Ruth C. Moore

Effects of 1-β-D-arabinofuranosylcytosine on chromosomes, depending upon the cell cycle stage at the time of exposure

1-beta-D-Arabinofuranosyl cytosine (ara-C) is a clinically important cytotoxic drug which is a potent inhibitor of DNA but which has a minimal effect on other cellular processes. The cytotoxic action of ara-C on mammalian cells has been suggested to be due to the chromosome aberrations induced by this compound. Using a marsupial cell line (JU56), the cells of which contain only 9 readily identified chromosomes, the different types of chromosome aberrations induced by a pulse of ara-C have been quantified, and the cell cycle dependence of the damage has been assessed. It was found that, for cells exposed in G2, both chromatid-type and chromosome-type lesions were produced. The frequency of these lesions was reduced by a chase of deoxycytidine, and there was some evidence that the initial lesions are gaps which may later be converted to true breaks. In early G2 and late S cells, lesions were produced chiefly at one chromosome locations; this location was not specifically late-replicating. At all stages of S, lesions were chiefly chromatid-type, and some exchanges occurred. The level of damage in S cells was not influenced by a deoxycytidine chase. There was negligible damage in cells exposed in G1. It is suggested that the reason previous investigators have obtained very different cell cycle dependence of chromosome damage is that the delaying effects of ara-C on cell cycle progression was not taken into account.

Dose relationships between different effects of aphidicolin in JU56 cells

Some effects of aphidicolin have been investigated in relationship to dose, in a permanent cell line, JU56. Inhibition of semi-conservative DNA synthesis occurred at concentrations greater than 3 X 10(-7) M. In this respect the cells were about as sensitive as L1210 and HeLa cells, and more than 10-fold more sensitive than PHA-stimulated human peripheral blood leucocytes. Delay of progress of cells through G2 occurred at concentrations which inhibited synthesis to about 2% of control levels. Chromatid aberrations appeared in cells at concentrations which decreased synthesis to 5%. Synergism with X-rays in the production of chromatid aberrations occurred at doses which reduced semi-conservative synthesis to 40% of control levels. Isochromatid aberrations appeared in cells continuously exposed to aphidicolin in G2 at concentrations which reduced synthesis to 5% of control units.

An investigation using inhibition of G2 repair of the molecular basis of lesions which result in chromosomal aberrations

Cultures of JU56 cells were irradiated with 2.5 Gy X-rays and 16 h later the cultures were exposed to a moderately inhibitory dose of 1-beta-D-arabinofuranosylcytosine (ara-C) or aphidicolin (APC) and to colcemid, for 2 h. The c-metaphases collected for examination had therefore been exposed to X-rays in G1 or early S, and to the repair inhibitors APC and ara-C during the latter half of G2. It was found that treatment of cells irradiated early in cell cycle, that is, in G1 and early S, with APC or ara-C in G2, (1) reduced the frequency of chromatid and chromosome exchanges below that of cells treated with X-rays alone, (2) produced no more chromatid breaks and gaps than were seen in unirradiated cells, (3) increased the number of chromosome fragments and gaps in a more than additive fashion, and (4) produced only an additive effect, by comparison with the effect of X-rays and drug given separately, on the total number of chromosomal aberrations.

Role of DNA polymerase α and δ in radiation clastogenesis

We here report the results of experiments in which human peripheral blood lymphocytes (HPBL) were X-irradiated in short-term in vitro culture during either the G 0 or the G 2 phases of the cell cycle, and then post-treated with BuPdG or BuAOMe

DNA polymerase α does not mediate G0–G1 increase in yield of X-ray-induced exchange aberrations in human peripheral blood lymphocytes

Abstract We report experiments to test the hypothesis that the increased yield of dicentric chromosomes observed in human peripheral blood lymphocytes treated with X-rays during the G 1 phase of their first cell cycle, as compared with the yield when the cells are treated in their G 0 phase prior to phytohemagglutinin stimulation, is a manifestation of the recently-reported conversion of an inactive form of DNA polymerase α to its active form as the PHA-stimulated cells pass from G 0 into G 1 (Sylvia et al., 1988). The specific polymerase α inhibitor butylphenyl deoxyguanosine was used as an X-ray post-treatment. The results show that polymerase α is not involved.

Dose relationship for different effects of aphidicolin in human peripheral blood leukocytes

Abstract We have previously reported upon dose-yield relationships for some effects of aphidicolin (APC) in cells of the permanent cell line JU56 (Moore et al., 1986). One of the effects which was measured was the frequency of chromosomal aberrations in cells exposed to APC for different lengths of time in G2. We observed that isochromatid aberrations appeared in substantial numbers after some conditions of exposure to APC. We report here upon the relationship between dose, exposure time, and different effects of APC in phytohemagglutinin-stimulated human peripheral blood lymphocytes.

Studies of X-ray-induced chromosome aberrations in cells of the black-tailed wallaby I. Non-random exchange in peripheral blood cells irradiated in G0

The distribution of exchanges between individual chromosome arms in mitotic peripheral blood cells following X-irradiation in Go has been measured. It was found that, although all arms exchanged with each other, there were small but significant departures from the frequencies expected on the basis of random breakage and exchange. It is suggested that non-randomness may reflect the non-uniform state of condensation of chromatin in Go lymphocytes.

DNA polymerase δ mediates increase in exchange production by X-radiation in human lymphocytes moving from G0 to G1

Earlier work of several laboratories established that the yields of radiation-induced ring and dicentric chromosomes are greater when human peripheral blood lymphocytes are irradiated in GH1 some hours after phytohemagglutinin stimulation than if they are irradiated in G0 before stimulation. Post-treatment of lymphocytes irradiated in G0 with the DNA polymerase inhibitor aphidicolin, which is effective against both pol alpha and pol delta, produces a similar increase in ring and dicentric yield. We found that aphidicolin post-treatment was much less effective in increasing ring and dicentric yield increases in cells irradiated in G1 four to five hours after stimulation. Because we had earlier found specific inhibitors of DNA pol alpha ineffective in producing increased yields in either G0 or G1 lymphocytes, we conclude that much of the G0 to G1 increase in yields is mediated by pol delta.

Synergism of 1-β-d-arabinofuranosylcytosine with itself and aphidicolin

Abstract JU56 cells have been exposed to 1-β- d -arabinofuranosylcytosine (ara-C) in S phase, and again to aphidicolin (APC) or ara-C during G 2 , and examined for chromosomal aberrations at c-metaphase. It was found that the two exposures acted synergistically in the production of chromosomal lesions of both the chromatid and isochromatid type. The results were interpreted as indicating that inhibition of the G 2 repair system prevented the repair of DNA single-strand regions produced by the incorporation of ara-C during semiconservative DNA synthesis.

The influence of growth medium on the yield of X-ray-induced chromatid exchanges in the presence and absence of aphidicolin

The frequency of exchanges in JU56 cells irradiated in the G2 phase in the presence and absence of the polymerase inhibitor aphidicolin (APC), and in the presence of a range of concentrations of cysteine was measured. It was found that in the absence of cysteine, incubation for 2 h with APC had no effect on the yield. Addition of cysteine at concentrations of 50-250 mg/l reduced the frequency of exchanges, and at these concentrations the frequency was increased by incubation with APC. At higher concentrations, the yield was reduced and incubation with APC did not elevate it. In following experiments, it was found that incubation with cysteine for a period of longer than 10 minutes was necessary before APC affected the yield of exchanges.