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M M Elkind

Publications and source records attributed to M M Elkind.

At least 19 recordsLinked to original sources
PubMed2001

Requirement for repair of DNA double-strand breaks by homologous recombination in split-dose recovery.

Utsumi, H., Tano, K., Takata, M., Takeda, S. and Elkind, M. M. Requirement for Repair of DNA Double-Strand Breaks by Homologous Recombination in Split-Dose Recovery. Radiat. Res. 155, 680-686 (2001). Split-dose recovery has been observed under a variety of experimental conditions in many cell systems and is believed to be the result of the repair of sublethal damage. It is considered to be one of the most widespread and important cellular responses in clinical radiotherapy. To study the molecular mechanism(s) of this repair, we analyzed the knockout mutants KU70-/-, RAD54-/-, and KU70-/-/RAD54-/- of the chicken B-cell line, DT40. RAD54 participates in the recombinational repair of DNA double-strand breaks (DSBs), while members of the KU family of proteins are involved in nonhomologous end joining. Split-dose recovery was observed in the parent DT40 and the KU70-/- cells. Moreover, the split-dose survival enhancement had all of the characteristics demonstrated earlier for the repair of sublethal damage, e.g., the reappearance of the shoulder on the survival curve with dose fractionation; cyclic fluctuation in cell survival at 37 degrees C; repair and no cyclic fluctuation at 25 degrees C. These results strongly suggest that repair of sublethal damage is due to DSB repair mediated by homologous recombination, and that these DNA DSBs constitute sublethal damage.

Animals↗
PubMed2001

Cell cycle-dependent effects of wortmannin on radiation survival and mutation.

Wortmannin, a known radiation sensitizer, has been used in experiments with synchronized cells to compare its effect on radiation survival and mutation induction within the cell cycle. PL61 cells (CHO cells with an inactivated HPRT gene containing a single active copy of a bacterial gpt gene) were synchronized by mitotic selection. Wortmannin administered before gamma irradiation caused a greater sensitization in G(1)-phase cells relative to late S/G(2)-phase cells. Preferential radiosensitization of G(1)-phase cells by wortmannin sets a limit to the proposed use of wortmannin in radiation therapy, since, in contrast to normal tissues, tumors usually have high proportions of S-phase cells. Wortmannin increased mutation frequencies in both G(1)- and S/G(2)-phase cells. Interestingly, relative increases in radiation-induced mutations in G(1) and S/G(2) phases were comparable. The results are discussed in terms of the contributions of different repair modes in the production of mutations.

Androstadienes↗
PubMed1999

p53 gene mutations in neoplastic transformation of C3H 10T1/2 and severe combined immunodeficiency fibroblasts.

The relevance of p53 mutations to the neoplastic malignant transformation of rodent fibroblasts by genotoxic physical and chemical agents is not clear. In the present study, we investigated p53 mutations (in exons 5-8) in non-transformed and neoplastically transformed C3H 10T1/2 and severe combined immunodeficiency (SCID) cells. No p53 mutations were detected in 15 neoplastically transformed (two spontaneous, one 3-methylcholanthrene-induced, seven gamma-ray-induced and five 'hot particle'-induced) and two non-transformed 10T1/2 cells. Wild-type p53 gene was also detected in all non-transformed (immortalized) SCID cell lines analyzed (four lines) whereas all three neoplastically transformed (two spontaneous, one gamma-ray-induced) cell lines displayed missense mutations in the p53 gene. These mutations were all transitions: A > G in codon 123, G > A in codon 152, and C > T in codon 238. We conclude that mutation in the p53 gene appears to be an infrequent event in 10T1/2 cells regardless of the transforming agent, but a frequent event in the neoplastic transformation of immortalized SCID cells. Non-transformed SCID cells are deficient in repair of DNA double-strand breaks, and neoplastically transformed cells are assumed to be deficient as well.

Animals↗
PubMed1999

The neoplastic transformation of SCID cells by radiation.

