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J J Roberts

Publications and source records attributed to J J Roberts.

At least 73 records · Page 4Linked to original sources
PubMed1978

Effects of cis-platinum(II) diamminedichloride on survival and the rate of DNA synthesis in synchronously growing Chinese hamster V79-379A cells in the absence and presence of caffeine inhibited post-replication repair; evidence for an inducible repair mechanism.

The mechanism by which cis-Platinum(II)diamminedichloride, (cis-Pt(II)), inhibited DNA synthesis was investigated using synchronously growing Chinese hamster V79-379A cells. The progression of G1 phase treated cells into S phase was not inhibited. However the rate of DNA synthesis throughout the S phase was depressed in a dose dependent manner compared to a control. The peak rate of DNA synthesis was delayed and the duration of the S phase was extended resulting in a dose dependent delay in the timing of mitosis in the first cell cycle after treatment. The delay in the timing of mitosis after the second S phase in treated cells was comparable to the delay observed after the first cycle. Post treatment incubation of cells in the presence of a non toxic concentration of caffeine resulted in an alteration in the pattern of DNA synthesis, leading to a reversal of the dose dependent delay in the timing of the peak rate of DNA synthesis to a position corresponding to the control. Caffeine potentiated the toxicity of cis-Pt(II) only if it was added during the first cell cycle after treatment. The patterns of DNA synthesis in synchronous cultures of Chinese hamster V79-379A cells after cis-Pt(II) treatment appeared to be similar to those previously demonstrated for difunctional alkylating agents. The results were discussed in relation to an existing model in which it was postulated that a DNA replication dependent repair mechanism enables cells to replicate DNA using a cis-Pt(II) damaged DNA template. This mechanism has been shown in Chinese hamster V79-379A cells to be sensitive to non toxic concentrations of caffeine resulting in an inhibition of the rate of ligation of nascent DNA which is followed by an increase in chromosome aberrations and cell death. The results presented support the proposed model and emphasise the importance of this replication repair mechanism in enabling cells to tolerate unexcised damage to their genome. In addition we propose that cis-Pt(II) damage stimulates the induction of a system which is involved in enhancing so called post replication repair in Chinese hamster V79 cells and that this induction might be inhibited by the presence of caffeine.

Animals↗
PubMed1978

Excision of hydrocarbon-DNA adducts and consequent cell survival in normal and repair defective human cells.

Survival curves for normal human cells and xeroderma pigmentosum variant cells (XP4BE) after ultraviolet radiation were indistinguishable. In comparison, cells from xeroderma pigmentosum complementation group A (XP12BE) were very sensitive to ultraviolet radiation. Complementation group C (XP2BE) cells were almost as sensitive as group A cells. These survival phenomena parallel to known unscheduled DNA synthesis responses of these cells to ultraviolet radiation, which, compared with normal cells, are: XP4BE, 100%; XP2BE, 20%; XP12BE, 2%. The relative capacities of these cells to excise 7-bromomethylbenz[a]anthracene-DNA adducts and to survive treatment with the carcinogen were similar to the responses to ultraviolet irradiation, except that the XP2BE cell line both excised and survived this damage far better than anticipated from its response to ultraviolet irradiation. Moreover, whilst in the normal cells and variant cells the ratio of hydrocarbon-adenine adduct to hydrocarbon-guanine adduct remaining in DNA decreased notably with excision, this ratio did not change significantly with excision in the XP2BE cell line. The relationship between greater excision capacity and increased cell survival in the experiments with the chemical carcinogen indicates that the unexcised damage is responsible for the cell-killing action of this agent. The different relative repair and survival responses of these cell lines to ultraviolet irradiation on the one hand, and to 7-bromomethylbenz[a]anthracene chemical carcinogen treatment on the other, indicate that in at least one of these cell lines (XP2BE), and possibly in all the lines, different cellular mechanisms are involved in the repair of DNA damage resulting from ultraviolet irradiation and that resulting from the chemical carcinogen treatment.

Benz(a)Anthracenes↗
PubMed1978

Increased sensitivity of UV-repair-deficient human cells to DNA bound platinum products which unlike thymine dimers are not recognized by an endonuclease extracted from Micrococcus luteus.

