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Biomedical subjects

J E Cleaver

Publications and source records attributed to J E Cleaver.

At least 19 recordsLinked to original sources
PubMed1996

Elevated DNA excision repair capacity in the extraembryonic mesoderm of the midgestation mouse embryo.

In order to determine whether there is differential cell-type-specific DNA repair we measured the nucleotide excision repair capacity of the four distinct cell lineages that comprise the extraembryonic yolk sac using the unscheduled DNA synthesis assay. Yolk sacs from mouse embryos at 11.5-12.5 days gestation were microdissected to yield purified trophoblast, parietal endoderm, mesoderm, and visceral endoderm, as well as fetal skin fibroblasts which were then grown as primary explants. At this midgestational stage of development, the yolk sac provides essential functions for the sustenance of the embryo while the complex process of organogenesis is proceeding in the liver, kidney, and gut. Trophoblast giant cells, parietal endoderm, and visceral endoderm all demonstrated low levels of unscheduled DNA synthesis consistent with levels measured in adult mouse skin fibroblasts. As has previously been documented, embryonic mouse skin fibroblasts were reproducibly 2- to 3-fold higher than adult mouse skin fibroblasts in levels of DNA excision repair. The extraembryonic mesoderm, however, displayed a statistically significant level of unscheduled DNA synthesis 10-fold higher than adult mouse skin fibroblasts or the other lineages of the midgestation yolk sac. Further, the S-indexes of these lineages were also determined to assess the possible relevance of differential repair to the proliferative status of the cells. These data demonstrate that DNA excision repair capacity is lineage-specific during embryogenesis in the mouse. These studies may begin to provide a context for understanding the perplexing developmental aspects such as the characteristic congenital abnormalities associated with the human heritable DNA repair deficiency diseases.

Animals↗
PubMed1996

Cockayne syndrome complementation group B associated with xeroderma pigmentosum phenotype.

Two siblings have been reported whose clinical manifestations (cutaneous photosensitivity and central nervous system dysfunction) are strongly reminiscent of the DeSanctis-Cacchione syndrome (DCS) variant of xeroderma pigmentosum (XP), a severe form of XP. Fibroblasts from the siblings showed UV sensitivity, a failure of recovery of RNA synthesis (RRS) after UV-irradiation, and a normal level of unscheduled DNA synthesis (UDS), which were, unexpectedly, the biochemical characteristics usually associated with Cockayne syndrome (CS). However, no complementation group assignment in these cells has yet been performed. We here report that these patients can be assigned to CS complementation group B (CSB) by cell fusion complementation analysis. To our knowledge, these are the first patients with defects in the CSB gene to be associated with an XP phenotype. The results imply that the gene product from the CSB gene must interact with the gene products involved in excision repair and associated with XP.

Cell Fusion↗
PubMed1996

Stimulation of repair replication by 3-aminobenzamide in human fibroblasts with ligase I deficiency.

A cell line deficient in DNA ligase I and sensitive to poly(ADP-ribose) inhibitors, 46BR, was used to examine the relationship between DNA ligation and a large stimulation of repair replication that is seen in cells grown in a poly(ADP-ribose) polymerase inhibitor, 3-aminobenzamide (3AB), after exposure to alkylating agents. Repair replication was stimulated at least 10-fold by 3AB in both normal and ligase-deficient cells. Despite increased 3AB toxicity, repair replication in ligase I-deficient cells was unchanged from that in normal cells. This evidence is consistent with previous observations that the enhancement of repair replication by 3AB is not a direct function of DNA break frequencies. The stimulation may instead result from alkylation damage to other cellular organelles that release nucleases that cause additional damage to DNA, which cells attempt to repair.

Benzamides↗
PubMed1996

Chromatid exchanges may be induced by damage in sites of transcriptional activity.

A conditional expression system has allowed us to vary the expression level of the xeroderma pigmentosum group A (XPA) photoproduct-specific DNA-binding protein in human cells and so control the response of cells to damage by UV light. Using a form of XPA that contains a single missense mutation (R207G) enabled us to study a lower range of function than that obtained with the wild-type sequence. This form of XPA has been previously shown to stimulate pyrimidine dimer excision preferentially in actively transcribed genes. We found that UV resistance increased as a linear function of XPA expression levels. Excision of (6-4) pyrimidine-pyrimidone photoproducts in the whole genome increased to a maximum at about the haploid level of XPA expression, but there was little pyrimidine dimer excision from the whole genome. SCE frequency induced by UV light was high in cells with no SPA expression and fell rapidly with increasing levels of SPA expression within 0-50% of the haploid level of expression. No further reduction in SCE frequency was produced at the highest levels of XPA expression, when repair replication extended to the overall genome. We speculate that a low level of repair, especially that occurring in actively transcribed genes, may selectively eliminate photoproducts that are particularly important in causing cell killing and SCEs.

