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Biomedical subjects

J E Cleaver

Publications and source records attributed to J E Cleaver.

At least 91 records · Page 5Linked to original sources
PubMed1989

Quiescent human lymphocytes do not contain DNA strand breaks detectable by alkaline elution.

On the basis of qualitative assays, quiescent lymphocytes have previously been reported to have numerous DNA strand breaks, which are thought to be repaired after mitogenic stimulation by a process associated with poly(ADP-ribosyl)ation. Using alkaline elution, a very sensitive assay for quantifying DNA single-strand breakage, we found no evidence for a high frequency of DNA strand breaks in unstimulated human peripheral blood lymphocytes. No differences in elution profiles were observed between unstimulated lymphocytes and lymphocytes 4 or 48 h after addition of the mitogen phytohemagglutinin (PHA). Furthermore, addition of 3-aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) synthetase, or aphidicolin, an inhibitor of DNA polymerase alpha, did not increase the amount of DNA eluting from the filter after PHA stimulation. In contrast to reported studies of mouse splenic lymphocytes, we found that human lymphocytes were able to replicate and divide in the presence of the ADP-ribosylation inhibitor. Human lymphocytes were also capable of proliferating in nicotinamide-free medium, with or without 3AB, indicating that ADP-ribosylation is not a requirement for lymphocyte differentiation. We therefore consider it unlikely that peripheral human lymphocytes contain significant numbers of strand breaks that play any role in their stimulation or differentiation in response to PHA.

Aphidicolin↗
PubMed1989

DNA damage and repair in normal, xeroderma pigmentosum and XP revertant cells analyzed by gel electrophoresis: excision of cyclobutane dimers from the whole genome is not necessary for cell survival.

Human fibroblasts were embedded in microscopic-sized agarose beads and lysed in situ to produce chromosome-sized DNA trapped inside the beads. Conventional alkaline agarose gel electrophoresis of trapped DNA from cells damaged by X-rays before embedding gave mol. wts similar to those obtained with alkaline sucrose gradients. When cells were irradiated with UV light before embedding in agarose and incubated with UV endonuclease to cleave cyclobutane pyrimidine dimer sites, UV damage was detectable by gel electrophoresis over a range of 2.6-52 J/m2 as a linear function of dose, and repair was detectable within 6 h of irradiation. Two independently derived UV-resistant xeroderma pigmentosum (XP) revertants did not remove cyclobutane dimers up to 48 h after irradiation. Incubation of normal and XP revertant cells with cytosine arabinoside after UV irradiation resulted in similar numbers of single-strand breaks; these breaks represent repair of non-dimer photoproducts. Evidently, excision of cyclobutane pyrimidine dimers from the whole genome is not necessary for survival of human cells after UV irradiation; rather, repair of non-dimer photoproducts such as photoproducts in the genome as a whole or cyclobutane dimers in a small number of genes may be more biologically important.

Cell Line↗
PubMed1989

DNA repair in man.

DNA repair in man can be described in general terms, but details are still obscure. Excision repair of base damage has a general similarity to the mechanism of the bacterial uvr ABC exonuclease, but the individual roles of at least 15 genes that regulate mammalian excision repair are as yet unknown. The differential repair of specific regions of DNA and of specific genes is highlighted by the clustered mode of repair characteristic of xeroderma pigmentosum group C and by the rapid repair of the dihydrofolate reductase gene. Cloning of genes that specify repair in man is proceeding slowly, in part, because of confusion by genes that produce only partial correction or nonspecific changes in sensitivity and by phenotypic reversion. In human cells, DNA damage-inducible genes are recognized that may overlap the spectra of other stress-induced proteins, but the relationship of these to any error-prone or recA-like system is unknown and unlikely. Four diseases, xeroderma pigmentosum, ataxia telangiectasia, Cockayne syndrome, and Fanconi anemia, have well-documented and significantly increased sensitivities to DNA-damaging agents, and each has recognizable though complex abnormalities in processing DNA damage. In addition, a wide variety of diseases and cellular processes have been ascribed to an association with DNA damage and repair, but the accuracy and significance of these associations are hard to identify.

Ataxia Telangiectasia↗
PubMed1988

Excision repair in xeroderma pigmentosum group C cells is regulated differently in transformed cells and primary fibroblasts.

Excision repair in xeroderma pigmentosum group C cells occurs at about 20-30% of normal levels. In confluent fibroblasts a unique characteristic of this low repair is that it is clustered, representing very efficient repair in a small region of the genome. In SV40-transformed fibroblasts and Epstein-Barr virus-transformed lymphocytes of complementation group C, however, excision repair is randomly distributed. This may be a consequence of the high rate of proliferation of both of these cell types, because random repair is also observed in rapidly proliferating group C fibroblasts. The distribution of sites that can be mended in group C cells, therefore, varies according to the transformed and proliferative state of the cells, demonstrating that transformed cells do not always exhibit repair characteristics identical to those of primary fibroblasts.

Cell Transformation, Neoplastic↗
PubMed1988

Rapid diagnosis of sensitivity to ultraviolet light in fibroblasts from dermatologic disorders, with particular reference to xeroderma pigmentosum.

