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Biomedical subjects

J E Cleaver

Publications and source records attributed to J E Cleaver.

At least 163 records · Page 9Linked to original sources
PubMed1982

Inactivation of ultraviolet repair in normal and xeroderma pigmentosum cells by methyl methanesulfonate.

Excision repair of ultraviolet damage in the DNA of normal and xeroderma pigmentosum (Groups C, D, and variant) cells was inactivated by exposure of cells to methyl methanesulfonate immediately before irradiation independent of the presence of 0 to 10% fetal calf serum. The inactivation could be represented by a semilog relationship between the amount of repair and methyl methanesulfonate concentration up to approximately 5 mM. The inactivation can be considered to occur as the result of alkylation of a large (about 10(6) daltons) repair enzyme complex, and the dose required to reduce repair to 37% for most cells types was between 4 and 7 mM. No consistent, large difference in sensitivity to methyl methanesulfonate was found in any xeroderma pigmentosum complementation group compared to normal cells, implying that reduced repair in these groups may be caused by small inherited changes in the amino acid composition (i.e., point mutations or small deletions) rather than by losses of major components of the repair enzyme complex.

Cells, Cultured↗
PubMed1982

Repair and replication of DNA in hereditary (bilateral) retinoblastoma cells after X-irradiation.

Fibroblasts from patients with hereditary retinoblastoma reportedly exhibit increased sensitivity to killing by X-rays. Although some human syndromes with similar or greater hypersensitivity to DNA-damaging agents (e.g., X-rays, ultraviolet light, and chemical carcinogens), such as xeroderma pigmentosum, are deficient in DNA repair, most do not have such clearly demonstrable defects in repair. Retinoblastoma cells appear to be normal in repairing single-strand breaks and performing repair replication after X-irradiation and also in synthesizing poly(adenosine diphosphoribose). Semiconservative DNA replication in these cells, however, is slightly more resistant than normal after X-irradiation, suggesting that continued replication of damaged parental DNA could contribute to the pathogenesis of the disease. This effect is small, however, and may be a consequence rather than a cause of the fundamental enzymatic abnormality in retinoblastoma that causes the tumorigenesis.

DNA↗
PubMed1982

Normal reconstruction of DNA supercoiling and chromatin structure in cockayne syndrome cells during repair of damage from ultraviolet light.

The chromatin of human cells undergoes structural rearrangements during excision repair of ultraviolet damage in DNA that were detected by transient relaxation of DNA supercoiling and increased staphylococcal nuclease digestibility of repaired sites. Inhibition of polymerization and/or ligation of repaired regions with inhibitors of DNA polymerase alpha (cytosine arabinoside and aphidicolin) resulted in the accumulation of single-strand breaks, delayed reconstruction of DNA supercoiling, and maintenance of the staphylococcal nuclease digestibility. These observations suggest that reconstruction of the native chromatin state requires completion of repaired regions with covalent ligation into the DNA strands. Although previous claims have been made that a late stage associated with ligation of repaired regions may be defective in cells from patients with Cockayne syndrome, complete reconstruction of the native chromatin occurred in cells from three unrelated patients after ultraviolet irradiation. No abnormality in repair was therefore detected in Cockayne syndrome cells. The hypersensitivity of cell survival and semiconservative DNA replication to damage by ultraviolet light in this human disorder must therefore be regarded as features of a primary defect in DNA metabolism unrelated to DNA repair.

Cell Survival↗
PubMed1981

Transient conformation changes in chromatin during excision repair of ultraviolet damage to DNA.

DNA labeled for 15 minutes during UV induced repair synthesis is two-fold more sensitive to micrococcal nuclease than the bulk nuclear DNA. As the length of the labeling period increases from 15 minutes to 4 hours the nuclease sensitivity of repair labeled DNA approaches that of bulk chromatin. Pulse-chase experiments indicate that the nuclease sensitivity of the repaired DNA labeled during a brief pulse decreases with a half-life of about 15 minutes. In contrast to previous interpretations, we consider these results to mean that immediately after synthesis, chromatin labeled during repair has a conformation which renders it more susceptible to nuclease digestion than the bulk chromatin. With time these repaired regions are assembled into a nucleosome structure with normal nuclease sensitivity.