We have studied the response of human cells in culture to cis platinum[II] diammine dichloride (cis Pt[II]) induced DNA damage. The survival data, measured as a function of cis Pt[II] dose were similar in a normal cell line (Human foetal lung) compared to a UV-sensitive, thymine dimer excision repair-deficient cell line (Xeroderma pigmentosum). However, there was a marked difference between the two cell lines when binding to DNA was plotted against dose of cis Pt[II] given for 1 h. When these findings were expressed as cell survival versus binding to DNA, a 4.1--fold difference between the slopes of the survival curves for the two cell lines was obtained. These findings are consistent with the notion that normal cells are able to excise cis Pt[II] induced damage from their genome and thus increase their ability to survive as compared to excision-deficient cells. An endonuclease preparation from Micrococcus luteus is able to recognise UV damage in DNA, but did not recognise cis Pt[II] induced damage. These results possibly indicate differences in the pathways of repair of damage caused by the two agents.

Cell Line↗
PubMed1978

Caffeine-inhibited DNA repair in 7-bromomethylbenz (a)-anthracene-treated Chinease hamster cells: formation of breaks in parental DNA and inhibition of ligation of nascent DNA as a mechanism for enhancement of lethality and chromosome damage.

The cytotoxic and clastogenic effects of 7-bromomethylbenz(a)anthracene (7-BMBA) are potentiated by post-treatment incubation of cells in the presence of an non-toxic concentration of caffeine. Under these conditions caffeine inhibits the rate of ligation of newly-synthesised DNA and induces breaks in the template strand of DNA. It is proposed that endonucleolytic attack occurs at the site of lesions in the template strand of DNA and that a later step(s) of excision-repair is (are) inhibited by the presence of caffeine-induced 'gap' in the nascent DNA opposite these lesions.

Benz(a)Anthracenes↗
PubMed1978

Caffeine inhibits excision of 7-bromomethylbenz (a) anthracene-DNA adducts from exponentially growing but not from stationary phase Chinese hamster cells.

Excision of 7-bromomethylbenz(a)anthracene (7-BMBA)-DNA adducts from exponentially growing cultures of Chinese hamster V79-379A cells followed logarithmic kinetics with a half of approximately 20 hrs. Post-treatment incubation in the presence of a sub-toxic concentration of caffeine markedly reduced this loss. Caffeine brought about a concomitant increase in overall DNA synthetic rate in treated exponential cultures. Excision in stationary, non-DNA-replicating cultures, was slower and caffeine did not affect this reduced rate of excision. These findings lend support to a previous proposition that the caffeine-induced inhibition of elongation of nascent DNA on a template containing chemical lesions results in an interference with the excision repair mechanism that removes these lesions.

Animals↗
PubMed1977

Excision of 7-bromomethylbenz[a]anthracene--DNA adducts in replicating mammalian cells.

The excision of 7-bromomethylbenz[a]anthracene--DNA adducts was studied in two cell lines (HeLa S-3 and Chinese hamster V-79379A). In both cell lines, carcinogen-modified adenine residues were excised more readily than the modified guanine residues and the percentage of the total products excised decreased after treatment with higher concentrations of carcinogen. At the highest concentrations used in the Chinese hamster cells, neither DNA synthesis nor excision was detected. The lowest concentration used for these cells permitted almost 100% survival and all the DNA was replicated in a 30-h interval even though 50% of the initial damage was still present. The two- to threefold lower sensitivity of the Chinese hamster cells (compared with the Hela cells) to the carcinogen is attributed to this capacity for replication of DNA on a damaged template since the two cell lines' capacities for excision of the chemical damage were found to be comparable.

Benz(a)Anthracenes↗
PubMed1976

Inhibition by caffeine of post-replication repair in Chinese hamster cells treated with cis platinum (II) Diamminedichloride: the extent of platinum binding to template DNA in relation to the size of low molecular weight nascent DNA.

