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Biomedical subjects

J E Cleaver

Publications and source records attributed to J E Cleaver.

At least 127 records · Page 7Linked to original sources
PubMed1985

Formation and repair of psoralen-DNA adducts and pyrimidine dimers in human DNA and chromatin.

DNA damage and repair in human cells exposed to ultraviolet light (254 nm) or to psoralen derivatives plus 360 nm light were compared by means of a variety of analytic techniques. The two kinds of damage show considerable structural similarity; both involve cyclobutyl bonds to 5,6 positions of pyrimidines as major products and have various minor products. In purified DNA, pyrimidine dimers, but not psoralen adducts, cause structural distortions that are substances for digestion with single-strand-specific nucleases. Whereas pyrimidine dimers are randomly produced in chromatin, psoralen adducts, are concentrated approximately 2- to 4-fold in linker regions of chromatin at doses that are not highly lethal. Chromatin shows considerable mobility; assignment of DNA to linker or core regions is not permanent, and psoralen adducts initially concentrated in linker regions become randomized after 10 hr. Pyrimidine dimers and psoralen adducts are excised by normal cells but not by repair-deficient xeroderma pigmentosum cells. This repair process requires DNA polymerase alpha, but its rate in ultraviolet-damaged cells is twice that in psoralen-damaged cells. Conversion of monoadducts to DNA-DNA crosslinks reduces the rate of repair because of the increased complexity of the damaged site.

Cell Line↗
PubMed1985

Do inhibitor studies demonstrate a role for poly(ADP-ribose) in DNA repair?

3-Aminobenzamide, an inhibitor of poly(ADP-ribose) synthesis, has been commonly used in attempts to demonstrate a regulatory role for the polymer during a late stage of repair. When a range of inhibitor concentrations was used paradoxical results were obtained. Up to 1 mM, 3-aminobenzamide appeared to reduce DNA break frequencies in cells damaged by methyl methane sulfonate; at doses of 2 mM and above, it appeared to increase break frequencies. In the high concentration range, many nonspecific side effects and cellular toxicity predominate. Evidence used to assert a role for poly(ADP-ribose) synthesis during ligation has usually been derived from experiments using high concentrations of 3-aminobenzamide, but these may be attributed to toxic side effects. 3-Aminobenzamide stimulates a large increase in repair replication which does not result from increased excision of damaged sites or an increased patch length but may be attributable to other cellular effects such as endogenous nuclease attack on DNA. The cellular effects of 3-aminobenzamide are therefore complicated by nonspecific effects over a commonly used concentration range and evidence for a specific regulatory role of poly(ADP-ribose) in DNA repair is weak.

Benzamides↗
PubMed1985

Increased repair replication in human lymphoid cells by inhibition of polyadenosine diphosphoribose synthesis with no increase in patch sizes.

Repair replication of alkylation damage in WIL-2 lymphoid cells is increased up to 7-fold by addition of 3-aminobenzamide, an inhibitor of polyadenosine diphosphoribose polymerase. This increase occurs without any change in the repair replication patch size and must therefore represent a large increase in the number of patches. The increase in the number of patches occurs without concomitant increase in the rate of excision of damaged sites. Therefore, it seems unlikely that 3-aminobenzamide plays any role in regulating ligation of repair patches, as commonly supposed. Instead, by inhibiting polyadenosine diphosphoribose polymerase or by other side effects, 3-aminobenzamide appears to elicit random nuclease attack of cellular DNA. The sites of attack are then repaired with patches of similar size, as are most other lesions. Nuclease attack may play a role in the increased cellular toxicity attendant on growth in 3-aminobenzamide.

Acrylamides↗
PubMed1985

Impaired immune function in patients with xeroderma pigmentosum.

The development of contact allergy in sun-exposed skin is markedly impaired in patients with xeroderma pigmentosum as compared to the responses in healthy control subjects. The degree of this immunological impairment is directly related to the severity of the cutaneous disease. These findings raise the possibility that sunlight-induced alterations of immune function may be involved in the marked susceptibility of these patients to the development of nonmelanoma skin cancer.

Adolescent↗
PubMed1985

Induction of DNA-DNA cross-link formation in human cells by various psoralen derivatives.