Cell Line, Transformed↗
PubMed1995

Overexpression of the XPA repair gene increases resistance to ultraviolet radiation in human cells by selective repair of DNA damage.

Overexpression of XPA genes, both wild type and a missense mutant, which code for a damage-specific, DNA-binding protein, increased the survival of repair-deficient and -competent human cells to levels above that of normal cells that did not overexpress XPA. The first 3 h after cells were damaged were most critical to achieving this increased survival. The dose at which 37% of the irradiated population survives could be restored to about one-half that of normal cells, with no detectable genome-wide repair of pyrimidine dimers or (6-4) photoproducts, suggesting that intermediate levels of XPA gene expression can direct repair to restricted critical regions of the genome. Current views of repair implicate transcriptionally active genes as a major component of such critical regions. Consistent with this interpretation, the repair of a transfected, actively expressed luciferase gene was higher than that of genomic DNA at intermediate and higher levels of XPA expression. High levels of XPA expression resulted in increased repair at early times after irradiation and extensive repair of (6-4) photoproducts but little, if any, pyrimidine dimer repair in the whole genome. At the highest level of expression, some clonal cell lines acquired resistance to radiation that corresponded to a dose at which 37% of the irradiated population survives that was about 1.5 to 2 times that of normal cells. The XPA gene product, therefore, can influence levels of DNA repair and radiation sensitivity quantitatively by contributing to selective repair at certain sites in the genome.

Cells, Cultured↗
PubMed1995

Xeroderma pigmentosum variant cells are resistant to immortalization.

Xeroderma pigmentosum (XP) is a human repair-deficient disorder that is caused by mutations in any of eight genes (A-G, V). The genes for complementation groups A-G have been cloned fully or in part, but the gene for the XP variant (XPV) has yet to be cloned. The lack of progress with XPV is in large part due to the rarity of stably transformed cell lines. We have attempted to immortalize fibroblasts from several XPV patients to obtain cell lines with which to characterize this disease and clone the appropriate gene. We have found, as have other investigators, that this XP group is very difficult to immortalize. We used a variety of approaches, including transfection with pSV ori- (a plasmid containing the simian virus (SV) 40 large T antigen) followed by spontaneous transformation, which provided stable immortal lines from Cockayne syndrome A and B, but not from XPV; transfection with pSV ori- and exposure to 3 Gy of X-rays; transfection with pSV ori-, exposure to 2 Gy of X-rays, and treatment with 1 mM ethyl methanesulfonate; transfection with human papilloma virus-16; and infection with SV40. Even though we used as many as 2 x 10(8) cells in some experiments, we were able to immortalize only one of our lines, XP30RO. Because the biochemical defect in XPV cell lines involves the capacity to replicate damaged DNA templates, perhaps the XPV gene product could be a replication factor that interacts with SV40 T antigen, and whose absence from XPV cell lines presents difficulties for the immortalization process to proceed.

Antigens, Polyomavirus Transforming↗
PubMed1995

Induction and repair of (6-4) photoproducts in normal human and xeroderma pigmentosum variant cells during the cell cycle.

The reduced rate of (6-4) photoproduct repair observed in some cell lines may represent a more severe repair deficiency in some cohort of the cell cycle, such as S-phase. Radioimmunoassay was used to determine the kinetics of (6-4) photoproduct repair in normal human fibroblasts and xeroderma pigmentosum variant cells fractionated into different phases of the cell cycle by counterflow centrifugal elutriation. Ultraviolet fluence response curves indicated that the same amount of (6-4) photoproduct damage was induced at all phases of the cell cycle. The extent of (6-4) photoproduct repair in asynchronous XP variant cells was significantly reduced compared to normal human cells. However, the rate and extent of (6-4) photoproduct repair was constant throughout the cell cycle in both normal and XP variant cells. Hence, the UV hypersensitive and hypermutable phenotypes observed in XP variant cells are not attributable to cell cycle-dependent deficiencies in excision repair nor the yield of photodamage through the cell cycle.

