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Biomedical subjects

D L Mitchell

Publications and source records attributed to D L Mitchell.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1997

Differentiation-dependent p53 regulation of nucleotide excision repair in keratinocytes.

The role of the tumor suppressor p53 in repair of ultraviolet light (UV)-induced DNA damage was evaluated using a host-cell reactivation (HCR) assay. HCR determines a cell's ability to repair UV-damaged DNA through reactivation of a transfected CAT reported plasmid. Most UV damage is removed through nucleotide excision repair (NER). Primary murine keratinocytes isolated from p53-deficient and wild-type p53 mice were used in the HCR assay. The NER was reduced in p53-/- keratinocytes as compared with p53+/+ keratinocytes. The reduced DNA repair in p53-/- mice was confirmed with a radioimmunoassay comparing cyclobutane dimers (CPDs) and (6-4) photoproducts in p53+/+ and p53-/- keratinocytes after the cells were exposed to UV irradiation. Our results demonstrate that wildtype p53 plays a significant role in regulating NER. Furthermore, as there is evidence that p53 protein levels decrease after keratinocytes become differentiated, we sought to determine whether p53 plays a role in NER in differentiated keratinocytes. Differentiation of the keratinocytes by increasing the Ca2+ concentration in the culture media resulted in a marked reduction in NER equally in both p53+/+ and p53-/- groups. This finding suggests that reduced DNA repair after differentiation is p53 independent. A similar reduction in HCR was confirmed in differentiated human keratinocytes. These data, taken together, indicate that p53 or p53-regulated proteins enhance NER in basal undifferentiated keratinocytes but not in differentiated cells. As nonmelanoma skin cancers originate from the basal keratinocytes, our findings suggest that loss of p53 may contribute to the pathogenesis of this common skin cancer.

Animals↗
PubMed1997

UV-sensitive rodent mutant cell lines of complementation groups 6 and 8 differ phenotypically from their human counterparts.

Rodent UV-sensitive mutant cell lines of complementation groups 6 and 8 are the genetic counterparts of human Cockayne syndrome CS-B and CS-A, respectively. The original mutant in this group, UV61, was described as defective in cyclobutane pyrimidine dimer removal after high doses of UV. We have examined the responses of several cell lines from group 6 to low doses of UV irradiation, and find that these mutants have wild-type capacity for DNA repair as indicated by incision, cyclobutane pyrimidine dimer, and (6-4) photoproduct removal. ERCC6, the product of the gene defective in CS-B and group 6 mutants, is implicated in the regulation of repair of actively transcribed genes in Cockayne syndrome; however, this protein clearly is not required for the processing of low levels of damage in CHO cells, which occurs remarkably efficiently, 40-50% of dimers being removed in both wild-type and group 6 mutants in 5 hours following 0.1 Jm(-2) of UV. The group 8 mutant cell line US31, on the other hand, is very deficient in repair of UV damage, showing a more extreme phenotype than is seen in the corresponding human syndrome CS-A. In both complementation groups, expression of mutations in a gene involved in regulation of DNA repair takes very different forms in human and rodent cells.

Animals↗
PubMed1996

A CHO mutant, UV40, that is sensitive to diverse mutagens and represents a new complementation group of mitomycin C sensitivity.

A new mitomycin C (MMC)-sensitive rodent line, UV40, has been identified in the collection of ultraviolet light- (UV-) sensitive mutants of Chinese hamster ovary (CHO) cells isolated at the previous Facility for Automated Experiments in Cell Biology (FAECB). It was isolated from an UV mutant hunt using mutagenesis of AA8 cells with the DNA intercalating frameshift mutagen ICR170. It is complemented by CHO-UV-1, irsl, irs3, irslSF, MC5, V-C8 and V-H4 with respect to its MMC sensitivity based on cell survival. Despite having approx. 4 X normal UV sensitivity and increased sensitivity to UV inhibition of DNA replication, it has near-normal incision kinetics of UV irradiated DNA, and normal (6-4) photoproducts removal. It also is not hypermutable by UV, and shows near normal levels of UV inhibition of RNA synthesis. UV40 also has approx. 11 x .10 x .5 x and 2 x AA8 sensitivity to MMC, ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), and X-rays, respectively. Thus, its defect apparently does not involve nucleotide excision repair but rather another process, possibly in replicating past lesions. The spontaneous chromosomal aberration frequency is elevated to 20% in UV40, and the baseline frequency of sister chromatid exchange is also approximately 4-fold increased. The phenotype of UV40 appears to differ from all other rodent mutants that have so far been described.

