Biomedical subjects

M I Kaplan

Publications and source records attributed to M I Kaplan.

11 recordsLinked to original sources

PubMed2001

Attenuation of radiation-induced genomic instability by free radical scavengers and cellular proliferation.

To investigate the mechanisms of radiation-induced chromosomal instability, cells were irradiated in the presence of the free radical scavengers DMSO, glycerol, or cysteamine, in the presence of DMSO while frozen, or held in confluence arrest post-irradiation to permit cells to repair potentially lethal DNA damage. Clones derived from single progenitor cells surviving each treatment were then analyzed for the subsequent development of chromosomal instability. The presence of scavengers (+/- freezing) during irradiation, and the recovery from potentially lethal damage after irradiation led to an increase in cell survival that was accompanied by a decrease in the initial yield of chromosomal rearrangements. Furthermore, analysis of over 400 clones and 80,000 metaphases indicates that these same treatments reduced the incidence of instability at equitoxic doses when compared to controls irradiated in the absence of scavengers at ambient temperature. Results suggest that preventing reactive species from damaging DNA, promoting chemical repair of ionized DNA intermediates, or allowing enzymatic removal of genetic lesions, represent measures that reduce the total burden of DNA damage and reduce the subsequent onset of radiation-induced genomic instability.

Animals↗

PubMed2000

Genomic instability induced by high and low LET ionizing radiation.

Genomic instability is the increased rate of acquisition of alterations in the mammalian genome, and includes such diverse biological endpoints as chromosomal destabilization, aneuploidy, micronucleus formation, sister chromatid exchange, gene mutation and amplification, variations in colony size, reduced plating efficiency, and cellular transformation. Because these multiple endpoints persist long after initial radiation exposure, genomic instability has been proposed to operate as a driving force contributing to genetic plasticity and carcinogenic potential. Many of these radiation-induced endpoints depend qualitatively and quantitatively on genetic background, dose and LET. Differences in the frequency and temporal expression of chromosomal instability depend on all three of the foregoing factors. On the other hand, many of these endpoints appear independent of dose and show bystander effects, implicating non-nuclear targets and epigenetic regulatory mechanisms. The present work will survey results concerning the LET dependence of genomic instability and the role of epigenetic mechanisms, with a particular emphasis on the endpoint of chromosomal instability.

Animals↗

PubMed1999

The DNA double-strand break repair gene hMRE11 is mutated in individuals with an ataxia-telangiectasia-like disorder.

We show that hypomorphic mutations in hMRE11, but not in ATM, are present in certain individuals with an ataxia-telangiectasia-like disorder (ATLD). The cellular features resulting from these hMRE11 mutations are similar to those seen in A-T as well as NBS and include hypersensitivity to ionizing radiation, radioresistant DNA synthesis, and abrogation of ATM-dependent events, such as the activation of Jun kinase following exposure to gamma irradiation. Although the mutant hMre11 proteins retain some ability to interact with hRad50 and Nbs1, formation of ionizing radiation-induced hMre11 and Nbs1 foci was absent in hMRE11 mutant cells. These data demonstrate that ATM and the hMre11/hRad50/Nbs1 protein complex act in the same DNA damage response pathway and link hMre11 to the complex pathology of A-T.

Acid Anhydride Hydrolases↗

PubMed1998

DNA double-strand breaks, chromosomal rearrangements, and genomic instability.

DNA double-strand breaks can lead to chromosomal rearrangements at the first mitosis after exposure to the DNA strand-breaking agent. The evidence suggests a number of different pathways for DNA double-strand break rejoining in mammalian cells, but it is unclear what factors determine the fate of the induced break and whether or not it will lead to chromosomal rearrangement. If a cell does survive and proliferate after DNA cleavage, delayed chromosomal instability can be observed in the clonal descendants of the exposed cell. Most, but not all DNA double-strand breaking agents are effective at inducing this delayed chromosomal instability. In this paper, we review the evidence for the role of the DNA double-strand break in directly induced and delayed chromosomal rearrangements.

Animals↗

PubMed1998

The evolution of chromosomal instability in Chinese hamster cells: a changing picture?

