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Biomedical subjects

J E Cleaver

Publications and source records attributed to J E Cleaver.

At least 109 records · Page 6Linked to original sources
PubMed1986

Antagonistic action of a tumor promoter and a poly(adenosine diphosphoribose) synthesis inhibitor in radiation-induced transformation in vitro.

Transformation of mouse C3H 10T1/2 cells by X-irradiation in vitro was blocked by the addition of 1 mM 3-aminobenzamide, an inhibitor of polyadenosine diphosphoribose (poly[ADP-ribose]) synthesis immediately after irradiation. 3-Aminobenzamide also inhibited an increase in the frequency of transformants caused by the addition of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, 7 days after irradiation. These results demonstrate a role for poly(ADP-ribose) synthesis during the initiation and promotion stages of transformation. From previous studies it is known that poly(ADP-ribose) synthesis is stimulated by the DNA damage caused by X rays during initiation. During promotion, however, 12-O-tetradecanoylphorbol-13-acetate acted as a mitogen but did not induce detectable DNA damage, and we could detect no stimulation of poly(ADP-ribose) synthetase. The roles of poly(ADP-ribose) during initiation and during promotion must, therefore, be significantly different.

Animals↗
PubMed1986

DNA repair and replication in xeroderma pigmentosum and related disorders.

Xeroderma pigmentosum (XP), ataxia telangiectasia (AT), and Cockayne syndrome (CS) are human diseases that exhibit increased sensitivity to environmental carcinogens [e.g., ultraviolet (UV) light, ionizing radiations, chemicals] because of genetic defects in the patient's capacity to repair and replicate damaged DNA accurately. The major defect in XP is a failure to repair UV damage to DNA; in AT, the failure is in repair or replication of double-strand breaks in DNA; in CS, the failure is in recovery of DNA replication after UV irradiation. Cancer is a major clinical feature of XP and AT, but not of CS. Each disease is complex, with multiple groups defined by complementation in cell-cell hybridization. Overlap is reported between some XP and CS groups. UV-sensitive hamster cell mutants are also known: most of these complement XP groups, and a human gene on chromosome 19 can correct the defects in hamster mutants, but not XP. XP group C is distinct from the other groups in exhibiting a strongly clustered mode of repair, as if only certain regions of the genome can be mended. This mode mainly occurs in confluent group C cells under conditions that permit much greater survival than in exponential growth, and therefore represents a more efficient mode of repair. These diseases all represent important examples of perturbation in the way carcinogen damage in DNA is metabolized, and further research aimed at identifying the kinds of molecular changes involved in the malignancy will be important.

Ataxia Telangiectasia↗
PubMed1986

Direct visualization of the sites of DNA methylation in human, and mosquito chromosomes.

Human and mosquito fixed chromosomes were digested with restriction endonucleases that are inhibited by the presence of 5-methylcytosine in their restriction sites (Hha I, Hin PI, Hpa II), and with endonucleases for which cleavage is less dependent on the state of methylation (Taq I, Msp I). Methylation-dependent enzymes extracted low DNA amounts from human chromosomes, while methylation-independent enzymes extracted moderate to high amounts of DNA. After DNA demethylation with 5-azacytidine the isoschizomers Hpa II (methylation-dependent) and Msp I (methylation-independent) extracted 12-fold and 1.4-fold amounts of DNA from human chromosomes, respectively. These findings indicate that human DNA has a high concentration of Hpa II and Msp I restriction sites (CCGG), and that the internal C of this sequence is methylated in most cases, while the external cytosine is methylated less often. All the enzymes tested released moderate amounts of DNA from mosquito chromosomes whether or not the DNA was demethylated with 5-azacytidine. Hpa II induced banding in the centromere chromosome regions. After demethylation with 5-azacytidine this banding disappeared. Mosquito DNA has therefore, moderate to high frequencies of nonmethylated CpG duplets. The only exception is the centromeric DNA, in which the high levels of C methylation present produce cleavage by Hpa II and the appearance of banding. Centromere regions of human chromosomes 1 have a moderately low concentration of Hpa II-Msp I restriction sites.

5-Methylcytosine↗
PubMed1986

Disturbances in DNA precursor metabolism associated with exposure to an inhibitor of poly(ADP-ribose) synthetase.

