Search PubMed⌕ Search

Biomedical subjects

J E Cleaver

Publications and source records attributed to J E Cleaver.

At least 37 records · Page 2Linked to original sources
PubMed1999

Increased ultraviolet sensitivity and chromosomal instability related to P53 function in the xeroderma pigmentosum variant.

The xeroderma pigmentosum (XP) variant (XPV) is a form of XP that has normal excision repair but shows defective DNA replication after UV irradiation. In developing various transformed fibroblast cell lines from these patients, we have found that there are significant phenotypic changes in transformed cells that seem to correlate with inactivation of p53. After transformation with SV40, XPV cell lines are only slightly UV sensitive, like their primary counterparts, but their sensitization with caffeine and the induction of sister chromatid exchanges (SCEs) by UV irradiation are greatly enhanced. After transformation by HPV16 E7, which targets the retinoblastoma cell cycle regulatory gene, there is no change in the UV sensitivity of XPV cells; but, when transformed by HPV16 E6 or E6 and E7 combined, there is a large increase in UV sensitivity and in the induction of SCEs. These changes are not associated with any detectable changes in the reactivation of an externally irradiated luciferase expression vector, the excision of cyclobutane pyrimidine dimers from bulk DNA, or unscheduled DNA synthesis and, therefore, do not involve excision repair. We suggest that if SCEs represent homologous recombination between sister chromatids, then in the absence of p53 function, the DNA chain arrest typical of UV-damaged XPV cells initiates strand exchange during recovery. In untransformed cells with normal p53, the preferred mode of recovery would then be replication bypass. The symptoms of elevated solar carcinogenesis in XPV patients may, therefore, be associated with increased genomic instability in cells of the skin in which p53 is inactivated by UV-induced mutations.

Caffeine↗
PubMed1999

A summary of mutations in the UV-sensitive disorders: xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy.

The human diseases xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy are caused by mutations in a set of interacting gene products, which carry out the process of nucleotide excision repair. The majority of the genes have now been cloned and many mutations in the genes identified. The relationships between the distribution of mutations in the genes and the clinical presentations can be used for diagnosis and for understanding the functions and the modes of interaction among the gene products. The summary presented here represents currently known mutations that can be used as the basis for future studies of the structure, function, and biochemical properties of the proteins involved in this set of complex disorders, and may allow determination of the critical sites for mutations leading to different clinical manifestations. The summary indicates where more data are needed for some complementation groups that have few reported mutations, and for the groups for which the gene(s) are not yet cloned. These include the Xeroderma pigmentosum (XP) variant, the trichothiodystrophy group A (TTDA), and ultraviolet sensitive syndrome (UVs) groups. We also recommend that the XP-group E should be defined explicitly through molecular terms, because assignment by complementation in culture has been difficult. XP-E by this definition contains only those cell lines and patients that have mutations in the small subunit, DDB2, of a damage-specific DNA binding protein.

Cockayne Syndrome↗
PubMed1999

Modification of the Comet assay for the detection of DNA strand breaks in extremely small tissue samples.

We modified the Comet assay to enable the quantification of DNA strand breakage in individual cells of extremely small tissue samples. This modification was used to analyze cells isolated from the ectoplacental cone and egg cylinder of mouse embryos at embryonic day 7.5. We detected more naturally occurring DNA strand breaks and a higher number of apoptotic cells in the ectoplacental cone compared with the egg cylinder.

Animals↗
PubMed1998

Distribution of mutations in the human xeroderma pigmentosum group A gene and their relationships to the functional regions of the DNA damage recognition protein.

