Biomedical subjects

G C Elliott

Publications and source records attributed to G C Elliott.

8 recordsLinked to original sources

PubMed1989

Lack of complementation between xeroderma pigmentosum complementation groups D and H.

The construction of permanent hybrid cell lines between xeroderma pigmentosum (XP) cells from different complementation groups allows analysis not only of the degree of repair correction but also of the restoration of biological activity to the UV-irradiated cells. With use of an immortal human cell line (HD2) that expresses excision repair defects typical of XP group D, a series of permanent hybrid cells has been produced with XP cells from groups A to H. Excision repair, as measured by incision analysis and unscheduled DNA synthesis, is restored to normal or near normal levels in crosses involving HD2 and cells from XP groups A, B, C, E, F, G, and I. All these hybrids show complementation for the recovery of normal UV resistance. As expected, hybrids expressing poor incision and hypersensitivity to UV were produced in crosses between HD2 and XPD fibroblasts, but they were also produced without exception when XPH was the partner. In the permanent HD2 x XPD or XPH hybrids, analysis of incision capacity reveals abnormally low activity and therefore that there has been no complementation. The true hybrid nature of HD2 x XPH cells has been confirmed by HL-A and -B tissue typing; moreover, detailed kinetic analysis of incision in these cells shows that the XPH phenotype, rather than the XPD, is expressed, i.e. breaks accumulate at low UV fluence of 1 J/m2. To help confirm these findings, another immortal XPD cell line was used in fusions involving HD2, XPH, or XPI. Cells resistant to ultraviolet were produced only with XPI fibroblasts. These data are discussed in terms of whether XPD and H mutations are likely to be allelic with respect to incision.

DNA Damage↗

PubMed1988

DNA-repair reactions by purified HeLa DNA polymerases and exonucleases.

PM2 duplex DNA substrates containing small gaps were utilized to study DNA repair reactions of extensively purified HeLa DNase V (a bidirectional double strand DNA exonuclease) and DNA polymerases beta, gamma (mitochondrial and extramitochondrial), and alpha holoenzyme, and delta as a function of ionic strength. At 50 mM NaCl, DNase V carried out extensive exonucleolytic degradation, and beta-polymerase exhibited strand displacement synthesis. However, at 150 mM NaCl, the DNase appeared only to remove damaged nucleotides from DNA termini while beta-polymerase catalyzed only gap-filling synthesis. When present in equimolar amounts, beta-polymerase and DNase V (which can be isolated as a 1:1 complex) catalyzed more degradation than synthesis at 50 mM NaCl; however, at 150 mM NaCl a coupled very limited nick translation reaction ensued. At physiological ionic strength DNA polymerase alpha holoenzyme was not active upon these substrates. In 15 mM KCl it could fill small gaps and carry out limited nick translation with undamaged DNA, but it could not create a ligatable substrate from UV-irradiated DNA incised with T4 UV endonuclease. Mitochondrial DNA polymerase gamma was more active at 150 mM NaCl than at lower ionic strengths. It readily filled small gaps but was only marginally capable of strand-displacement synthesis. The extramitochondrial form of gamma-polymerase, conversely, was less sensitive to ionic strength; it too easily filled small gaps but was not effective in catalyzing strand displacement synthesis. Finally, DNA polymerase delta was able to fill gaps of several to 20 nucleotides in 0.05 M NaCl, but at higher NaCl concentrations there was little activity. DNA polymerases delta did not demonstrate strand displacement synthesis. Therefore, at physiological ionic strength, it appears that either DNA polymerase beta or extramitochondrial DNA polymerase gamma might aid in short patch DNA repair of nuclear (or transfecting) DNAs, whereas mitochondrial gamma-polymerase might fill small gaps in mitochondrial DNA.

DNA↗

PubMed1987

Slow DNA rejoining in ultraviolet-irradiated human diploid fibroblasts treated with the mitogens trypsin and insulin.

Normally in mammalian cells the postincision steps of UV-induced excision repair are much more rapid than the recognition of damage and incision. This means that at any one time the level of repair-generated single-stranded DNA breaks is very low. Here we report that detectable levels of DNA breaks accumulate in quiescent human fibroblasts which are UV irradiated a few hours after replating in conditions that stimulate progress through the cell cycle. Most DNA breaks accumulate in cultures trypsinized and seeded in medium supplemented with insulin, and irradiated in early G1. Because trypsin and insulin have no effect on UV-induced incision rates, as measured by DNA break accumulation in the presence of DNA synthesis inhibitors, we argue that our ability to detect incomplete repair-sites is due to a significant reduction in the rate of gap sealing indicative of a shift in the steady state of excision repair. Provision of DNA precursors prevents the enhancing effect of trypsin and insulin on the accumulation of DNA breaks, implying that these agents affect DNA precursor metabolism. Perturbation of the repair process, which leads to the accumulation of 1500-2000 DNA breaks/genome, is also associated with other effects including increased lethality, the appearance of double-strand breaks and the loss of NAD, the last effect presumably arising as a consequence of break-stimulated poly(ADPR) transferase activity. Addition of 3-amino-benzamide, an inhibitor of poly(ADPR) synthesis, completely blocks the decline in NAD levels, but does not change the rate of sealing of the accumulated DNA breaks. These results strongly suggest that ligation is largely, if not entirely, independent of ADP ribosylation in this system.

Adenosine Diphosphate Ribose↗

PubMed1987

DNA repair under stress.