Severe combined immunodeficiency (SCID) cells are hypersensitive to killing by ionizing radiation because of deregulation of DNA-dependent protein kinase (DNA-PK) and a concomitant deficiency in the repair of DNA double-strand breaks. The effect of this condition on the neoplastic transformation of SCID fibroblasts, designated SCID 3T1, has been investigated. The spontaneous transformation rate was approximately 2 x 10(-5) at early passages and increased up to approximately 7 x l0(-3) at later passages. The radiation survival curves of transformed cells had thresholds and therefore appeared to be qualitatively similar to the survival curves of C3H 10T(1/2) mouse fibroblast cells, but the initial slopes were steeper. In contrast, per unit dose, SCID cells were more sensitive to transformation than 10T(1/2) cells. Eight transformed clones were tested for tumorigenicity, and all produced fibrosarcomas in athymic nude mice. Properties associated with the tumor suppressor Trp53 (formerly known as p53) were examined in three of the clones. In these clones, although Trp53 protein was overexpressed, a lower expression of Cdkn1a (formerly known as p21, Cip1) protein was observed compared to parental cells. The expression of Trp53 and Cdkn1a and the G(1)-phase arrest (one set of data on G(1)-phase delay is included as an example) was not induced by ionizing radiation in these transformed clones; each clone carried a point mutation in Trp53. This suggests that the deficiency in the repair of DNA double-strand breaks increased the tumorigenicity and the genomic instability of transformed SCID cells.

Animals↗
PubMed1999

Wortmannin sensitizes mammalian cells to radiation by inhibiting the DNA-dependent protein kinase-mediated rejoining of double-strand breaks.

Wortmannin has been shown to be an efficient radiosensitizer. Since wortmannin is able to inhibit DNA-dependent protein kinase (DNA-PK) and double-strand break (DSB) rejoining, it is believed that its mechanism of radiation sensitization is through the inhibition of DNA-PK-mediated repair of DSBs. However, since wortmannin is not a specific inhibitor, the possibility that other kinases are inhibited and thereby may contribute to radiosensitization cannot be ruled out. Here we present data confirming the radiosensitizing effect of wortmannin on cells of different cell lines. In the same range of wortmannin concentrations, survival after exposure to ionizing radiation correlated well with DSB rejoining and the induction of micronuclei, suggesting that the inhibition of the processing of DSBs is involved in the sensitizing effect. Pretreatment with wortmannin enhanced the radiosensitivity of ataxia telangiectasia (AT) cells, thereby precluding the participation of ATM protein in the radiation sensitization by wortmannin. At the same time, irradiated DNA-PK-deficient cells were not significantly affected by pretreatment with wortmannin. These observations support a likely mechanism; that is, wortmannin sensitizes cells to radiation through inhibition of the DNA-PK-mediated rejoining of DSBs.

Androstadienes↗
PubMed1998

Phase transitions in the growth of C3H 10T1/2 cells.

In systems used to express transformation using focus formation as the end point, nontransformed cells generally express a down-regulation of cell growth and division made evident by the formation of a monolayer of cells that completely covers the growth surface. In C3H 10T1/2 cells, down-regulation is thought to be progressively effected principally by cell-to-cell communication via gap junctions. Starting with a sparse population in asynchronous growth--e.g. containing cells in all phases of the growth cycle--as the area density increases, cells are progressively lost from the distribution in the order M phase, G2 phase, S phase and G1 phase, leading to the accumulation of viable cells out of cycle in so-called G0 phase. We have measured the progressive phase transitions as a function of inoculum size and time. The influence of a promoter and an antipromoter was also examined as well as the expression of the cyclin/cyclin-dependent kinase inhibitors p21Waf1/Cip1 and p27Kip1 as the cells grew into confluence. Using cells synchronized in mitosis, we found that with increasing cell density the expression of p27 increased and concomitantly p21 decreased.

Animals↗
PubMed1997

Cell-cycle sensitivity, recovery from radiation damage and a new paradigm for risk assessment.

