent report that pretreatment of mice with glucose prior to hyperthermia decreased the growth rate in a similar fibrosarcoma tumor. The intercellular tumor pH dropped from its resting value of 7.1 to a value of 6.6 at 1.5 hr after a single injection of glucose. While treatment with either glucose alone or heat alone produced very little detectable damage, the combination of these two agents resulted in marked degradation of tumor DNA isolated immediately after treatment. DNA degradation was accompanied by a simultaneous decrease in cell viability; thus, direct cell killing and subsequent nuclease or lysosomal enzyme activity are probably involved. We also tested whether hyperglycemia combined with hyperthermia influenced the DNA-DNA crosslinking induced by subsequent cyclophosphamide (Cy) treatment. Because of the extensive degradation caused by the pretreatment alone under the conditions used in these initial experiments, we were not able to quantitate with validity the amount of Cy-induced crosslinking. However, damage in viable cells may presumably interact with the subsequent Cy treatment to produce further selective cell killing in the tumor.

Animals↗
PubMed1984

Effect of misonidazole pretreatment on nitrogen mustard-induced DNA cross-linking in mouse tissues in vivo.

In the present study we have used the alkaline elution technique to study the effect of misonidazole (MISO) on the initial amount of DNA cross-linking in various normal and neoplastic tissues of C3H mice treated with nitrogen mustard (HN2) in vivo. Tissue samples for analysis of the cross-links were prepared 1 h after injection with HN2 to minimize the effect of subsequent repair processes on the yield of lesions. For mice receiving HN2 alone, the greatest level of cross-linking was found in spleen and jejunum, with the liver showing the lowest level. In animals that had been pretreated with MISO (1 mg g-1, i.p.) for 0.5 h prior to injection with HN2, the amount of cross-linking in the spleen and jejunum was not affected by MISO; however, in all other tissues that were examined, cross-linking was enhanced by MISO to a varying extent depending on the specific tissue. The greatest enhancement was observed in the liver (X 6) and kidney (X 3.1), both of these tissues showing a greater enhancement than either of the two fibrosarcomas. The potentiation of HN2 cross-linking in a particular tissue correlated well with two cellular processes that are known to be nitroreduction-dependent in vitro, namely, the degree of MISO-induced GSH depletion and the binding of MISO to cellular macromolecules. Thus, the potentiation of cross-linking in normal tissues such as liver and kidney, and by inference in tumours, may be intimately related to the generation and/or accumulation of nitro-reduced MISO metabolites in those tissues.

Animals↗