Cell Cycle↗
PubMed1995

Sister chromatid exchanges in cells defective in mismatch, post-replication and excision repair.

Three processes associated with DNA damage and genomic instability have been defined experimentally as operating during or soon after DNA replication: mismatch repair, post-replication repair and sister chromatid exchange. All these processes appear to operate on damage and/or errors in newly replicated DNA. Both mismatch repair and post-replication repair involve resynthesis of up to 1 kb of newly synthesized DNA: mismatch repair operates on single-base or slippage errors; post-replication repair operates on persistent gaps in newly synthesized DNA caused by damage on parental strands. Using colon cancer cells with different mismatch repair capacity, together with normal cells and excision-repair-defective and post-replication-repair-defective xeroderma pigmentosum (XP) cells, we analysed possible interactions between these processes. No evidence for overlap of mismatch repair with excision or post-replication repair was found. However, post-replication-repair-defective XP variant cells that were SV40 transformed showed higher UV-induced sister chromatid exchange frequencies than did untransformed cells. This suggests that sister chromatid exchanges in the XP variant are closely involved with UV-induced replication errors that are enhanced by transformation.

Caffeine↗
PubMed1995

Nucleotide excision repair: variations associated with cancer development and speciation.

Nucleotide excision repair requires the action of multiple interacting proteins that locate damage in DNA, remove it as a short oligonucleotide and synthesize a replacement patch. Mutations in genes coding for these proteins give rise to a wide range of diseases involving skin carcinogenesis, neuronal decline and developmental disorders of bone and central nervous system. Complex clinical symptoms of more than one clinical disorder may occur because of mutations that influence protein-protein interactions. Significant differences in repair occur between individuals and species for which the molecular basis and phenotypic consequences have yet to be explained.

Animals↗
PubMed1994

Genetic testing.

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Genetic Diseases, Inborn↗
PubMed1994

Prenatal diagnosis of xeroderma pigmentosum and Cockayne syndrome.

In a study of fetal cells from a series of 12 pregnancies in ten families at risk for the ultraviolet light-sensitive, DNA repair-deficient diseases xeroderma pigmentosum (XP) and Cockayne syndrome (CS), we detected one XP and two CS homozygote fetuses. The diagnoses were confirmed by analysis of fetal skin fibroblasts or second amniotic samples after termination of the pregnancies. The measurement of ultraviolet light sensitivity and DNA repair depended on properties common to the seven excision repair-deficient XP complementation groups (A-G) and the two CS complementation groups (A, B). No XP variant families were included in the study, because the variant requires different testing techniques. Reliable and rapid diagnosis proved possible in all but one of the 12 pregnancies, supporting the use of these methods until the spectrum of mutations in the various XP and CS genes of the U.S. population is fully characterized and a DNA sequence-based diagnostic procedure becomes available.

Amniocentesis↗
PubMed1994

Mutation and expression of the XPA gene in revertants and hybrids of a xeroderma pigmentosum cell line.

A series of ultraviolet (UV)-resistant cell lines have been generated from a UV-sensitive XP group A cell line homozygous for a stop codon (TGA) in the chromosome 9 XPA gene. Three lines generated by chemical mutagenesis acquired the ability to excise (6-4) photoproducts but not cyclobutane dimers from the whole genome; two lines generated by a fusion procedure with hamster cells acquired the ability to excise both (6-4) photoproducts and cyclobutane dimers from the whole genome. A central region of the hamster XPA gene was cloned and sequenced. With the use of species-specific primers in the polymerase chain reaction, we found that the hybrid cell lines do not contain a hamster XPA gene. Sequence analysis showed that all of the UV-resistant cell lines contain reversions of the human stop codon, resulting in missense mutations (glycine or leucine for arginine) or wild-type sequences. The concentration of XPA protein in revertant cell lines was about one-half that in normal cells, which would be expected from heterozygous cells; there was no evidence that the mutant proteins were less stable than the wild-type proteins. These results are consistent with the idea that the XPA protein initiates repair by binding to damaged sites with various affinities, depending on the photoproduct and the transcriptional state of the region. A concentration of XPA protein near 50% is needed before repair can proceed into nontranscribed regions of the genome. The revertant cell lines represent a class of missense mutations in the XPA gene that may have altered specificity and that can be used to understand some of the regulatory differences in repair of photoproducts in various regions of the genome.

Amino Acid Sequence↗