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M M Elkind

Publications and source records attributed to M M Elkind.

At least 19 recordsLinked to original sources
PubMed1993

Identification of a cytochrome P450 gene by reverse transcription--PCR using degenerate primers containing inosine.

A cytochrome P450-like gene, tentatively named P450CMEF, was amplified by a mixed oligonucleotide-primed amplification of cDNA from C3H mouse embryo fibroblast cells, designated 10T1/2, that had been treated with 7,12-dimethylbenz[a]anthracene (DMBA) or benz[a]anthracene (BA). A set of inosine-containing degenerate primers that were targeted to two conserved regions of known cytochrome P450 cDNAs were used. One primer was coded for the well-described and conserved heme-binding region of P450 enzymes, and the second was designed based upon other considerations of homology among P450 molecules. One of the four PCR-amplified cDNA products hybridized to two major RNA bands, 4.2 and 5.3 kb, that were induced by DMBA or BA. The amino acid sequence of the fragment deduced from the base-sequence data indicate that the amplified cDNA has a 50-55% identity with the cytochrome P450 subfamily 1A. The induction of P450CMEF mRNA preceded the induction of aryl hydrocarbon hydroxylase activity after DMBA or BA treatment, suggesting that the product of P450CMEF is involved in the metabolism of these polycyclic aromatic hydrocarbons in 10T1/2 cells. From the partial sequence of the cDNA identified by this procedure, we propose that P450CMEF is a member of the P450 superfamily, possibly in a subfamily of family 1, that is induced in 10T1/2 cells by DMBA and BA. This method should be useful in identifying additional P450 genes and genes in other gene families.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1992

Enhanced sensitivity to neoplastic transformation by 137Cs gamma-rays of cells in the G2-/M-phase age interval.

C3H mouse 10T1/2 cells, exposed to low doses of fission-spectrum neutrons, have an enhanced frequency of neoplastic transformation if protracted exposures are used (Hill et al. 1982, 1984a, 1985). To explain this anomaly, a biophysical model was proposed (Elkind 1991a,b) having the following essential features: (1) a narrow age interval exists in the growth cycle of 10T1/2 cells in which cells have high sensitivities to transformation; (2) in the latter age interval, cells are also sensitive to killing; (3) with increasing dose, cells at ages earlier in the growth cycle are progressively delayed from entering the sensitive age window; and (4) with increasing dose, the transformation sensitivity of cells in the sensitive window is not expressed due to increased killing. Protracted low doses result in elevated frequencies because of less killing, and reductions in delays in cell progression. Therefore, transformation-sensitive cells can progress into the sensitive interval to replace those that have progressed out of it. The unique shape and radiobiological properties of cells in and around mitosis, led to the proposal that the sensitive window is mitosis and possible cells just preceding or just following M phase (Elkind 1991a,b). Because of the likelihood that the properties of the cells in a sensitive window would not be evident only when fission-spectrum neutrons are used, this study was undertaken using 137Cs gamma-rays. We have found that late G2- to M-phase 10T1/2 cells have a maximal sensitivity to neoplastic transformation as well as to killing by 137Cs gamma-rays.

Animals↗
PubMed1992

Abrogation of killing and neoplastic transformation of C3H10T1/2 cells due to 7,12-dimethylbenz[a]anthracene.