Adenine↗
PubMed1996

Evidence for a novel DNA damage binding protein in human cells.

We describe a novel DNA damage binding activity in nuclear extracts from a normal human fibroblast cell strain. This protein was identified using electrophoretic mobility shift assays of immunopurified UV-irradiated oligonucleotide substrates containing a single, site-specific cyclobutane pyrimidine dimer or a pyrimidine (6-4) pyrimidinone photoproduct. Compared with the (6-4) photoproduct, which displayed similar levels of binding in double and single-stranded substrates, the protein showed somewhat lower affinity for the cyclobutane dimer in a single-stranded oligonucleotide and negligible binding in double-stranded DNA. The specificity and magnitude of binding was similar in cells with normal excision repair (GM637) and repair-deficient cells from xeroderma pigmentosum groups A (XP12RO) and E (XP2RO). An apparent molecular mass of 66 kDa consisting of two subunits of approximately 22 and approximately 44 kDa was determined by Southwestern analysis. Cell cycle studies using centrifugal cell elutriation indicated that the binding activity was significantly greater in G1 phase compared with S phase in a human lymphoblast cell line. Gel supershift analysis using an anti-replication protein A antibody showed that the binding protein was not antigenically related to the human single-stranded binding protein. Taken together, these data suggest that this activity represents a novel DNA damage binding protein that, in addition to a putative role in excision repair, may also function in cell cycle or gene regulation.

Base Sequence↗
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↗
PubMed1996

Decreased DNA repair but normal apoptosis in ultraviolet-irradiated skin of p53-transgenic mice.

p53 tumor suppressor plays a vital role in the cellular responses to genotoxic stress. It is believed that p53 regulates the cell cycle by activating the G1 checkpoint after exposure to agents like ionizing radiation, ultraviolet (UV) radiation, or genotoxic chemicals. Recently, it is conjectured that p53 may have additional functions in DNA repair and apoptosis. Previously, we demonstrated that p53-transgenic mice that carry mutant alleles of a p53 gene developed twice as many skin tumors as control mice after UV exposure. To elucidate the molecular mechanisms of mutant p53 in skin cancers, we studied DNA repair efficiency and the rate of apoptosis in murine keratinocytes after UV irradiation. In this report, we show that mutant p53-transgenic mouse skin has reduced repair of UV-induced DNA damage in both in vivo and in vitro radioimmunoassays. In control mice, DNA repair is associated with increased amounts of wild-type P53 protein. Unexpectedly, mutant p53-transgenic mice had slightly increased apoptosis after UV irradiation, suggesting that the wild-type p53 protein in the cells still functions in inducing apoptosis, or that this cell death results from p53-independent mechanisms. These results suggest that mutant p53 interferes with wild-type p53 in the repair of UV-induced DNA damage but not in apoptosis.

Animals↗
PubMed1996

Teaching managers to appraise performances.

To accomplish effective performance reviews and appraisals that respect employee dignity, managers must cultivate a wide range of competencies. Systems are required to observe and collect data, cue and measure observations, then interpret behavior.

Education, Nursing, Continuing↗
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

Repair of UV-induced (6-4) photoproducts in nucleosome core DNA.

Using radioimmunoassays, we examined rates of removal of UV-induced pyrimidine-pyrimidone (6-4) photoproducts ((6-4)PDs) and cyclobutane pyrimidine dimers (CPDs) from 146-base pair nucleosome core DNA (and 166-base pair chromatosome DNA) of confluent human diploid fibroblasts. Dose-response experiments indicate that the yield of (6-4)PDs in core DNA is about 30% that of CPDs in the UV dose range of 0-200 J/m2. Repair experiments indicate that, at 40 J/m2, (6-4)PDs are removed much faster (approximately 75% in 2 h) from nucleosome core (and chromatosome) DNA than CPDs (10-15% in 2 h). A slow rate of removal of CPDs is also observed when the UV dose is reduced to 10 J/m2 (i.e. even when the level of CPDs is less than that of (6-4)PDs at 40 J/m2). These results indicate that (a) the accessibility of repair proteins to (6-4)PDs in nucleosomes is markedly different than their accessibility to CPDs and/or (b) repair enzymes are much more efficient at incising and removing (6-4)PDs than CPDs in human chromatin.