A series of xeroderma pigmentosum group A cell lines from 19 patients and cell lines from 13 other family members were examined for XPA mutations to find previously unidentified mutations from American and European patients, to establish pedigrees in represented families, and to develop a database for XPA diagnosis. Most mutations were deletions and splice site mutations observed previously in other XPA patients, in exon III, intron III, or exon IV, that resulted in frameshifts within the DNA binding region-including an Afl III RFLP (G to C) in four unrelated families. One new mutation was a point mutation within intron III (A to G) creating a new splice acceptor site that may compete with the original splice acceptor site. Missplicing at this new site inserts 11 nucleotides in the mRNA creating a frameshift. A small amount of normal splicing to give wild-type XPA protein is the likely molecular mechanism for the relatively mild clinical features of this patient. In another patient, a new 2 bp deletion in the RPA70 binding region was identified in the same region as a 20 bp deletion previously characterized in an unrelated patient. Mutations in the DNA binding region of XPA were from patients with the more severe disease often associated with neurological complications, whereas mutations in the C-terminal end of the protein, which interacts with the TFIIH transcription factor, were from patients with milder skin disease only. The rarity of naturally occurring missense mutations in the DNA binding region of XPA suggests that amino acid changes might be sufficiently tolerated that patients would have mild symptoms and escape detection.

Alternative Splicing↗
PubMed1998

Xeroderma pigmentosum group C splice mutation associated with autism and hypoglycinemia.

A 4 y old boy of Korean ancestry had xeroderma pigmentosum (XP) with sun sensitivity, multiple cutaneous neoplasms, and inability to speak. Neurologic examination revealed hyperactivity and autistic features without typical XP neurologic abnormalities. Cultured skin fibroblasts (XP22BE) showed decreased post-UV survival, reduced post-UV plasmid host cell reactivation and defective DNA repair (16% of normal unscheduled DNA synthesis in intact cells and undetectable excision repair in a cell free extract). In vitro and in vivo complementation assigned XP22BE to XP group C (XPC) and a markedly reduced level of XPC mRNA was found. Two XPC cDNA bands were identified. One band had a deletion of 161 bases comprising the entire exon 9, which resulted in premature termination of the mutant XPC mRNA. The larger band also had the same deletion of exon 9 but, in addition, had an insertion of 155 bases in its place (exon 9a), resulting in an in-frame XPC mRNA. Genomic DNA analysis revealed a T-->G mutation at the splice donor site of XPC exon 9, which markedly reduced its information content. The 155 base pair XPC exon 9a insertion was located in intron 9 and was flanked by strong splice donor and acceptor sequences. Analysis of the patient's blood showed persistently low levels of glycine (68 microM; NL, 125-318 microM). Normal glycine levels were maintained with oral glycine supplements and his hyperactivity diminished. These data provide evidence of an association of an XPC splice site mutation with autistic neurologic features and hypoglycinemia.

Alternative Splicing↗
PubMed1997

The DNA damage-recognition problem in human and other eukaryotic cells: the XPA damage binding protein.

The capacity of human and other eukaryotic cells to recognize a disparate variety of damaged sites in DNA, and selectively excise and repair them, resides in a deceptively small simple protein, a 38-42 kDa zinc-finger binding protein, XPA (xeroderma pigmentosum group A), that has no inherent catalytic properties. One key to its damage-recognition ability resides in a DNA-binding domain which combines a zinc finger and a single-strand binding region which may infiltrate small single-stranded regions caused by helix-destabilizing lesions. Another is the augmentation of its binding capacity by interactions with other single-stranded binding proteins and helicases which co-operate in the binding and are unloaded at the binding site to facilitate further unwinding of the DNA and subsequent catalysis. The properties of these reactions suggest there must be considerable conformational changes in XPA and associated proteins to provide a flexible fit to a wide variety of damaged structures in the DNA.

Animals↗
PubMed1997

Quantification of XPA gene expression levels in human and mouse cell lines by competitive RT-PCR.