A rapid and simple method for determining the sensitivity of human fibroblasts to ultraviolet light is described. As an alternative to the colony formation assay, this method can be used for the rapid diagnosis of ultraviolet light sensitivity in fibroblasts from photosensitive disorders. The method is based on growth of small numbers of cells in 1-cm wells of culture trays for 4 or more days after irradiation and determination of cell survival by the incorporation of [3H]hypoxanthine. D37 values (the dose at which 37% of the control level of incorporation remains) obtained from this procedure showed the same relative sensitivity of normal and xeroderma pigmentosum fibroblasts as was obtained by colony formation. Untransformed and SV40-transformed fibroblasts, which have different growth rates and different responses to high cell densities, gave different D37 values by this assay in culture trays as compared with colony formation. Comparison of relative sensitivities to irradiation should therefore be made only between cell types with similar growth characteristics. The similar sensitivity of normal and xeroderma pigmentosum cells to mitomycin C was also determined by this culture tray method. By increasing cell density at the beginning of the experiments, a greater capacity of group C compared with group D fibroblasts for recovery from potentially lethal damage was also detected.

Cells, Cultured↗
PubMed1988

Proximity of repair patches to persistent pyrimidine dimers in DNA of normal human and xeroderma pigmentosum cells.

The proximity of repair patches to persistent pyrimidine dimers in normal human cells and xeroderma pigmentosum group C and D cells was analyzed by sequential digestion of repaired DNA with Micrococcus luteus UV-endonuclease and Escherichia coli DNA polymerase I. Although this enzymatic digestion removed one-third of the pyrimidine dimers, less than 3% of the label associated with repair patches and a similar amount of uniformly labeled DNA were removed. The repair patches therefore appear to be similarly distant from persistent dimers in all cell types, and, in particular, are not adjacent to unexcised dimers in xeroderma pigmentosum group D cells. A previous model that suggested that patches are inserted adjacent to dimers in xeroderma pigmentosum group D cells receives no support from these results.

DNA Repair↗
PubMed1987

Relative importance of incision and polymerase activities in determining the distribution of damaged sites that are mended in xeroderma pigmentosum group C cells.

Those pyrimidine dimers that are repaired in confluent xeroderma pigmentosum Group C cells are clustered together in the genome. Although the average level of repair in this complementation group is of the order of 25% of normal, this percentage represents normal levels of repair in one quarter of the genome and little repair in the remainder. The factors that regulate this clustering process have been investigated using inhibitors of the initial incision step of repair (novobiocin) and of the polymerization step (aphidicolin). Novobiocin at a concentration that permitted 30% of repair to continue reduced the clustering of mended sites only slightly. Aphidicolin, in contrast, at a concentration that permitted 30 to 60% of repair to continue caused the mended sites to be distributed randomly. The clustering of repair sites seen in xeroderma pigmentosum Group C cells, therefore, is produced by an excision repair mechanism in which an aphidicolin-sensitive DNA polymerase, presumably alpha, plays an important regulatory role in determining which damaged sites are mended.

Aphidicolin↗
PubMed1987

Correction of excision repair in xeroderma pigmentosum by hamster chromosome fragments involves both classes of pyrimidine dimers.

The ultraviolet light-sensitive phenotype of xeroderma pigmentosum (XP) has been corrected by the incorporation into XP cells of small chromosome fragments from Chinese hamster ovary cells. Like normal human and hamster cells, these XP-hamster hybrids are able to excise both of the photoproducts produced by ultraviolet light: cyclobutane pyrimidine dimers and the minor photoproduct, (6-4) pyrimidine-pyrimidone dimers. This excision capacity contrasts with that of an XP revertant, of the same cell line used in this study, which is able to excise only the (6-4) photoproducts. The excision defect of XP has been fully corrected in the hybrids; therefore, the small hamster chromosome fragments they contain should carry the gene for complementation group A of XP.

Animals↗
PubMed1987

3-Aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase, is a stimulator, not an inhibitor, of DNA repair.

An inhibitor of poly(ADP-ribose) synthesis, 3-aminobenzamide (3AB), at low concentrations (0.01-0.1 mM) was found to reduce strand-break frequencies and increase repair replication in human lymphoid cells damaged by methyl methanesulfonate. A concentration of 0.1 mM 3AB was adequate to produce a maximum effect on strand-break frequencies and repair replication. This evidence, together with our previous measurements, demonstrates that 3AB cannot be regarded as an inhibitor of DNA repair; rather, it actually accelerates the ligation of DNA repair patches. Previous considerations of 3AB as a repair inhibitor may have derived from the use of excessive concentrations above 1 mM that may have stimulated additional damage and from the use of ethyl alcohol as a solvent for 3AB. Interpretations of the role of single-strand breaks and poly(ADP-ribose) in DNA repair, differentiation, and gene activity may need reevaluation because they have frequently been based on an erroneous notion of 3AB as a repair inhibitor, when its mode of action is, in fact, more complex.