Furocoumarin-induced DNA damage, monoadducts, and cross-links were measured in normal human, xeroderma pigmentosum, and Fanconi's anemia cells after exposure to near-UV (356 nm). At similar concentrations and near-UV doses, photoaddition by 8-methoxypsoralen was twice that by angelicin and the substitution of bromodeoxyuridine for thymidine in one strand of DNA did not alter the binding. The rate of cross-linking by 8-methoxypsoralen was twice that of 5-methoxypsoralen. Low frequencies of cross-links were detected from angelicin and 3-carbethoxypsoralen but none were detected from 5-geranoxypsoralen at concentrations up to 25 micrograms/ml and near-UV doses up to 45,000 J/m2.

Cells, Cultured↗
PubMed1985

Repair of psoralen-induced cross-links and monoadducts in normal and repair-deficient human fibroblasts.

SV40-transformed normal, xeroderma pigmentosum (XP) and Fanconi's anemia (FA) fibroblasts have distinct repair capacities for monoadducts and DNA interstrand cross-links produced by exposure to near-UV (320-400 nm) light in the presence of 8-methoxypsoralen or angelicin. Excision repair of monoadducts occurred rapidly in normal and FA cells after exposure but not in XP cells. Cross-links were repaired in normal cells with a t1/2 of about 10 h but not in XP or FA cells. When the total number of adducts induced by 8-methoxypsoralen in normal cells was kept constant, the amount of repair replication decreased as the ratio of cross-links to monoadducts increased. This suggests either that cross-link repair is significantly different from monoadduct repair, involving smaller patches and a much slower rate of patching or that cross-links can inhibit monoadduct repair. Our results show that XP group A and FAH12 cell lines are deficient in cross-link repair. The data also suggest that the mechanism of cross-link repair in human cells involves several enzymes and that different ones may be deficient in XP and FA cells.

DNA↗
PubMed1984

DNA repair deficiencies and cellular senescence are unrelated in xeroderma pigmentosum cell lines.

Senescence of skin fibroblast cultures from normal individuals occurred after 23.9 +/- 6.3 (S.D.) passages; senescence in DNA repair-deficient cell lines from xeroderma pigmentosum patients occurred at 22.9 +/- 5.5 passages. Cells from xeroderma pigmentosum variant and Cockayne syndrome patients reached senescence at similar passage numbers. Xeroderma pigmentosum patients contract skin cancer as a consequence of their repair deficiencies but show no symptoms of premature ageing; neither do their cells age prematurely in vitro. The clinical spectrum and the life-span of fibroblasts in culture therefore lend no support for a correlation between ageing and the DNA repair or DNA replication deficiencies found in xeroderma pigmentosum and Cockayne syndrome cells.

Cell Line↗
PubMed1984

Sensitivity of mitomycin C and nitrogen mustard crosslinks to extreme alkaline conditions.

DNA-DNA crosslinks in cells treated with mitomycin C, nitrogen mustard, or decarbamoyl mitomycin C were measured in alkaline isopycnic gradients as a function of pH. Crosslinks from cells treated with mitomycin C and nitrogen mustard, which react with DNA purines, could be detected at pH 12.5 but not at pH 14. No crosslinks from cells treated with decarbamoyl mitomycin C were detected at either pH. Previous studies with cells exposed to psoralen derivatives plus 360 nm light, which produce DNA-DNA crosslinks with pyrimidines, demonstrated stable crosslinks at pH 14. These studies indicate that DNA-DNA crosslinks involving DNA purines are much less stable at high pH than those involving pyrimidines, and that methods involving exposure to extreme alkaline conditions may give inaccurate information for some agents.

Antibiotics, Antineoplastic↗
PubMed1984

Inhibitors of poly(adenosine diphosphate-ribose) synthesis: effect on other metabolic processes.

3-Aminobenzamide and benzamide, purported to be specific inhibitors of the synthesis of poly(adenosine diphosphate-ribose), were used to elucidate possible functions of this biopolymer. These compounds, at frequently used experimental concentrations, not only inhibited the action of poly(adenosine diphosphate-ribose) synthetase but also affected cell viability, glucose metabolism, and DNA synthesis. Thus, the usefulness of 3-aminobenzamide and benzamide may be severely restricted by the difficulty of finding a dose small enough to inhibit the synthetase without producing additional metabolic effects.

Benzamides↗
PubMed1984

The action of ultraviolet light on the patterns of banding induced by restriction endonucleases in human chromosomes.