Expression of the mammalian photoproduct-specific DNA-binding protein XPA has been quantified by competitive reverse transcription-polymerase chain reaction, a method that provides relative numbers of mRNA molecules per cell. Human primary and simian virus (SV)40-transformed fibroblasts had 4.8 and 8.4 transcripts per cell, respectively; mouse embryonic and SV40-transformed fibroblasts had 6.7 and 5.5 transcripts per cell, respectively. None of these differences are significant, and the mean value of 5 to 8 transcripts per cell indicates that XPA is expressed as a low-abundance mRNA. Two cell lines transfected with XPA on a conditional promoter showed different numbers of XPA mRNA molecules, consistent with their respective responses to an inducer and their sensitivity to ultraviolet light. The similarity of results in human and mouse cells shows that a difference in XPA expression cannot account for the greater repair of nontranscribed DNA in human cells.

Animals↗
PubMed1997

DNA repair characteristics and mutations in the ERCC2 DNA repair and transcription gene in a trichothiodystrophy patient.

Patient TTD183ME is male and has typical trichothiodystrophy characteristics, including brittle hair, ichthyosis, characteristic face with receding chin and protruding ears, sun sensitivity, and mental and growth retardation. The relative amount of NER carried out by a TTD183ME fibroblast cell strain after ultraviolet (UV) exposure was approximately 65% of normal as determined by a method that converts repair patches into quantifiable DNA breaks. UV survival curves show a reduction in survival only at doses greater than 4 J/m2. Nucleotide sequence analysis of the ERCC2 (XPD) DNA repair and transcription gene cDNA revealed both a Leu461-to-Val substitution and a deletion of amino acids 716-730 in one allele and an Ala725-to-Pro substitution in the other allele. The first allele has also been reported in one xeroderma pigmentosum group D patient and two other trichothiodystrophy patients, while the second allele has not been previously reported. Comparisons suggest that the mutation of Ala725 to Pro correlates with TTD with intermediate UV sensitivity.

Amino Acid Sequence↗
PubMed1997

Photocarcinogenesis: an overview.

Photocarcinogenesis represents the sum of a complex of simultaneous and sequential biochemical events that ultimately lead to the occurrence of skin cancer. These events, initiated by UV radiation of appropriate wavelength, include the formation of DNA photoproducts: DNA repair; mutation of proto-oncogenes and tumor suppressor genes; UV-production of radical species with subsequent effects on mutation and extra-nuclear function; and other epigenetic events that influence the course of carcinogenesis. The epigenetic influences may include immunological responses, antioxidant defenses, and dietary factors. This review represents an effort to provide current research results in the aforementioned areas and an attempt to meld these events into a comprehensive overview of photocarcinogenesis. If effective prevention and intervention strategies for skin cancer are to developed, a more thorough understanding of the disease process is imperative.

Animals↗
PubMed1997

Ultraviolet irradiation produces novel endonuclease III-sensitive cytosine photoproducts at dipyrimidine sites.

Ultraviolet light irradiation of DNA in vitro and in vivo induces cyclobutane dimers, (6-4) pyrimidine-pyrimidone photoproducts and a variety of minor products. Using a defined DNA fragment, we have identified two classes of sites that can be cleaved by Escherichia coli endonuclease III: single cytosines whose heat lability corresponds to that of cytosine hydrates and more heat-stable dipyrimidines containing cytosine. The dipyrimidine products are induced at sites suggestive of (6-4) photoproducts but are not recognized as (6-4) photoproducts by radioimmunoassay. Use of oligonucleotides containing a single cyclobutane thymine dimer, a (6-4) photoproduct or the Dewar photoisomer of the (6-4) photoproduct also indicated that these products are not substrates for endonuclease III. We have therefore identified a minor UV photoproduct that has the same sequence specificity as the two major dipyrimidine photoproducts; it may be a minor isomer, a unique derivative or an oxidative lesion confined to dipyrimidine sites. Its biological significance is not yet known but may be masked by the preponderance of major products at the same sites. Its occurrence at the particular site in dipyrimidine sequences involved in the mutagenic action of UV photoproducts suggests that it may play a role in generating C to T transitions that are common UV-induced mutations.

Animals↗
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↗