Animals↗
PubMed1981

Sensitivity of excision repair in normal human, xeroderma pigmentosum variant and Cockayne's syndrome fibroblasts to inhibition by cytosine arabinoside.

Inhibition of the gap-filling, polymerizing step of excision repair by 1-beta-D-arabinofuranosylcytosine (ara-C) after irradiation with ultraviolet light in human diploid fibroblasts resulted in the formation of persistent DNA strand breaks in G1, G2, and plateau phase cells, but not in S phase cells. Addition of hydroxyurea to ara-C resulted in partial inhibition of repair in S phase cells. These observations can be explained either in terms of changing roles in repair for different DNA polymerases throughout the cell cycle or by the presence of a pool of deoxycytidine nucleotides during S phase equivalent to be an external source of deoxycytidine at 50 microM concentration. A similar concentration dependence on ara-C was observed for inhibition of repair in normal human, xeroderma pigmentosum (XP) variant, and Cockayne's syndrome cells but slightly more in XP variant cells. Exonuclease III and S1 nuclease independently both degraded about 50% of the 3H-thymidine incorporated into repaired regions in the presence of ara-C. Sequential digestion with both enzymes degraded nearly 90% of the repaired regions. These observations can be explained if excision repair proceeds by displacing the damaged strand so that both the 3H-labeled patch and the damaged region are still ligated to high molecular weight DNA and compete for the same complementary strand during in vitro incubation with the nucleases. The amount of 3H-thymidine incorporated in DNA by repair decreased with increasing concentrations of ara-C and hydroxyurea, suggesting that the incomplete patches became shorter under these conditions. Extrapolation of the digestion kinetics with exonuclease III permits an estimate of the normal patch size of about 100 nucleotides, consistent with previous estimates.

Cockayne Syndrome↗
PubMed1981

Repair of ultraviolet damage in human cells also exposed to agents that cause strand breaks, crosslinks, monoadducts and alkylations.

Excision repair of UV damage in human cells was measured by the incorporation of new bases into DNA after exposure to UV light and variety of other carcinogens including X-rays, furocoumarins plus 360 nm light (8-methoxypsoralen (8-MOP), 4'-aminomethyl 4,5',8-trimethylpsoralen hydrochloride (AMT) and angelicin), methyl methanesulfonate (MMS), dimethyl sulfate (DMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Repair of UV damage was unaffected by concomitant exposure to X-rays. Furocoumarin adducts and alkylating agents, however, interacted with UV repair and reduced the amount of repair replication observed. The interaction between repair of furocoumarin and UV damage is consistent with the involvement of a common DNA repair pathway: its saturation with respect to repair of UV damage also results in saturation with respect to other lesions involving that pathway. The observed effect of alkylating agents on UV repair, however, cannot be due to saturation of a common pathway because damage from these agents are repaired by different mechanisms than UV damage. Instead, it appears likely that the effect is due to alkylation damage to repair enzymes. From a consideration of the degree of protein alkylation at millimolar concentrations of alkylating agents, we estimate that the UV repair system could consist of an extremely large complex of protein subunits in the order of 1 million molecular weight. Whereas some previous studies have used concomitant exposures to pair of different agents to determine whether the damage they produce is repaired by common or independent pathways, our results indicate that such a method is an unreliable indicator of the number of repair pathways.

Alkylating Agents↗
PubMed1981

Inhibition of DNA replication by hydroxyurea and caffeine in an ultraviolet-irradiated human fibroblast cell line.