The irradiation of metaphase spreads of human cells with ultraviolet (UV) light blocked the chromosome banding induced by Alu I, Mbo I, Dde I, Hinf I, Hae III, and Rsa I restriction endonucleases. At 13 J/m2 there was moderate inhibition of the nuclease action, which was detected as an increase in the stain intensity of chromosomes (Alu I, Mbo I, Dde I, Rsa I) or as a change in the banding pattern (Hinf I, Hae III). AT 70-300 J/m2 the UV-induced blockage was complete; the chromosomes showed no banding, and stain intensity was similar to that of control slides incubated with buffer. BrdU substitution and the irradiation of BrdU-substituted chromosomes with 313 nm light at 1800-15000 J/m2 did not block the action of restriction nucleases. On the other hand, UV irradiation of BrdU-substituted chromosomes inhibited the action of restriction enzymes at the same fluences that blocked the nuclease action in unsubstituted chromosomes. The data indicate that DNA-protein crosslinkage is the factor inhibiting DNA extraction and chromosome banding.

Cells, Cultured↗
PubMed1984

Inhibition of malignant transformation in vitro by inhibitors of poly(ADP-ribose) synthesis.

Malignant transformation in vitro of hamster embryo cells and mouse C3H 10T 1/2 cells by x-rays, ultraviolet light, and chemical carcinogens was inhibited by benzamide and by 3-aminobenzamide at concentrations that are specific for inhibition of poly(ADP-ribose) formation. These compounds slow the ligation stage of repair of x-ray and alkylation damage but not of ultraviolet light damage. At high concentrations they also inhibited de novo synthesis of DNA purines and DNA methylation by S-adenosylmethionine. The suppression of transformation by the benzamides is in striking contrast to their reported effectiveness in enhancing sister chromatid exchange, mutagenesis, and killing in cells exposed to alkylating agents. Our results suggest that mechanisms regulating malignant transformation are different from those regulating DNA repair, sister chromatid exchange, and mutagenesis and may be associated with changes in gene regulation and expression caused by alterations in poly(ADP-ribosyl)ation.

Animals↗
PubMed1984

Methylating and ethylating carcinogens have different requirements for poly(ADP-ribose) synthesis during malignant transformation.

Transformation of mouse C3H 10T1/2 cells by various alkylating carcinogens can be modulated by inhibiting poly(ADP-ribose) synthesis with a low concentration of 3-amino-benzamide, which induces no additional toxicity or reported side effects. Transformation by methylating agents was decreased by 3-aminobenzamide, whereas transformation by ethylating agents was increased. These results confirm earlier work on transformation by methylating agents, X-rays and u.v. light. Transformation by ethylating agents, however, appears to proceed by a different mechanism.

Alkylating Agents↗
PubMed1984

Completion of excision repair patches in human cell preparations: identification of a probable mode of excision and resynthesis.

Excision repair of u.v. damage in human fibroblasts is more sensitive to inhibitors of DNA polymerase alpha (cytosine arabinoside, aphidicolin) than to an inhibitor of polymerase beta (dideoxythymidine), which indicates a greater role in repair for polymerase alpha than for polymerase beta. These inhibitors all generate shortened patches with free 3' termini; the detailed structure of these patches was investigated in permeable cells or isolated nuclei by degradation of DNA with exonuclease III and by resynthesis with DNA polymerase I (Klenow fragment) and T4 DNA ligase. The structure of the shortened patches appears to be a short stretch of DNA synthesized in the 5'----3' direction within a longer single-strand gap. The single-strand gap ahead of the 3' terminus can be bridged only by the combined action of polymerase and ligase. This structure implies that excision must involve removal of an oligonucleotide or widening of a gap by 5'----3' exonuclease action to produce a single-strand region wide enough to be a substrate for polymerase alpha. There is no evidence for structures generated by nick translation or strand displacement.

Aphidicolin↗
PubMed1984

Repair of psoralen adducts in human DNA: differences among xeroderma pigmentosum complementation groups.

Angelicin and 5-methylangelicin formed photoadducts in DNA after illumination with 360-nm radiation that were excised rapidly from normal cells; 80-90% of the initial angelicin adducts and 65% of the initial 5-methylangelicin adducts were excised within 24 h. Xeroderma pigmentosum group A cells excised about 20% of the angelicin adducts, group D cells excised 55-60%, and group E, 80%. This extent of excision resembles that reported for pyrimidine dimers in these complementation groups, except for group D. Repair of psoralen adducts may not, therefore, be identical in every respect to repair of pyrimidine dimers. Group D cells seem exceptionally able to repair angelicin adducts in comparison to their repair of pyrimidine dimers, suggesting that these cells lack a gene product that is required to a greater extent for the repair of pyrimidine dimers than for the repair of angelicin adducts.

DNA↗