Benzamides↗
PubMed1987

Unique cross-link and monoadduct repair characteristics of a xeroderma pigmentosum revertant cell line.

Monoadducts and cross-links formed in DNA of human cells by a psoralen derivative, 4'-hydroxy-methyl-4,5',8-trimethylpsoralen (HMT), have been measured by a new, simple method, based on S1 nuclease digestion of 3H-labeled adducts in DNA, that provides rapid information on the repair of both classes of lesions. Normal human fibroblasts and cells from patients with dyskeratosis congenita and xeroderma pigmentosum (XP) group C were capable of removing both monoadducts and cross-links, whereas XP groups A and D failed to remove either. An XP revertant, isolated from a group A cell line on the basis of an acquired mutagen-induced resistance to ultraviolet light, has the unique property of being capable of removing cross-links but not monoadducts. Consistent with this property, the XP revertant was found to be resistant to cell killing by the cross-linking psoralen derivative, HMT, but as sensitive as its parental cell line to a monofunctional psoralen derivative, 5-methylisopsoralen.

Cell Line↗
PubMed1987

Differences in the stimulation of repair replication by 3-aminobenzamide in lymphoblastoid cells damaged by methylmethanesulfonate or ultraviolet light.

Human lymphoblastoid cells damaged by u.v. light accumulated DNA breaks in the presence of cytosine arabinoside and hydroxyurea at a frequency similar to that of cells damaged by methylmethanesulfonate. 3-Aminobenzamide (1 mM) reduced the net strand-break frequency detected after either kind of damage. Repair replication, however, was stimulated only in methylmethanesulfonate-damaged cells. This stimulation is therefore not related directly to the DNA strand-break frequencies and concomitant poly(ADP-ribose) synthesis, but depends on some other cellular response specific to alkylating agents.

Benzamides↗
PubMed1987

Purified scrapie prions resist inactivation by UV irradiation.

The development of effective purification protocols has permitted evaluation of the resistance of isolated scrapie prions to inactivation by UV irradiation at 254 nm. Prions were irradiated on ice with doses of UV light ranging up to 120,000 J/m2. UV dosimetry experiments, performed with Saccharomyces cerevisiae plasmid DNA or eucaryotic cells, indicated that under these experimental conditions an incident UV dose of 10 J/m2 formed 2 thymine dimers per 5.1 X 10(6) daltons of eucaryotic cell DNA. The D37 values for scrapie prions ranged from 17,000 to 22,000 J/m2; D37 values were also determined for virus, viroid, and enzyme controls. The number of pyrimidine dimers formed was correlated with the D37 values obtained for irradiated prions and target nucleic acids. The D37 value for bacteriophage M13, 6.5 J/m2, occurred at a dose that would form 0.56 dimers per target genome; the D37 for potato spindle tuber viroid, 4,800 J/m2, occurred at a dose that would form about 24 dimers per target viroid. The D37 value for an EcoRI restriction site, a target of 12 bases, occurred at a dose that would correspond to the formation of 0.89 thymine dimers per target site. The D37 value for prions occurred at a dose that would form 1 dimer in every 4 bases of single-stranded target nucleic acid. If the putative scrapie nucleic acid were double-stranded and readily repairable after UV damage, then the prion D37 value could reflect a nucleic acid molecule of 30 to 45 base pairs. While the D37 value for prions fell within the range of pure protein targets, our experiments cannot eliminate the possibility that a prion contains a small, highly protected nucleic acid molecule.

Animals↗
PubMed1987

Unique DNA repair properties of a xeroderma pigmentosum revertant.

A group A xeroderma pigmentosum revertant with normal sensitivity was created by chemical mutagenesis. It repaired (6-4) photoproducts normally but not pyrimidine dimers and had near normal levels of repair replication, sister chromatid exchange, and mutagenesis from UV light. The rate of UV-induced mutation in a shuttle vector, however, was as high as the rate in the parental xeroderma pigmentosum cell line.

4-Nitroquinoline-1-oxide↗
PubMed1986

DNA repair in human xeroderma pigmentosum group C cells involves a different distribution of damaged sites in confluent and growing cells.

Xeroderma pigmentosum is a human disease consisting of several complementation groups that are deficient in excision repair. Group C is one in which excision repair occurs at about 20-30% of normal levels. The distribution of mended sites in relation to unrepaired sites has been determined by cutting remaining unrepaired pyrimidine dimers with Microccocus luteus UV endonuclease. The mended sites have been found clustered together in a fashion that depended on cell proliferation. In confluent group C cells, the mended sites were clustered in regions where dimer excision was as efficient as excision in the DNA of normal cells. In proliferating group C cells, however, mended sites were randomly dispersed. The total amount of repair replication was the same in confluent and proliferating cells. Since previous work has shown that confluent group C cells show more extensive recovery from the lethal effects of UV irradiation than some other groups, clustered repair may correlate with a more efficient mechanism of restoring cell viability. The different distribution of repaired sites during DNA replication may be the result of changes in the state of the substrate for repair or changes in the metabolic priorities of DNA polymerases.

Cell Cycle↗