3-Aminobenzamide (3AB) is widely used as an inhibitor of poly(ADP-ribose) synthetase to study the effect of protein ribosylation on various cellular processes, but the specificity of its inhibition has not been demonstrated. We found that 3AB has a wide range of effects on DNA precursor metabolism, as determined by high-performance liquid chromatographic separation of deoxynucleosides derived from enzymatic digestion of cellular DNA. 3AB (10-20 mM) significantly reduced cell growth in human lymphoblastoid cells. Furthermore, the incorporation of [3H]deoxycytidine into DNA was significantly enhanced relative to incorporation of [3H]deoxythymidine, [3H]deoxyguanosine, and [3H]deoxyadenosine. Incorporation of fragments of [3H]glucose into the pyrimidine fraction of DNA was significantly inhibited relative to incorporation into the purine fraction. At only 1 mM, 3AB had a major inhibitory effect on the incorporation of the methyl group from [3H]methionine into deoxyguanosine, deoxyadenosine, and deoxycytidine, with 50% inhibition into deoxyguanosine and deoxyadenosine and 90% inhibition into deoxycytidine. The specificity of 3AB inhibition to poly(ADP-ribose) synthetase is therefore doubtful in view of this variety of metabolic effects, involving pyrimidine synthesis and de novo synthesis via the one-carbon pool.

Benzamides↗
PubMed1986

Enhanced ligation of repair sites under conditions of inhibition of poly(ADP-ribose) synthesis by 3-aminobenzamide.

The rate of intracellular ligation of excision-repair patches has been measured under conditions of inhibition of poly(ADP-ribose) synthesis by 3-aminobenzamide. Excision-repair patches in DNA of cells damaged by methyl methanesulfonate were labeled with [3H]thymidine and blocked at an intermediate stage by aphidicolin, an inhibitor of DNA polymerase alpha. Removal of [3H]thymidine and aphidicolin permitted the intracellular ligation rate to be determined by rapid digestion of [3H]-labeled 3' termini with exonuclease III. Contrary to previous conclusions from more indirect experiments, inhibition of poly(ADP-ribose) synthesis by 3-aminobenzamide actually facilitates rapid ligation.

Aphidicolin↗
PubMed1986

Evaluation of homology between cloned Escherichia coli and yeast DNA photolyase genes and higher eukaryotic genomes.

Repair of ultraviolet-induced pyrimidine dimers by photoreactivation is catalyzed by a single enzyme, DNA photolyase. However, the process of photoreactivation is difficult to detect reproducibly in cultured mammalian cells. We have used clones containing yeast and Escherichia coli DNA photolyase genes to determine whether their sequences are conserved and whether there is homology between either cloned sequence and chick or human genomic DNA and mRNA sequences. The cloned sequences failed to hybridize to each other even under nonstringent conditions, indicating little conservation of sequence between the yeast and E. coli genes. Furthermore, only weak hybridization under nonstringent conditions was found between the cloned photoreactivating genes and human or chick genomic DNA or mRNA. This indicates that there is negligible homology between the cloned probes and mammalian DNA, but we are unable to conclude whether this indicates sequence divergence for prokaryotic and eukaryotic photoreactivation genes or the absence of such genes from the mammalian genome.

Animals↗
PubMed1986

Excision repair in xeroderma pigmentosum group C but not group D is clustered in a small fraction of the total genome.

DNA repair in xeroderma pigmentosum complementation groups C and D occurs at a low level. Measurements of pyrimidine dimers remaining in bulk DNA from the whole genome indicated very little excision in either complementation group. The repair sites in group C cells were, however, clustered together in small regions of the genome which appeared to be mended nearly as efficiently as the whole genome is mended in normal cells, while repair in group D cells was randomly distributed. Growth of normal cells in cycloheximide or 3-aminobenzamide neither inhibited repair nor altered the distribution of repair sites. Growth of normal cells in novobiocin or aphidicolin inhibited excision but repair remained randomly distributed. On the basis of these observations, and consideration of other cellular features of group C and D, we suggest that group C may represent a mutation which results in a low level of repair enzymes with normal function. Group D, on the other hand, may represent a mutation resulting in functionally defective repair enzymes.

Aphidicolin↗
PubMed1986

Some protease inhibitors are also inhibitors of poly(ADP-ribose) polymerase.