Treatment of Chinese hamster lung V79-379A cells with the anti-tumour agent cis platinum (II) diamminedichloride, (cis Pt(II)), resulted in an immediate recuction in the rate of DNA synthesis. Sedimentation of newly synthesised DNA through alkaline sucrose gradients revealed it to be approximately the same size as that obtained from untreated cells. In contrast, in the presence of 0.75 mM caffeine, the rate of DNA synthesis rapidly returned to control levels, although sedimentation analysis showed the DNA synthesised in cis Pt(II)-treated cells to be of lower molecular weight than in untreated cells. The reduction in molecular weight was directly proportional to the initial dose of the platinum compound. Furthermore, the results of separate binding studies suggested that at several levels of reaction the new DNA was synthesised up to a size approximately equal to the interplatinum distance in the template strand. This has been interpreted as being the result of the formation of a gap in the daughter DNA strand opposite every DNA-platinum product in the template strand. If caffeine was removed from the culture medium, there was a rapid increase in the molecular weight of the nascent DNA strands. However, if caffeine remained in the medium, the DNA remained of lower molecular weight than in untreated cells. It is proposed that this effect of caffeine is the result of the inhibition of a post-replicative DNA repair process which allows the eventual synthesis of a continuous DNA strand on a template containing unexcised lesions. It is further proposed that inhibition of this post-replicative DNA repair process provides a molecular basis for the previously observed potentiation by caffeine of cis Pt(II)-induced chromosomal aberrations and lethality.

Binding Sites↗
PubMed1975

Inactivation of the DNA template in HeLa cells treated with chlorambucil.

The effect of chlorambucil on the colony-forming ability of HeLa cells following either treatment for 1 h or continuous treatment has been measured. Concentrations of chlorambucil which had only minimal effects on cell survival inhibited the rate of DNA but not RNA and protein synthesis within 1 h of treatment. Nevertheless, cells continued to synthesize DNA for many hours after this treatment in the absence of cell division. Synchronous populations of HeLa cells treated prior to DNA synthesis, in the G1 phase of the cell cycle, were not delayed in their progression into the S phase where they exhibited a marked dose-dependent inhibition of the rate of DNA synthesis. Cells in which DNA synthesis had been depressed showed a prolongation of the S phase and this was accompanied by a corresponding dose-dependent mitotic delay. Treatment during the G2 phase of the cell cycle did not induce any delay or block in the next mitosis, but did inhibit the rate of DNA synthesis in the following cell cycle in a dose-dependent manner; this depression of DNA synthesis was followed by a delay in the next mitosis. Cross-linking of either isolated DNA or DNA present in treated HeLa cells was demonstrated, and in the latter case calculated to be of the same order as that which occurred with other difunctional agents at comparable toxic concentrations.

Cell Division↗
PubMed1975

Investigations into the mechanism of action of anti-tumour platinum compounds: time- and dose-dependent changes in the alkaline sucrose gradient sedimentation profiles of DNA from hamster cells treated with cis-platinum (II) diamminedichloride.

Under the conditions of low speed centrifugation used in this study, the proportion of radioactively labelled DNA from Chinese hamster V79-379A cells sedimenting to the 700S region of an alkaline sucrose gradient was increased in a dose-dependent manner by prior treatment of the cells for 2 h with cis-platinum (II) diamminedichloride [cis-Pt(II)]. This increase was at the expense of material sedimenting in the 400-650S region. This profile was not modified by a 2-h post-treatment incubation prior to centrifugation. 6 h after treatment, the DNA from treated cells sedimented in a narrow band at a position corresponding to 350S. 21 h after exposure to the drug, a dose-dependent restitution of the DNA species sedimenting in the 450-650S range was observed. These results combined with other data relating to platinum binding allow the following conclusions to be reached: (1) cis Pt(II) treatment does not lead to the rapid formation of single-stranded breaks or alkali-labile sites in cellular DNA. (2) The time-dependent changes in sedimentation rate of DNA from treated cells may reflect the transient appearance of gaps following endonuclease attack at platinum-bound sites in DNA. (3) The likely ratio of inter to intra-strand DNA-platinum interactions suggests that such endonuclease attack is primarily at platinum induced inter-strand DNA cross-links.

Animals↗
PubMed1975

Post-replication repair of DNA in Chinese hamster cells treated with cis platinum (II) diamine dichloride. Enhancement of toxicity and chromosome damage by caffeine.

The anti-tumor agent cis platinum (II) diammine dichloride (cis Pt(II)) caused chromosomal abnormalities in Chinese hamster V79-379A cells. The time of appearance of these abnormalities suggested that they arise as a consequence of DNA synthesis on a damaged template. The yield and severity of chromosomal abnormalities was greatly enhanced by a non-toxic concentration of caffeine, and this enhancement was associated with a potentiation of cis Pt(II) induced cell death. These results suggest that damage to DNA which arises from cis Pt(II) treatment can be repaired in this cell line by a caffeine-sensitive post-replication repair process.

Animals↗