When the excision repair process of eukaryote cells is arrested by inhibitors of repair synthesis including hydroxyurea (HU), 1-beta-D-arabinofuranosylcytosine (araC) or aphidicolin, major cellular changes follow the accumulation of repair-associated DNA breaks. These changes, each of which reflects more or less severe cellular stress, include cycle delay, chromosome behaviour, fall in NAD level, the development of double-stranded DNA breaks, rapid chromosome fragmentation and cell killing. Disruption of the repair process by agents such as araC after therapeutic DNA damage may, therefore, have some potential value in cancer treatment. The extreme cellular problems associated with the artificial arrest of repair may have their subtler counterparts elsewhere, and we discuss several systems where delays in the completion of excision repair in the absence of repair synthesis inhibitors have marked repercussions on cell viability. We also show that the average completion time of an excision repair patch varies according to the state of cell culture, and that completion time is extended after treatment with insulin or following trypsin detachment. Under certain growth conditions ultraviolet irradiation followed by mitogenic stimulation results in double-stranded DNA breakage and additional cell killing, and we discuss these data in the light of protocols that have been used successfully to transform human or rodent cells in vitro. Finally, we consider whether the rejoining of DNA breaks accumulated by repair synthesis inhibitors is a valid model system for studying ligation, and show that this protocol provides an extremely sensitive assay for most incision events and, thereby, a means for discriminating between normal human cells on the one hand, and Cockayne's Syndrome cells and their heterozygotes on the other.

Aphidicolin↗

PubMed1986

Qualitative differences between replicative and repair synthesis of DNA in normal and transformed mouse cells as measured by precursor discrimination.

Inhibitors of DNA polymerase alpha such as aphidicolin (APC) or 1-beta-D-arabinofuranosyl-cytosine (araC) cause DNA-strand breaks to accumulate after UV-irradiation, at sites where repair resynthesis is inhibited. Transformed cells accumulate fewer such breaks than normal cells do; this may be due to differences in the extent, or the nature, of excision-repair synthesis in transformed and in normal cells. We have looked for differences in the nature of repair synthesis, comparing the labelling of DNA by deoxycytidine (dC) and araC through UV-induced repair in normal and transformed mouse cells. We have made parallel determinations of precursor discrimination in replicative synthesis, and find that normal cells discriminate better against araC in replicative synthesis than do transformed cells. But repair synthesis discriminates against araC less than normal replicative synthesis does, to a similar extent in both cell types. Thus, there are qualitative differences between the DNA polymerases engaged in UV excision repair and replication in normal and transformed mouse cells; but there is no evidence for a predominantly araC-insensitive repair synthesis in transformed cells, such as might account for the difference in break accumulation.

Animals↗

PubMed1986

Analysis of DNA repair in XP-HeLa hybrids; lack of correlation between excision repair of u.v. damage and adenovirus reactivation in an XP(D)-like cell line.

Hybrids formed between HeLa cells and fibroblasts from xeroderma pigmentosum group D show either HeLa sensitivity or XPD-like hypersensitivity to u.v. radiation and corresponding high or low excision repair capability. Hybrids with low repair are presumed to have lost, via chromosome segregation, the HeLa wild type D alleles. In this paper we analyse the u.v. sensitivity and excision repair capability of another hybrid, HD1A, derived spontaneously from the normally sensitive hybrid HD1. While HD1A closely resembles the XPD phenotype in terms of u.v. sensitivity and excision repair it differs from XPD because of its ability to reactivate u.v.-irradiated adenovirus 2 to an extent similar to that of its HeLa parent. This capacity functionally dissociates excision repair of chromatin-based damage from damage in a viral environment. Moreover, on the basis of complementation studies the excision repair of genomic damage by HD1A is subtly different from that of a true XPD-like hybrid, HD2. The data are discussed in terms of a second change in the defective D allele of the HD1A cell.

Adenoviridae↗

PubMed1985

DNA repair in mouse embryo fibroblasts. II. Responses of nontransformed, preneoplastic and tumorigenic cells to ultraviolet irradiation.

Ultraviolet light-induced excision repair, as measured by single-strand DNA-break accumulation in the presence of hydroxyurea and 1-beta-D-arabinofuranosylcytosine, undergoes an apparent decline concomitant with spontaneous transformation of mouse cells in vitro. This decline is seen in preneoplastic transformed cells as well as tumorigenic cells, suggesting that it is an early event in transformation. The difference between nontransformed and transformed mouse cells in apparent incision rates is greatest at short times after irradiation when nontransformed cells show a transient phase of rapid incision. No gross differences in the effects of UV on replicative DNA synthesis, bulk RNA synthesis, cell proliferation or clonal survival in nontransformed and transformed cells were seen, in spite of the reduced incision capacity of the latter. Taken together the results suggest that transformed cells are capable of growth in the presence of significantly increased amounts of DNA damage. A decreased ability of nontumorigenic cells to remove DNA lesions, coupled with unrestricted growth, may be responsible for genetic alterations which increase the probability of a cell becoming tumorigenic.

Animals↗

PubMed1985

Xeroderma pigmentosum D-HeLa hybrids with low and high ultraviolet sensitivity associated with normal and diminished DNA repair ability, respectively.

Fusion between HeLa and fibroblasts from complementation group D xeroderma pigmentosum (XPD) followed by challenge with small doses of ultraviolet light (u.v.) results in the production of hybrid cells expressing either HeLa (HD1) or XPD-like (HD2) sensitivity to u.v. and related repair capacity. Assays used included unscheduled DNA synthesis (UDS), DNA break accumulation in the presence of inhibitors of DNA repair synthesis and host cell reactivation of irradiated adenovirus. Complementation assay in heterokaryons reveals limited ability of HD2 to restore UDS in XPD nuclei. We believe this complementation is more apparent than real since proliferating hybrids of HD2 and XPD parentage are without exception u.v.-sensitive and express limited excision repair. On the other hand hybrids between HD2 and XPC, XPE or XPF fibroblasts show true complementation resulting in a return to normal u.v. sensitivity and elevated repair ability.

Cells, Cultured↗