The combined action of 7,12-dimethylbenz[a]anthracene (DMBA) and alpha-naphthoflavone (alpha NF) on the survival and neoplastic transformation of C3H10T1/2 mouse embryo fibroblasts has been examined and correlated with DNA adduct formation and removal. When a 24 h DMBA treatment of asynchronously growing cells was followed for the next 24 h by a treatment with alpha NF + DMBA, both killing and transformation per viable cell were abrogated to a large extent. In some instances, transformation was completely abrogated--i.e. reduced to control frequencies--even at nontoxic concentrations of DMBA, indicating that changes in survival were not the reason for the reduction in transformation. Even at toxic concentrations of DMBA, post-treatment with alpha-NF + DMBA resulted in 10-fold reductions in transformation frequency. 3-Methylcholanthrene (3MC) also reversed DMBA cytotoxicity but with a dependence on 3MC concentration that was qualitatively different from that for alpha NF. The abrogation of cell killing occurred at lower molar ratios of alpha NF:DMBA than the abrogation of transformation; less than or equimolar concentrations resulted in maximal abrogation of killing, but about equal concentrations were required to abrogate transformation. Although the preceding findings suggest that different mechanisms may be involved in these endpoints, taken together they suggest that second treatments make apparent the repair of lesions due to a first treatment with DMBA alone. To test this hypothesis, the formation and removal of DMBA-DNA adducts were measured. Adducts were not removed when the second treatment was growth medium alone, but enhanced removal was observed when second treatments consisted of DMBA alone or DMBA plus one of several other polycyclic aromatic hydrocarbons (PAHs). Relative to killing and neoplastic transformation, these results suggest DMBA induces a repair process that limits its own effectiveness--a process that can be sustained by other PAHs.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1991

Aryl hydrocarbon hydroxylase activity assayed in whole cell lysates using synchronous fluorescence spectroscopy.

Synchronous fluorescence spectrophotometry has been used to measure induced and constitutive levels of aryl hydrocarbon hydroxylase activity in lysates of C3H 10T1/2 mouse embryo fibroblasts. Without compromising sensitivity, the method was reproducible, eliminated the need to extract metabolites, and made the procedure simpler and less time consuming than other methods. Moreover, since the assay was tailored to directly measure 3-hydroxybenzo(a)pyrene, a metabolite produced by several cytochrome P-450s, it may be more generally applicable than dealkylation assays, which apparently detect only P-450-IA1.

Animals↗
PubMed1991

Amsacrine-induced lesions in DNA and their modulation by novobiocin and 2,4-dinitrophenol.

The cancer chemotherapeutic agent amsacrine, 4'-(9-acridinylamino)-methanesulfon-m-anisidide (mAMSA), is thought to effect cytotoxicity by inhibiting the ATP-dependent enzyme topoisomerase II in the act of its duplex strand-passing action. Upon protein denaturation, the arrested "cleavable complex" that results gives rise to double- and single-strand breaks (dsbs and ssbs) and DNA-protein cross-links (dpcs). Simultaneous cotreatments with 2,4-dinitrophenol (DNP) or novobiocin (novo) abrogates mAMSA cytotoxicity in Chinese hamster cells (H. Utsumi et al., Cancer Res., 50:2577-2581, 1990). Pulsed-field gel electrophoresis was used to estimate dsbs, velocity sedimentation in alkaline sucrose gradients for ssbs, and alkaline elution without protease digestion for dpcs. Although cotreatment with DNP or novo modulated somewhat the yield of DNA lesions due to mAMSA, quantitatively these changes did not correlate at all with, and therefore could not account for, the reduced lethality that resulted from cotreatments. For example, DNA cotreatment markedly increased the yields of dsbs, ssbs, and dpcs, even though cell killing was appreciably reduced. Furthermore, neither DNP nor novo cotreatment affected the rate, or the completeness of, the repair of mAMSA-induced DNA damage, and neither cotreatment lowered total cellular ATP. Hence, the arresting of the cleavable complex by mAMSA, made evident by lesions in DNA, did not correlate with cytotoxicity. However, cotreatment with either DNP or novo resulted in an enhanced recovery of the mAMSA-induced inhibition of replicative DNA synthesis. Because DNP and novo (transiently) slow down DNA synthesis, it is proposed that these compounds abrogate mAMSA killing of S phase cells by reducing the disorganization of the processing of replicated DNA by topoisomerase II.

2,4-Dinitrophenol↗
PubMed1991

Caffeine and D2O medium interact in affecting the expression of radiation-induced potentially lethal damage.