The low-molecular-weight peptide protease inhibitors, tosyl-lysine-chloromethyl ketone, antipain and leupeptin, inhibited poly(ADP-ribose) [poly(ADP-Rib)] polymerase in permeable cells. The concentrations required for 50% inhibition were 3.6, 5 and 29 mM, respectively. Two peptides without protease inhibitor activity, fibrinopeptide A and phenylalanine-leucine-(glutamine)2-leucine, also inhibited poly (ADP-Rib) synthesis; doses required for 50% inhibition were 0.37 and 11.2 mM, respectively. These concentrations lie within a range bracketed by the 50% inhibition concentrations of the strong and weak poly(ADP-Rib) synthesis inhibitors, 3-amino-benzamide (0.15 mM) and caffeine (greater than 100 mM), respectively. N-Ethylmaleimide also inhibited poly(ADP-Rib) synthesis, at a 50% inhibitory dose of 0.3 mM, in the absence of exogenous thiol reagents. High-molecular-weight protease inhibitors, such as soybean (including Bowman-Birk reagent) and lima bean trypsin inhibitors and human alpha 1-protease inhibitor, had no effect on poly(ADP-Rib) synthesis up to 2 mg/ml. Interference with transformation and other cellular effects that have been reported in carcinogen-damaged cells treated with low-molecular-weight peptide protease inhibitors may therefore involve common mechanisms with poly(ADP-Rib) inhibitors. Similar effects of high-molecular-weight protease inhibitors presumably involve different mechanisms.

Antipain↗
PubMed1986

DNA ligation and changes in chromatin structure associated with repair patches under conditions of inhibition of poly(ADP-ribose) synthesis.

The rate of intracellular ligation of excision repair patches has been measured under conditions of inhibition of poly(ADP-ribose) synthesis by 3-aminobenzamide. Excision repair patches in DNA of cells damaged by methyl methanesulfonate were labeled with [3H]thymidine and blocked at an intermediate stage by aphidicolin, an inhibitor of DNA polymerase alpha. Nearly half of the [3H]thymidine label in the repair patches was sensitive to rapid digestion by exonuclease III, indicating that the label was at unligated 3' termini of repair sites. Removal of [3H]thymidine and aphidicolin permitted the intracellular ligation rate to be determined. From analysis of chromatin, ligation appeared to occur rapidly, independent of the effect of 3-aminobenzamide. Analysis of purified DNA, however, indicated that high doses of methyl methanesulfonate resulted in slow ligation rates but that 3-aminobenzamide accelerated the rates of ligation. The analysis of chromatin, therefore, indicates that unligated repair sites are sites of protein accretion which block exonuclease III action. The results from analysis of DNA indicate that poly(ADP-ribose) synthesis and associated pool depletion inhibits ligation rates; 3-aminobenzamide prevents poly(ADP-ribose) synthesis, maintains pool levels high and facilitates rapid ligation.

Aphidicolin↗
PubMed1986

Mutation frequencies from X-rays, ultraviolet light, and methyl methanesulfonate in Chinese hamster ovary cells incubated with 3-aminobenzamide.

An inhibitor of polyadenosine diphosphoribose polymerase, 3-aminobenzamide, had no effect on survival or mutagenesis in Chinese hamster ovary cells exposed to X-rays or u.v. light. After exposure to methyl methanesulfonate, 3-aminobenzamide increased cell killing and mutagenesis at the 6-thio-guanine-resistance locus, but had no effect on mutagenesis at the ouabain-resistance locus. These results are consistent with the greater role played by polyadenosine diphosphoribose in cells damaged by alkylating agents rather than by radiations.

Animals↗
PubMed1986

Repair-deficient xeroderma pigmentosum cells made UV light resistant by fusion with X-ray-inactivated Chinese hamster cells.

Xeroderma pigmentosum (XP) is an autosomal recessive human disease, characterized by an extreme sensitivity to sunlight, caused by the inability of cells to repair UV light-induced damage to DNA. Cell fusion was used to transfer fragments of Chinese hamster ovary (CHO) chromosomes into XP cells. The hybrid cells exhibited UV resistance and DNA repair characteristics comparable to those expressed by CHO cells, and their DNA had greater homology with CHO DNA than did the DNA from XP cells. Control experiments consisted of fusion of irradiated and unirradiated XP cells and repeated exposure of unfused XP cells to UV doses used for hybrid selection. These treatments did not result in an increase in UV resistance, repair capability, or homology with CHO DNA. The hybrid cell lines do not, therefore, appear to be XP revertants. The establishment of these stable hybrid cell lines is an initial step toward identifying and cloning CHO DNA repair genes that complement the XP defect in human cells. The method should also be applicable to cloning genes for other diseases, such as ataxia-telangiectasia and Fanconi's anemia.