PURPOSE: To investigate the kinetics of chromosomal instability induced in clones of Chinese hamster cells following X-irradiation. MATERIALS AND METHODS: X-irradiated clones of GM10115, human-hamster hybrid cells containing a single human chromosome 4 (HC4), have been previously established. These clones were defined as unstable if they contained > or = three subpopulations of cells with unique rearrangements of HC4 as detected by FISH. Stable and unstable clones were analysed by FISH and Giemsa staining at various times post-irradiation. RESULTS: While most of the stable clones continued to show chromosomal stability of HC4 over time, one became marginally unstable at approximately 45 population doublings post-irradiation. Clones exhibiting chromosomal instability had one of several fates. Many of the unstable clones were showed similar levels of instability over time. However, one unstable clone became stable with time in culture, while another became even more unstable over time. Cytogenetic analyses of all clones after Giemsa staining indicated that in some clones the hamster chromosomes were rearranged independent of HC4, demonstrating increased frequencies of chromatid breaks and dicentric chromosomes. The majority of the unstable clones also had higher yields of chromatid gaps. CONCLUSIONS: These data demonstrate the dynamic nature of chromosomal instability as measured by two different cytogenetic assays.

Animals↗

PubMed1998

The nucleus is the target for radiation-induced chromosomal instability.

We have previously described chromosomal instability in cells of a human-hamster hybrid cell line after exposure to X rays. Chromosomal instability in these cells is characterized by the appearance of novel chromosomal rearrangements multiple generations after exposure to ionizing radiation. To identify the cellular target(s) for radiation-induced chromosomal instability, cells were treated with 125I-labeled compounds and frozen. Radioactive decays from 125I cause damage to the cell primarily at the site of their decay, and freezing the cells allows damage to accumulate in the absence of other cellular processes. We found that the decay of 125I-iododeoxyuridine, which is incorporated into the DNA, caused chromosomal instability. While cell killing and first-division chromosomal rearrangements increased with increasing numbers of 125I decays, the frequency of chromosomal instability was independent of dose. Chromosomal instability could also be induced from incorporation of 125I-iododeoxyuridine without freezing the cells for accumulation of decays. This indicates that DNA double-strand breaks in frozen cells resulting from 125I decays failed to lead to instability. Incorporation of an 125I-labeled protein (125I-succinyl-concanavalin A), which was internalized into the cell and/or bound to the plasma membrane, neither caused chromosomal instability nor potentiated chromosomal instability induced by 125I-iododeoxyuridine. These results show that the target for radiation-induced chromosomal instability in these cells is the nucleus.

Animals↗

PubMed1997

Chromosomal instability and its relationship to other end points of genomic instability.

Chromosomal destabilization is one end point of the more general phenomenon of genomic instability. We previously established that chromosomal instability can manifest in clones derived from single progenitor cells several generations after X-irradiation. To understand the potential relationship between chromosomal destabilization and the other end points of genomic instability, we generated a series of chromosomally stable and unstable clones by exposure to X-rays. All clones were derived from the human-hamster hybrid line GM10115, which contains a single copy of human chromosome 4 in a background of 20-24 hamster chromosomes. These clones were then subjected to a series of assays to determine whether chromosomal instability is associated with a general "mutator phenotype" and whether it modulates other end points of genomic instability. Thus, we analyzed clones for sister chromatid exchange, delayed reproductive cell death, delayed mutation, mismatch repair, and delayed gene amplification. Statistical analyses performed on each group of chromosomally stable and unstable clones indicated that, although individual clones within each group were significantly different from unirradiated clones for many of the end points, there was no significant correlation between chromosomal instability and sister chromatid exchange, delayed mutation, and mismatch repair. Delayed gene amplification was found to be marginally correlated to chromosomal instability (P < 0.1), and delayed reproductive cell death (the persistent reduction in plating efficiency after irradiation) was found to be significantly correlated (P < 0.05). These correlations may be explained by chromosomal destabilization, which can mediate gene amplification and can result in cellular lethality. These data implicate multiple molecular and genetic pathways leading to different manifestations of genomic instability in GM10115 cells surviving exposure to DNA-damaging agents.

Animals↗

PubMed1997

Differential induction of chromosomal instability by DNA strand-breaking agents.

