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C L Limoli

Publications and source records attributed to C L Limoli.

31 records · Page 2Linked to original sources
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↗
PubMed1997

Photochemical production of uracil quantified in bromodeoxyuridine-substituted SV40 DNA by uracil DNA glycosylase and a lysyl-tyrosyl-lysine tripeptide.

Exposure to UVA radiation of SV40 DNA substituted with bromodeoxyuridine (BrdU) in the presence of Hoechst dye 33258 results in the production of uracil. The yield of uracil was determined by measuring the increase in the single-strand break (SSB) yield after incubation of the photolyzed DNA with uracil-DNA glycosylase (UDG) in the presence of the tripeptide lysyl-tyrosyl-lysine (KYK). UDG removes uracil to leave an abasic site which is then cleaved to a SSB by KYK. The SSB yield was quantified by digital video imaging of ethidium fluorescence after separation of the I, II and III forms of SV40 DNA by agarose gel electrophoresis. Uracil is not detected when photolysis is carried out in the absence of the dye nor when unsubstituted DNA is used as the substrate. Without UDG or KYK treatment, the F0 for the loss of form I DNA is 100 J/m2. This falls to 13 J/m2 after incubation with UDG and KYK, indicating that uracil formation is approximately 5-fold more efficient than SSB formation. Formation of uracil suggests a mechanism for the high cellular toxicity of the dye-BrdU-UVA treatment.

Bromodeoxyuridine↗
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↗
PubMed1995

Mechanisms of radiosensitization in iododeoxyuridine-substituted cells.

The radiosensitization caused by iododeoxyuridine (IdU)-substitution of thymidine in V79-171 cells is decreased by the presence of acetone during irradiation. Acetone, at 1 mol dm-3, removes almost all the increase in double strand breaks (dsbs) caused by IdU substitution, but removes only about two-thirds of the enhancement in killing. Similar observations were made with BrdU-substituted cells. The decrease in cell radiosensitization coincides with the removal of the additional dsbs. The protection afforded by acetone is assumed to be due to its scavenging of hydrated electrons, thought to be the active species causing enhanced DNA damage in the presence of halogenated pyrimidines. The residual component of IdU radiosensitization, which could not be removed by treatment with acetone, is manifest largely as a shoulder effect (Dq) and may be due to either a subset of non-scavengable, lethal dsbs and/or the influence of IdU on the fixation of potentially lethal damage. This study further demonstrates that halogenated pyrimidine-mediated radiosensitization consists of at least distinct components each associated with a different phenomenon.

Acetone↗
PubMed1995

Photochemical production of double-strand breaks in cellular DNA.

In a recent publication we described a novel route for the introduction of DNA double-strand breaks (DSBs) into cellular DNA. This involved the labelling of cellular DNA with bromodeoxyuridine (BrdU) and exposure to UVA light in the presence of Hoechst dye No. 33258. Here, we report an extension of that work to the use of iododeoxyuridine (IdU); cells substituted with known levels of IdU were subjected to a similar photolysis treatment and analyzed for strand breaks by elution assays. Results indicate that both single-strand breaks (SSBs) and DSBs depend linearly on the level of IdU substitution and fluence of UVA light. The yields of SSBs and DSBs were found to be 3.5 x 10(-5) and 9.5 x 10(-7)/IdU moiety/kJm-2, respectively. These results indicate that approximately 15-fold less SSBs and 5-fold less DSBs are produced per IdU than per BrdU moiety.

Animals↗
PubMed1994

Response of bromodeoxyuridine-substituted Chinese hamster cells to UVA light exposure in the presence of Hoechst dye #33258: survival and DNA repair studies.

Previous work has established that DNA double-strand breaks (DSBs) are formed when Chinese hamster cells are substituted with 5-bromo-2'-deoxyuridine (BrdU) and exposed to UVA light in the presence of Hoechst dye #33258. Double-strand breaks produced by this treatment (5.1 x 10(-6) DSBs/BrdU residue/kJ m-2) were found to depend linearly on the level of BrdU substitution, Hoechst dye and fluence of UVA light. To examine the biological consequences of these novel DSBs, clonogenic assays were used to score cell survival, and elution assays were used to measure strand break levels at various times after photolysis. Using this system, marked cell killing was observed; photosensitivity could be increased by four orders of magnitude compared to cells without BrdU and dye. Decreases in the F0 value and the shoulder of survival curves followed increasing levels of BrdU substitution. In addition, the results indicate that DSBs produced by this photolysis protocol are two to three times more effective in causing cell killing than the DSBs produced by the action of ionizing radiation. To investigate the cause of the toxicity, repair of DSBs after photolysis was measured. Unexpectedly, DSB levels increased two- to threefold over 1 h at 37 degrees C, then decreased to initial damage levels over the next 2 h. The implications of this break induction are discussed in terms of mechanism and cell killing.