Tikvah Alper's interest in science was broad, from scrapie to mammalian cells and cancer. Much of her own work focused on cell lethality, like that of many other radiobiologists, but this was natural because of the simplicity of the endpoint cell survival and its relevance to cancer therapy. Tikvah had broader interests, however, that included the effects of radiation on living systems in general like the induction of cancer and the cellular and molecular processes contributing to it. In this essay, some ideas are developed that lie in the mainstream of her interests. Starting with functional measures of the recovery or repair from radiation damage, a role for repair is illustrated in connection with mutagenesis and neoplastic transformation both discussed in the context of radiation-induced cancer. These topics are central to a model explaining the anomalous enhanced neoplastic transformation and cancer observed when low doses of a high-LET radiation are protracted in time. Under particular circumstances, the formalism of the model predicts application to protracted low-LET exposures as in the instance of repair-deficient target cells and sporadic breast cancer. The latter discussion leads to the proposal that the paradigm in current use for evaluating cancer risk should be broadened: from a simple dose-effect relation to one that includes cell kinetics (during protracted exposures), cell-cycle dependencies, and the influence of cellular repair or the lack thereof.

Animals↗
PubMed1994

Enhanced cytochrome P450 (Cyp1b1) expression, aryl hydrocarbon hydroxylase activity, cytotoxicity, and transformation of C3H 10T1/2 cells by dimethylbenz(a)anthracene in conditioned medium.

The basal and benz(a)anthracene-induced aryl hydrocarbon hydroxylase activities of C3H 10T1/2 mouse embryo fibroblasts have been shown to vary with population growth. We report here that, in the case of dimethylbenz(a)anthracene, cytotoxicity and transformation (neoplastic/morphological transformation and focus formation) increased as a consequence of population growth and, at high cell densities, DNA adduct formation was elevated. Among the factors that may contribute to these changes, we have found that conditioning of the medium with population growth plays a significant role. Cells treated with medium conditioned by several days of cell growth supported increases in the dimethylbenz(a)anthracene induction of mRNA expression of a new mouse cytochrome P450 gene designated Cyp1b1, aryl hydrocarbon hydroxylase activity, cytotoxicity, and the frequency of neoplastic transformation. These results suggest a cause and effect relationship between the enhanced expression of Cyp1b1 due to medium conditioning and the enhanced expression of the cellular endpoints cytotoxicity and transformation.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1994

Inhibitors of poly (ADP-ribose) synthesis inhibit the two types of repair of potentially lethal damage.

PURPOSE: The purpose of this study was to examine whether 3-amino-benzamide (3ABA), an inhibitor of poly (ADP-ribose) synthesis, inhibits the two types of potentially lethal damage (PLD) repair, termed slow and fast. METHODS AND MATERIALS: The fast-type PLD repair was measured by the decrease in survival of V79 Chinese hamster cells by postirradiation treatment with 3ABA. The slow-type PLD repair was measured by the increase in survival by posttreatment with conditioned medium (CM), which became conditioned by growing a crowed culture of cells and supports the slow-type PLD repair. RESULTS: Up to 1 mM, 3-ABA inhibited the slow type repair; at doses of 2 mM and above, it inhibited the fast type of PLD repair. CONCLUSION: There are quantitative differences in cellular effects of 3ABA dependent on concentration. Poly (ADP-ribose) appears to play an important role in the PLD repairs and has little effect on the repair of sublethal damages.

Animals↗
PubMed1994

Radon-induced cancer: a cell-based model of tumorigenesis due to protracted exposures.

In 1982, results with C3H mouse embryo cells showed that the frequency of neoplastic transformation was enhanced when exposures to fission-spectrum neutrons were protracted in time. This finding was unexpected because the opposite was found with low-LET radiations. Similar neutron enhancements were reported with normal life-span Syrian hamster embryo cells, and with human hybrid cells. Because other studies did not confirm the preceding, in 1990--at a conference convened by the US Armed Forces Radiobiological Research Institute--a biophysical model was proposed to explain the basis for the enhancement observed in some experiments but not in others. The model attributed special sensitivities, related to killing and neoplastic transformation, to cells in and around mitosis. Subsequently, it was shown that late G2/M phase cells constituted this window of sensitivity. In the instance of tumorigenesis, the model predicted that protracted exposures to a high-LET radiation would result in enhanced frequencies of transformation providing that susceptible cells were cycling or could be induced to cycle. The model explained data on lung tumour induction in rats breathing radon at different concentrations, and uranium miners working in atmospheres containing different concentrations of radon. The model also explains the anomalous finding that lung cancer deaths are often sublinearly correlated with indoor radon concentration.