Earlier work (Ben-Hur et al. 1980) has been extended to compare the killing of log-phase V79 Chinese hamster cells by ionizing radiation when they are treated immediately after irradiation with medium containing either caffeine or 90% D2O. The object was to determine if the enhanced killing due to post-treatment with caffeine, or D2O, resulted from action on the same sector of potentially lethal damage as appeared to be the case for hypertonic shock and D2O medium. The treatments by themselves were not toxic to unirradiated cells. We found that the enhanced expression of potentially lethal damage by post-treatment with caffeine or D2O medium is similar. For example, the kinetics of the repair of the potentially lethal damage expressible by either post-treatment was similar, and an additive enhancement of potentially lethal damage occurred when the two treatments were administered sequentially. These findings suggest that caffeine and D2O medium affect the same sector of potentially lethal damage. When the two treatments were combined, however, they competed with each other. That is, exposures to caffeine, which by themselves did not enhance killing (up to 1 mM for 2 h), decreased the enhanced killing due to D2O medium, Reciprocally, D2O medium reduced the enhanced killing due to high concentrations of caffeine (greater than 1 mM). Thus, although caffeine and D2O medium act on the same sector of potentially lethal damage they do so differently, suggesting that more than one pathway of the expression of radiation damage can result in the same phenotypic effect.

Animals↗
PubMed1991

Bleomycin induces similar survival variations through the cell cycle as do X rays with and without hypertonic shock.

In an earlier report [H. Utsumi and M. M. Elkind, Radiat. Res. 119, 534-541 (1989)], it was shown that the survival of V79 Chinese hamster cells treated with bleomycin was significantly reduced by a posttreatment with anisotonic phosphate-buffered saline in a manner that was qualitatively similar to what had been observed with X rays [H. Utsumi and M. M. Elkind, Radiat. Res. 77, 346-360 (1979)]. This similarity suggested that similarities might exist in the cyclic variation in the suppression of the repair of potentially lethal damage following treatment with bleomycin or X rays. Accordingly, the age-response variations of survival, with or without a posttreatment challenge with hypertonic buffer, were compared in the same experiment when cells were treated with either agent. Although a significant difference was observed near the G1/S-phase border, in general the damage induced by the two agents showed a similar dependence on cell age, and posttreatment with hypertonic buffer enhanced cell killing appreciably following either treatment. The results support the inference that bleomycin is a radiomimetic agent.

Animals↗
PubMed1991

Physical, biophysical, and cell-biological factors that can contribute to enhanced neoplastic transformation by fission-spectrum neutrons.

In radiobiology, fission-spectrum neutrons frequently have been used as a surrogate for other high-LET radiations, particularly when thick absorbers were involved as in animal studies. However, the spectrum of proton secondaries, plus the gamma rays generated in the absorption processes, suggests that a characterization of such a beam, based upon an average LET alone, may not adequately account for the spectrum of biological properties that it may have. Conflicting results have been reported on the relative effectiveness of reduced dose rates of fission-spectrum neutrons, and other high-LET radiations, for the induction of noeplastic transformation of cells in culture. Enhanced rates of neoplastic transformation were reported for C3H 10T1/2 mouse cells, Syrian hamster embryo cells, and human hybrid cells-all with the same beam of fission-spectrum neutrons generated by the JANUS reactor at the Argonne National Laboratory. No enhancement was observed with C3H 10T1/2 cells exposed to the beam from the TRIGA reactor at the Armed Forces Radiobiological Research Institute, or to maximally effective alpha particles. The recent report that an enhancement was also observed when human hybrid cells were exposed at a low dose rate to the TRIGA beam indicated that physical factors alone were not responsible for the differences observed with C3H 10T1/2 cells exposed to these various beams. To resolve the lack of consistency in the results that had been reported, a biophysical model was developed based, in part, on the existence of a narrow age interval in the growth cycle of a cell during which it is particularly sensitive to radiation neoplastic transformation. Because of the special physical and biological properties of cells in M phase, and/or in late G2 phase or early G1 phase, these cohorts of cells were proposed as those that are hypersensitive to neoplastic transformation by radiation.