Animals↗
PubMed1994

DNA damage in bromodeoxyuridine substituted SV40 DNA and minichromosomes following UVA irradiation in the presence of Hoechst dye 33258.

Bromodeoxyuridine-substituted SV40 DNA and SV40 minichromosomes were prepared from infected CV1 cells, and exposed to UVA light in the presence of different concentrations of Hoechst dye 33258. Following agarose gel electrophoresis, the yields of ssbs and dsbs were determined. In DNA these yields are dependent on the level of BrdU substitution (0.038 ssb and 0.00022 dsb per BrdU residue/kJm-2), and on the mode of dye binding (type I versus II). The yields of both ssbs and dsbs were found to be lower by factors of 1.25 and 2 respectively in minichromosomes. UVA irradiation in the presence of the thiol cysteamine results in lower strand break yields, presumably due to the repair of DNA radicals formed during photolysis. The thiol was found to react seven times more slowly with the DNA radicals than oxygen. The implications of these results are discussed in light of prior cellular studies.

Bisbenzimidazole↗
PubMed1993

A new method for introducing double-strand breaks into cellular DNA.

A novel method is used to introduce double-strand breaks into cellular DNA containing controlled levels of 5-bromo-2'-deoxyuridine (BrdU). Chinese hamster V79 cells substituted with BrdU are treated with Hoechst dye #33258 and then exposed to UVA light. Using neutral elution (pH 7.2) the yield of DNA double-strand breaks is found to be linearly dependent on the level of BrdU substitution (0.36-7.5%), concentration of Hoechst dye (0-100 micrograms cm-3), and fluence of UVA light (0.2-8 kJ m-2). The yield of double-strand breaks produced by this photolysis treatment is 5.1 x 10(-6) breaks/BrdU residue/kJ m-2, regardless of whether one or both strands of the DNA polymer contain BrdU. No double-strand breaks are detected in the absence of Hoechst dye, BrdU, or UVA light. The formation of double-strand breaks appears to involve strand cleavage at a BrdU site on one strand with cleavage in the opposite strand not necessarily requiring the presence of BrdU. The utility of this photolytic regimen in modeling the biological significance of double-strand break lesions and some putative mechanisms for their formation are discussed.

Animals↗
PubMed1991

An examination of the repair saturation hypothesis for describing shouldered survival curves.

The hypothesis that after irradiation a competition exists between fixation of radiation damage and its repair and that this competition determines cell survival was to be tested. Postirradiation temperature of holding was employed as a means of modulating rate of damage repair, and the postirradiation rates of repair of DNA strand breaks (both single and double) were monitored using elution assays. At temperatures below 37 degrees C following irradiation the rates of rejoining were decreased markedly, although rejoining of single-strand breaks was seen even at 10 degrees C and rejoining of double-strand breaks still occurred at 16 degrees C. However, 3 h incubation of cells at these lowered temperatures had no observable effect on cell survival parameters. It is concluded that either damage fixation and damage repair have the same dependence on temperature, or simple measurements of rejoining of breaks are insufficient to detect the details of the competition between repair and fixation (some measure of fidelity of repair is needed).

Animals↗
PubMedDate not supplied

DNA replication in the face of (In)surmountable odds.

We describe here a model for sequential recruitment of various enzymatic systems that maintain DNA replication fidelity in cells with damaged bases, especially those formed by ultraviolet (UV) irradiation. Systems of increasing complexity but decreasing fidelity are recruited to restore replication of damaged DNA. The first and most accurate response is nucleotide excision repair (NER) that is cell cycle-independent; next come various delaying cell cycle checkpoints that provide an extended time window for NER. These delay the onset of the S phase at the G1/S boundary, and inhibit the initiation of individual replicating units (replicons and clusters of replicons) within the S phase. When checkpoints fail to operate completely, DNA replication forks must negotiate damage and the loss of coding information on the parental DNA strands. Replication can be resumed using bypass polymerases, or alternative bypass mechanisms. Finally, if all else fails, replication forks may degrade to double strand breaks and recombinational processes then allow their reconstruction. A network of signaling kinases modulates the efficiency of many damage responsive proteins to tailor their activities and subcellular localizations by phosphorylation and dephosphorylation.

Animals↗