Animals↗
PubMed1994

cDNA cloning, sequence analysis, and induction by aryl hydrocarbons of a murine cytochrome P450 gene, Cyp1b1.

C3H mouse embryo fibroblast cells, designated 10T1/2, can be transformed by physical and chemical agents including polycyclic aromatic hydrocarbons. In a previous report (Shen et al., Proc. Natl. Acad. Sci. USA 90, 11483-11487, 1993), we identified a cytochrome P450 gene induced by polycyclic aromatic hydrocarbons (PAHs) that is different from 1A1 or 1A2, and which we tentatively named P450CMEF. Here, we report the entire cDNA sequence of P450CMEF (5,128 bp) and the amino acid sequence deduced from it (543 residues). A comparison of the latter sequence with known cytochrome P450s indicates that P450CMEF is in a new subfamily of family 1 of the P450 superfamily. Accordingly, the Committee on Standardized Cytochrome P450 Nomenclature designated the gene Cyp1b1. Exposure to various aryl hydrocarbons (2.5 hr) induced Cyp1b1 mRNA in 10T1/2 cells to different degrees: 2,3,7,8-tetrachlorodibenzo-p-dioxin, 7,12-dimethylbenz[a]anthracene, benz[a]anthracene, benzo[a]pyrene, and beta-naphthoflavone were strong inducers; alpha-naphthoflavone and 3-methylcholanthrene, were moderate inducers; and benzo[e]pyrene was a weak inducer.

Amino Acid Sequence↗
PubMed1994

Enhanced tumorigenesis by small, protracted doses of densely ionizing radiation.

Starting with observations that were first published in 1982, a series of additional findings led to the discovery of an important property of cells in late G2/mitosis. In addition to being the most sensitive to killing, cells in this age-interval were also shown to be the most sensitive to radiation-induced neoplastic transformation. In this work, C3H mouse cells, designated 10T1/2, were irradiated with fission-spectrum neutrons and assayed in vitro via the endpoint focus formation on a monolayer of normal cells. From these observations, a biophysical model was developed to explain the anomalous finding that the frequency of transformation by low doses was enhanced when the exposure was protracted. In contrast to transformation by X- and gamma-rays where repair and kinetics during exposure play dominant roles, with radiations like reactor neutrons and alpha-particles repair has a minimal effect; only cell kinetics acts significantly to modify transformation due to protracted doses. In this report, the lack of responsiveness of mitotic cells to promotion by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate is shown further to agree with the findings with fission-neutrons and the model that, at low dose rate, enhanced transformation is due principally to the progression of cells into the window of sensitivity. Implications of this model for lung cancer due to environmental radon are also discussed.

Animals↗
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↗
PubMed1993

Neoplastic transformation of C3H mouse embryo cells, 10T1/2: cell-cycle dependence for 50 kV X-rays and UV-B light.

The variation of neoplastic transformation induced by 50 kV X-rays, and by solar-simulating UV-B light, was studied through the cell cycle of C3H mouse embryo cells designated 10T1/2. A mitotic shake-off method was used to harvest mitotic cells. The progression through the cell cycle of initially mitotic cells was followed as a function of time by flow cytometry, DNA labelling for passage through S-phase, and growth curves for cell number. At 2-3 h after shake-off, about 90% of the cells were in early G1-phase and by 15 h 60-70% of cells had reached S-phase. For 2.5 Gy, the transformation frequency per viable cell in M-phase was some five times higher than in S-phase. In contrast, at similar survival levels, UV-B light is less efficient in transforming mitotic cells. For both types of radiation, the frequency of neoplastic transformation per viable cell was roughly inversely proportional to survival.

Animals↗