Animals↗
PubMed1990

Abrogation by novobiocin of cytotoxicity due to the topoisomerase II inhibitor amsacrine in Chinese hamster cells.

Using cultured V79 Chinese hamster cells, we found that novobiocin (or 2,4-dinitrophenol) can abrogate, almost completely, the cytotoxicity due to the topoisomerase II inhibitor amsacrine (mAMSA). V79 cells were sensitive to mAMSA killing at all stages in the cell cycle but mainly in S phase followed by late G1 phase; however, novo rescued cells of all ages. The properties of two kinds of radiation-sensitive Chinese hamster cells were also examined, i.e., the line of V79 cells that can be rescued by caffeine, designated S-10 (H. Utsumi and M.M. Elkind, Radiat. Res., 96: 348-358, 1983); and Chinese hamster ovary cells (P.A. Jeggo and L.M. Kemp, Mutat. Res., 112: 313-327, 1983) which are also sensitive to other DNA-damaging agents. As is the case for exposure to radiation, after mAMSA treatment caffeine rescued V79/S-10 cells. Although Jeggo's Chinese hamster ovary cells were more responsive to mAMSA, novo still abrogated mAMSA toxicity in the mutant cells as well as in the parental Chinese hamster ovary cells 2,4-Dinitrophenol acted similarly to novo with respect to mAMSA killing, but neither compound reduced the ATP content of V79 cells. We propose that one reason for the rescue from mAMSA killing of at least S-phase cells by novo or 2,4-dinitrophenol is their ability transiently to inhibit replicative DNA synthesis.

2,4-Dinitrophenol↗
PubMed1990

The "recall effect" in radiotherapy: is subeffective, reparable damage involved?

It has been proposed that lethal mutations among the progeny of a surviving cell could be the basis for the recall effect when chemotherapy is applied subsequent to the repair of normal-tissue injury resulting from a course of radiation therapy. Because radiotherapy is usually multifractionated, the possibility exists that repair of heritable injury of this type could occur between fractions as is the case for sublethal damage. To examine this possibility, the endpoint small-colony formation was used--an endpoint which integrates the effects of a number of radiation-induced aberrancies including lethal mutations--and low-dose-rate irradiation. It was found that, even after net surviving fractions comparable to those sought in radiotherapy were reached, little damage remained expressible as a deficiency in the size of the colony generated from a surviving cell. We conclude that the damage expressible as a lethal mutation is reparable and therefore the recall effect must be attributed to some other cellular mechanism.

Animals↗
PubMed1990

Additivity of cytotoxic damage due to dimethylbenz(a)anthracene and X rays.

7,12-Dimethylbenz(a)anthracene is one of a group of polycyclic aromatic hydrocarbons that are known to be indirectly acting carcinogens. As a product of the incomplete combustion of complex hydrocarbons, dimethylbenzanthracene is present in the environment and may therefore act on living systems in conjunction with ionizing radiation. We have studied the cytotoxic effects of dimethylbenzanthracene by itself, together with other polycyclic aromatic hydrocarbons, and combined with X radiation. Pre- or postirradiation treatment of mouse C3H 10T1/2 cells with dimethylbenzanthracene progressively removes the shoulder of the X-ray survival curve and, consistent with that observation, the survival sparing from dose fractionation is progressively lost. The cotreatment of cells with 3-methylcholanthrene and dimethylbenzanthracene largely abrogates the killing due to the latter compound alone and, accordingly, returns the shoulder to the survival curve. The application of dimethylbenzanthracene, or similar compounds, between X-ray dose fractions, separated by 4 h, is without effect quite likely because of the need for metabolic activation of the compound for effectiveness. Dimethylbenzanthracene is believed to be genotoxic because, after it is activated, it forms bulky adducts with DNA. Hence these results suggest that bulky adducts are a form of DNA damage operationally equivalent to sublethal X-ray damage.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1990

Cellular NAD+ and ATP levels in alkylation-induced cytotoxicity enhanced by an inhibitor of poly(ADP-ribose) synthesis.