DNA replication in human fibroblasts with normal excision repair was investigated after ultraviolet irradiation and incubation with caffeine or hydroxyurea. The DNA synthesized soon after irradiation had a reduced size, but that synthesized later was near normal size. When caffeine was present before labeling, it reduced the size of DNA synthesized but when added after labeling it was without effect. When irradiated cells were allowed to grow, labeled DNA increased in size steadily for 60 min to a maximum that was below control and dose-dependent. Further growth resulted in a transition of some label to parental DNA sized, but a large fraction remained permanently blocked at smaller sizes, producing bimodal distributions of DNA. The steady increase in size was inhibited by hydroxyurea. Removing cells from hydroxyurea resulted in increases similar to or slightly slower than those observed immediately after labeling, and this protocol did not permit cells to acquire any induced or enhanced capacity to replicate damaged DNA.

Caffeine↗
PubMed1981

Inhibition of excision-repair of ultraviolet damage in human cells by exposure to methyl methanesulfonate.

Unscheduled DNA synthesis and excision of pyrimidine dimers in human cells exposed to ultraviolet let were inhibited by exposure to methyl methanesulfonate (MMS, 1-2 mM), but repair of MMS damage was not inhibited by UV light. Because the pathways for excision of pyrimidine dimers and alkylation damage have previously been shown to be different, this observation implies a direct effect of alkylation on repair enzymes. We estimate that if inhibition is due to protein alkylation, the UV repair system must present an extremely large target to alkylation and may involve a complex of protein subunits in the order of 1 million daltons such that 1 or more alkylations occur per complex at the concentrations used. These results also indicate that the method of exposing cells to 2 DNA-damaging agents to determine whether they are repaired by common or different pathways can be quite unreliable because of other effects on the repair systems themselves.

Animals↗
PubMed1981

Protease inhibitors neither damage DNA nor interfere with DNA repair or replication in human cells.

Human fibroblasts were exposed to antipain or leupeptin at concentrations up to 2.5 mM and the presence of DNA damage and repair was assayed by several different methods. These did not reveal DNA damage or repair after exposure to either antipain or leupeptin, even in the presence of rat-liver microsomal S9 mix. Antipain also had no significant effects on the repair or replication of DNA after ultraviolet or X-irradiation. The demonstrated potentiation of radiation-induced transformation of human cells and the selective killing of repair-deficient cells by antipain must therefore occur by mechanisms that do not involve direct interaction of antipain with DNA.

Antipain↗
PubMed1981

Xeroderma pigmentosum patients from Egypt: II. Preliminary correlations of epidemiology, clinical symptoms and molecular biology.

Xeroderma pigmentosum (XP) occurs with high frequency in Egypt and a continuation of our field studies has identified representatives of the 3 major complementation groups A, C, and variant. Group A patients, with one exception, showed very early onset of sun sensitivity and development of skin cancers, and microcephaly and mental retardation. The exceptional group A patient was 35 yr old, with normal stature and intelligence who had 2 normal children. DNA repair was as low in his cells as in other group A cases. Group C patients showed a slightly slower onset of sun sensitivity and had no central nervous system disorders. The variants showed later onset of sun sensitivity and no skin cancers evident at the time of observation (about 20 yr of age). No sun sensitivity was present in the 25 heterozygotes we observed, nor reportedly in the additional 60 not yet observed. This indicates that only homozygosity for XP genes increases risk of skin cancer. Cell cultures from both normal persons and these XP patients reached in vitro "senescence" at similar passage levels. Groups A and C appear to have lost different major gene products that are involved in the excision of UV damage from DNA, but the residual repair in XP-C cells facilitates more recovery of DNA synthesis than in other groups. This may contribute to the higher in vitro survival in culture and milder clinical symptoms in group C as compared to group A. XP variants appear to have lost a gene product that permits normal cells to replicate, uninterrupted by DNA damage, and consequently synthesize DNA in smaller pieces than normal.

Adult↗
PubMed1980

Ultraviolet radiation inhibits replicon initiation in S phase human cells.

DNA replication was examined in ultraviolet-irradiated human fibroblasts and HeLa cells. The principal effect of exposures to low radiation fluences (less than 1.3 J/m2) was a reduced synthesis of molecules about one-half replicon in size resulting from an inhibition of replicon initiation. As the fluence of radiation was increased, inhibitory effects on strand elongation and joining masked the effect on replicon initiations.

Cell Cycle↗