Animals↗
PubMed1985

Chromatin dynamics. Fast and slow modes of nucleosome movement revealed through psoralen binding and repair.

Psoralen adducts, when formed in DNA at low frequencies that permit extensive survival of normal and repair-deficient cells, are found in both linker and core regions of nucleosomes, but are slightly enriched in the linker sites. The relative frequencies of adducts obtained with 5-methylisopsoralen and angelicin, which form only monoadducts, and 8-methoxypsoralen and trimethylpsoralen, which form monoadducts and cross-links, represent an enrichment in linker DNA that is approx. 2-3-fold higher per nucleotide than in core DNA. 5-Methylisopsoralen monoadducts, which are initially in linker DNA, become randomized during 12 h of growth. This suggests a slow lateral movement of nucleosomes with respect to DNA and implies that linker and core regions of DNA are not permanent assignments. Randomization of 5-methylisopsoralen adducts is independent of the synthesis of DNA, RNA, protein, or poly(ADP-ribose) and is also independent of DNA repair. Excision repair of these adducts, in contrast, causes rapid local changes in nucleosome conformation and an initial increase in staphylococcal nuclease sensitivity that reverts to the sensitivity of bulk chromatin in less than 1 h. Chromatin, therefore, can undergo at least two distinct dynamic changes under physiological conditions: a slow randomization of the nucleosomes with respect to DNA, and a rapid but transient local rearrangement to facilitate repair.

Cell Nucleus↗
PubMed1985

The pattern of restriction enzyme-induced banding in the chromosomes of chimpanzee, gorilla, and orangutan and its evolutionary significance.

The pattern of banding induced by five restriction enzymes in the chromosome complement of chimpanzee, gorilla, and orangutan is described and compared with that of humans. The G banding pattern induced by Hae III was the only feature common to the four species. Although hominid species show almost complete chromosomal homology, the restriction enzyme C banding pattern differed among the species studied. Hinf I did not induce banding in chimpanzee chromosomes, and Rsa I did not elicit banding in chimpanzee and orangutan chromosomes. Equivalent amounts of similar satellite DNA fractions located in homologous chromosomes from different species or in nonhomologous chromosomes from the same species showed different banding patterns with identical restriction enzymes. The great variability in frequency of restriction sites observed between homologous chromosome regions may have resulted from the divergence of primordial sequences changing the frequency of restriction sites for each species and for each chromosomal pair. A total of 30 patterns of banding were found informative for analysis of the hominid genealogical tree. Using the principle of maximum parsimony, our data support a branching order in which the chimpanzee is more closely related to the gorilla than to the human.

Animals↗
PubMed1985

Single-strand breaks in DNA of hamster and human cells exposed to methyl methanesulfonate and ultraviolet light.

The number of single-strand breaks produced in DNA after exposure to UV light or to methyl methanesulfonate (MMS) was additive when cells were exposed to both agents in close succession. Repair of the damage from either agent was partially inhibited by cytosine arabinoside, resulting in higher break frequencies under all conditions of exposure. Exposure to both agents followed by growth in cytosine arabinoside resulted in break frequencies that were approximately the same as the sum of those from each agent individually. These findings contrast with previous results in which pyrimidine dimer excision and repair replication after exposure to UV light were inhibited by MMS. These observations are not due to cell permeability changes after alkylation, but can be explained if the complex of excision-repair proteins is only partially inactivated by alkylation. Initial incisions to start repair would still occur but only limited amounts of repair replication would ensue without actual removal of the pyrimidine dimers.

Animals↗
PubMed1985

Relationship of ultraviolet light-induced DNA-protein cross-linkage to chromatin structure.

The production of banding patterns in metaphase chromosomes by restriction enzymes is inhibited by ultraviolet (UV) irradiation. Irradiation of fixed chromatin produces a 15-fold decrease in DNA extraction by restriction enzymes in comparison with that observed by irradiation before fixation. Alcohol-acid fixation of chromatin produces two major changes, the extraction of histones and dehydration. The effect of UV light is probably the result of a net increase in the yield of DNA-protein cross-links at comparable fluences of UV light and of the stabilization of the structural changes in the fixed chromatin fibril induced by the photoadducts. The X-irradiation of cells before fixation, as well as the rehydration of fixed chromatin, increases the extraction of DNA from fixed chromatin irradiated with UV light to levels similar to or even higher than those obtained with living cells. The effect of UV light before and after fixation on the extraction of DNA by restriction enzymes and proteinase K can be related to changes in chromatin structure and DNA conformation.

Animals↗