Alkylating agents cause a marked depletion of cellular NAD+ levels by activating nuclear ADP-ribosyl transferase (ADPRT), which utilizes NAD+ as a substrate in the synthesis of poly(ADP-ribose). As a consequence of NAD+ depletion, it is possible that cellular ATP pools could be depleted. Because of this, exogenously supplied NAD+ had been proposed as a way to counteract some of the effects of an alkylator. We found that exogenously supplied NAD+ significantly increased intracellular levels of NAD+ in MMS- and MNNG-treated V79 Chinese hamster cells. Cytotoxicity was not changed by the exogenously supplied NAD+, however. 3-Aminobenzamide (3-ABA), an ADPRT inhibitor, prevented the depletion of intracellular NAD+ by MMS or MNNG treatment and potentiated cytotoxicity. As was the case without 3-ABA, exogenously supplied NAD+ plus 3-ABA did not change the cytotoxicity, even though NAD+ levels were increased. Intracellular ATP levels were also measured and were found to be unaffected following MMS treatment, and only slightly depleted following MNNG treatment. Exogenously supplied NAD+ raised these levels above those for their respective controls. Because survival was unaffected by elevated levels of NAD+ and ATP, our results suggest that depletion of cellular NAD+ pools following MMS and MNNG treatment is not a critical factor in determining cytotoxicity for these V79 cells. The energy reserves of V79 cells, at doses of MMS or MNNG which kill 99% of the cells, are apparently adequate to maintain normal levels of ATP.

Adenosine Triphosphate↗
PubMed1990

Novobiocin inhibits the repair of potentially lethal damage but not the repair of sublethal damage.

Because of the critical role of the DNA topoisomerases in the synthesis and conformation of DNA, and the well-known observation that radiation inhibits replicative DNA synthesis, we have examined the possibility that inhibitors of these enzymes might influence radiation lethality. In particular, using protocols involving the administration of either fresh or conditioned medium, we examined the ability of intercalative and nonintercalative inhibitors to affect the expression of potentially lethal damage and/or sublethal damage. The inhibitors examined were amsacrine, teniposide, etoposide, and novobiocin; only the latter compound was clearly effective in a selective way at nontoxic concentrations, and this was observed specifically in reference to the repair of potentially lethal damage effected by incubation in conditioned medium. These results are another example of differences between the repair of sublethal versus potentially lethal damage that further support distinctions between the two. At a mechanistic level, these and other data suggest that the property of novobiocin that is relevant in the foregoing is its metabolic inhibition of replicative DNA synthesis, a process which may be more important in the repair of potentially lethal damage as opposed to sublethal damage.

Amsacrine↗
PubMed1989

Fixation and repair of radiation-induced potentially mutagenic damage sensitive to hypertonic treatment in human diploid fibroblasts.

The effect of hypertonic salt treatment on the repair of potentially lethal damage and potentially mutagenic damage in X-irradiated asynchronous and synchronous human diploid fibroblasts (IMR91) have been studied. Resistance to 6-thioguanine was used for the mutagenic end point. When cells in late-S-phase were treated with hypertonic salt solution immediately after X-irradiation, both cell killing and mutation induction were enhanced, as compared to X-irradiation alone. This suggests that X-irradiation of cells in late S phase induces both potentially lethal damage and potentially mutagenic damage and that both are sensitive to hypertonic salt solution. When cells were allowed 2 h for repair after exposure to X-rays, both types of damage were completely repaired. Almost the same results were obtained with asynchronous cells. These results are discussed in terms of the relationship between radiation damage leading to cell lethality and mutagenesis.

Aphidicolin↗