To investigate the role of DNA strand breakage as the molecular lesion responsible for initiating genomic instability, five different strand-breaking agents, bleomycin, neocarzinostatin, hydrogen peroxide, restriction endonucleases, and ionizing radiation, were examined for their capacity to induce delayed chromosomal instability. These studies used GM10115 human-hamster hybrid cells, which contain one copy of human chromosome 4 in a background of 20-24 hamster chromosomes. Chromosomal instability was investigated using fluorescence in situ hybridization to visualize chromosomal rearrangements involving the human chromosome. Rearrangements are detected multiple generations after treatment, in clonal populations derived from single progenitor cells surviving treatment of the specified DNA-damaging agents. Clastogenic and cytotoxic activities of all agents were tested by examining chromosome aberration yields in first-division metaphases and by clonogenic survival assays. Analysis of over 250 individual clones representing over 50,000 metaphases demonstrates that when compared at comparable levels of cell kill, ionizing radiation, bleomycin, and neocarzinostatin are equally effective at eliciting delayed genomic instability. These observations document, for the first time, the persistent destabilization of chromosomes following chemical treatment. In contrast, the analysis of nearly 300 clones and 60,000 metaphases, involving treatment with four different restriction endonucleases and/or hydrogen peroxide, did not show any delayed chromosomal instability. These data indicate that DNA strand breakage per se does not necessarily lead to chromosomal instability but that the complexity or quality of DNA strand breaks are important in initiating this phenotype.

Adenine Phosphoribosyltransferase↗

PubMed1997

Perpetuating radiation-induced chromosomal instability.

Chromosomal instability is characterized by an increased rate of chromatid and chromosome rearrangements occurring multiple generations after exposure to ionizing radiation. Although chromosomal instability has been observed in many different cell types after exposure to ionizing radiation, the mechanisms perpetuating the manifestation of this instability phenotype are unknown. This review explores the current evidence concerning the role of dicentric-mediated bridge-breakage-refusion cycles and the role of recombination at interstitial telomere bands in mediating chromosomal instability. In addition, the contribution of genetic and epigenetic factors are discussed with regard to the predisposition of cells to chromosomal instability.

Animals↗

PubMed1996

Genomic instability induced by ionizing radiation.

Genomic instability is characterized by the increased rate of acquisition of alterations in the mammalian genome. These changes encompass a diverse set of biological end points including karyotypic abnormalities, gene mutation and amplification, cellular transformation, clonal heterogeneity and delayed reproductive cell death. The loss of stability of the genome is becoming accepted as one of the most important aspects of carcinogenesis, and the numerous genetic changes associated with the cancer cell implicate genomic stability as contributing to the neoplastic phenotype. Multiple metabolic pathways govern the accurate duplication and distribution of DNA to progeny cells; other pathways maintain the integrity of the information encoded by DNA and regulate the expression of genes during growth and development. For each of these functions, there is a normal baseline frequency at which errors occur, leading to spontaneous mutations and other genomic anomalies. This review summarizes the current status of knowledge about radiation-induced genomic instability. Those events and processes likely to be involved in the initiation and perpetuation of the unstable phenotype, the potential role of epigenetic factors in influencing the onset of genomic instability, and the delayed effects of cellular exposure to ionizing radiation are discussed.

Animals↗

PubMed1994

Stabilizing and destabilizing effects of placing beta-branched amino acids in protein alpha-helices.

In order to gain greater insight into the effects of beta-branched amino acids on protein alpha-helices, hydrophobic amino acids with varying degrees of beta-branching, including the fully beta-substituted L-2-amino-3,3-dimethylbutanoic acid (ADBA), were incorporated into the protein T4 lysozyme. The unnatural and natural amino acids were substituted at two solvent-exposed alpha-helical sites, Ser 44 and Asn 68, in the protein using the technique of unnatural amino acid mutagenesis. The stabilities of the mutant proteins were determined by using a heat of inactivation assay and from their circular dichroism thermal denaturation curves. Surprisingly, while substitution of the amino acid with the greatest degree of beta-branching, ADBA, destabilizes the protein by 2.5 +/- 0.1 degrees C (0.69 +/- 0.03 kcal/mol) relative to Ala at site 44, the same substitution stabilizes the protein by 1.0 +/- 0.1 degree C (0.27 +/- 0.03 kcal/mol) at site 68. The difference observed at these two positions illustrates the extent to which the local context can mediate the impact of a particular mutation. Molecular dynamics simulations were carried out in parallel to model the structures of the mutant proteins and to examine the energetic consequences of incorporating ADBA. Together, these results suggest that the conformationally restricted beta-branched amino acids are destabilizing, in part, because the beta-branched methyl groups can cause distortions in the local helix backbone. In addition, it is proposed that in some contexts the conformational rigidity of beta-branched amino acids may be stabilizing because it lowers the entropic cost of forming favorable side-chain van der Waals interactions.

Acetonitriles↗