Cells, Cultured↗
PubMed1995

Evidence for a basalt-free surface on Mercury and implications for internal heat.

Microwave and mid-infrared observations reveal that Mercury's surface contains less FeO + TiO2 and at least as much feldspar as the lunar highlands. The results are compatible with the high albedo (brightness) of Mercury's surface at visible wavelengths in suggesting a rock and soil composition that is devoid of basalt, the primary differentiate of terrestrial mantles. The occurrence of a basalt-free, highly differentiated crust is in accord with recent models of the planet's thermal evolution and suggests that Mercury has retained a hot interior as a result of a combination of inefficient mantle convection and minimal volcanic heat loss.

Ferrous Compounds↗
PubMed1995

Enhanced photoproduct repair: its role in the DNA damage-resistance phenotype of human malignant melanoma cells.

A fundamental issue in understanding melanoma is to seek the basis for the cellular resistance to DNA damaging agents, which is manifested in vivo as pronounced tumor resistance to therapeutic agents. The published consensus on melanoma has been that exaggerated postreplication recovery (PRR), rather than excision repair, underlies the unusual damage-resistance phenotype. We examined the resistance to the model DNA damaging agent, UV-C, of subclones derived from a human metastatic melanoma cell line. The clones essentially fall into two groups: one with normal and the other with enhanced resistance. We exploited this range to investigate the interrelationships between replication, transcription, and repair of DNA after UV irradiation. Subclones resistant to UV killing were indeed found to possess enhanced rates of PRR and were coresistant to cisplatin. However, we now report an overall elevation of photoproduct repair in both melanoma groups compared to nonmelanoma controls and conclude that this accounts for the resistant melanoma phenotype, including that of enhanced PRR. Repair enhancement may explain chemoresistance, while loss of efficiency of certain functions, such as PRR, due to the intrinsic genetic lability of tumor cells, may generate the class of melanoma subclones exhibiting only normal resistance.

Cisplatin↗
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↗
PubMed1994

Excision of cyclobutane dimers in genomic and episomal DNA in human cells.

Direct determination has been made of cyclobutyl pyrimidine dimer induction and excision repair in an episomal SV40 DNA population in vivo. Maintaining SV40-transformed human (GM637) cells in confluent culture results in amplification of a mutant SV40 episome to high copy number. T4 endonuclease V was used to quantify the induction and repair of cyclobutane dimers in the SV40 episome and genomic DNA of the same cells. Differences in both parameters were observed; cyclobutane dimers were induced at 1.5-2-fold greater frequency in episomal DNA and excised at a reduced rate compared to genomic DNA in the host cells.

Cell Line, Transformed↗
PubMed1993

New patterns of bulk DNA repair in ultraviolet irradiated mouse embryo carcinoma cells following differentiation.

Mouse embryocarcinoma stem cells differentiate in culture, given the appropriate induction. We examined whether these cells could provide information about the regulation of nucleotide excision repair in relation to differentiation by measuring the rate-limiting incision step, the removal of cyclobutane dimers and (6-4) photoproducts from the genome as a whole and the effect of the bacteriophage T4 endonuclease (denV) gene on repair in differentiated cells. It was found that differentiation is accompanied by a marked decline in the early incision ability after UV irradiation (sixfold for P19, fourfold for PCC7 and twofold for F9), and we measured, in parallel, the loss of two common UV photoproducts [cyclobutane dimers and (6-4) photoproducts] from P19 cells. After differentiation, the excellent overall cyclobutane dimer repair capacity of proliferating cells (84% removal in 24 h) is lost (no removal in 24 h), while removal of (6-4) photoproducts, although normal at 24 h (94%), is much slower than in undifferentiated P19 at 3 h (no removal versus 64%). The presence of the denV gene greatly stimulates the repair of cyclobutane dimers in undifferentiated P19 cells (94% removal at 3 h versus 40%) and also in differentiated cells (50% removal at 24 h versus no removal). The denV gene also stimulates the early repair of (6-4) photoproducts in both differentiated and undifferentiated cells.

Animals↗