Developmental psychology. Arithmetic in the cradle.
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Humans↗
Biomedical subjects
P E Bryant
Publications and source records attributed to P E Bryant.
Induction of chromosomal damage by restriction endonuclease in CHO cells porated with streptolysin O.
A procedure is described for the poration of living CHO cells with the bacterial cytotoxin streptolysin O (SLO) which allows the introduction into cells of the restriction endonuclease Pvu II to mimic and model the effects of ionising radiation in causing chromosomal damage. The dependence of this clastogenic effect of Pvu II on SLO concentration was measured by assaying the formation of micronuclei in cytokinesis-blocked binucleate cells. The optimum concentration was found to be 0.045 U/ml. Using the micronucleus assay, the time-course of expression of chromosome damage was investigated and found to show a biphasic kinetic with time. Using a sampling time of 30 h, a dose-effect curve for micronucleus induction by Pvu II was generated. Using this optimized SLO treatment protocol, the frequency of metaphase chromosome damage was subsequently investigated and found to be also linearly related to Pvu II concentration and total aberrations were approximately double the frequency of micronuclei. The induction and repair kinetics of DNA double-strand breaks were investigated in CHO cells treated with SLO and Pvu II using the neutral filter elution technique at pH 9.6. The data presented show that SLO can be used as an alternative method for porating cells to allow the introduction of restriction endonucleases into cells.
Animals↗
Effects of adenine arabinoside and coformycin on the kinetics of G2 chromatid aberrations in X-irradiated human lymphocytes.
The kinetics of chromatid aberrations have been studied in human lymphocytes exposed to X-rays in the G2 phase of the cell cycle and incubated with or without the nucleoside analogue 9-beta-D-arabinofuranosyladenine (ara A), known to inhibit the repair of DNA double-strand breaks. In the absence of ara A an exponential decrease in frequencies of chromatid breaks occurred which we interpret as repair. Few breaks were observed if samples were harvested immediately following irradiation. The frequency of chromatid breaks at 1 h after X-irradiation (442 per 100 cells/Gy) was similar to that previously observed in Chinese hamster ovary (CHO) K1 cells. However, the exponential decrease of chromatid breaks between X-irradiation and sampling occurred with a t1/2 of 0.87 h, a faster rate than we have previously observed in CHO K1 cells and was not inhibited by 200 microM ara A alone, in contrast with our previous findings in a human fibroblast line. However, in the presence of the ADA inhibitor coformycin, inhibition of break repair was already observed at an ara A concentration of 100 microM indicating that the apparent unresponsiveness to ara A of lymphocyte chromatid break rejoining results from the deamination of this nucleoside analogue. This deamination effect was confirmed by measurements of DNA synthesis which showed stable inhibition of synthesis by ara A only when coformycin was present. Frequencies of chromatid exchanges in irradiated cells remained constant except at the sampling time directly after irradiation, consistent with the view that chromosomal radiosensitivity remained constant throughout the G2 phase, except for the period immediately prior to mitosis.(ABSTRACT TRUNCATED AT 250 WORDS)
Chromatids↗
Mimicking radiation with restriction endonucleases.
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Animals↗
Developmental psychology. Face to face with babies.
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Humans↗
Lack of interference of DNA single-strand breaks with the measurement of double-strand breaks in mammalian cells using the neutral filter elution assay.
In this study, the effect of DNA single strand breaks (ssb) on the neutral (pH 9.6) filter elution of DNA from Chinese hamster ovary (CHO K1) cells containing DNA double strand breaks (dsb) was investigated. Protein associated ssb were induced by the inhibition of DNA topoisomerase I with camptothecin (cpt). Protein associated dsb were introduced by treating cells with the DNA topoisomerase II poison; etoposide (VP-16). Protein associated ssb and dsb were converted to ssb and dsb by proteinase K present in the lysis solution. In some experiments dsb were generated by the restriction endonuclease Pvu II. It was found that elution of DNA in the presence and absence of ssb was similar under neutral conditions. This finding is consistent with the view that the fast component of the bi-phasic repair kinetics observed in irradiated mammalian cells with the neutral filter elution technique is not attributable to the interference of ssb with the measurement of dsb, and thus suggests that the two components of repair observed with the neutral filter elution elution technique may represent two different types of dsb or modes of repair of dsb.
Animals↗
Elevated levels of DNA double-strand breaks (dsb) in restriction endonuclease-treated xrs5 cells correlate with the reduced capacity to repair dsb.
Recently we have reported the kinetics of DNA double-strand breaks (dsb) induced in electroporated mammalian (CHO) cells that had been treated with the restriction endonuclease PvuII, as measured by the filter elution assay at the non-denaturing pH of 9.6. A gradual accumulation of dsb was observed over a 24-h incubation period following the restriction endonuclease (RE) treatment and this was attributed to a competition between incision of the DNA by PvuII and dsb repair. In order to test this 'competition' hypothesis we have carried out similar experiments in the radiosensitive xrs5 mutant cell line, which has been shown to be deficient in dsb repair. The levels of dsb monitored by the non-denaturing filter elution assay in the xrs5 cell line treated with PvuII was found to be 3-4 times higher than that found for the wild-type CHO K1 cell line. Levels of dsb were also significantly raised in xrs5 cells treated with BamHI, as compared with the background levels observed in the CHO line. These data lend strong support to the competition hypothesis of simultaneous incision and repair of RE-induced dsb.
Animals↗
Differences in accumulation of blunt- and cohesive-ended double-strand breaks generated by restriction endonucleases in electroporated CHO cells.
Restriction endonucleases (RE) have been used in cytogenetic studies to mimic the DNA double-strand break (dsb)-inducing action of radiation. In the experiments presented here, we have treated electroporated CHO cells with RE and have measured the resulting dsb using the filter elution technique under non-denaturing conditions (pH 9.6). PvuII, which generates blunt-ended dsb, gave rise to a significant number of measurable dsb. The frequency of the dsb induced by PvuII is shown to increase over a 3-12-h post-treatment incubation period, which implies that the RE is active in the cell for a considerable length of time. We postulate that the accumulation of dsb reflects a competition between enzymatic incision and repair of the DNA. The presence of araA, a known inhibitor of DNA synthesis, did not affect the frequency of PvuII-induced breaks indicating a lack of an inhibitory effect of araA on the repair of RE-induced dsb. Two RE which cause cohesive-ended dsb, namely BamHI and EcoRI, were found to be ineffective in giving rise to measurable dsb. Our interpretation of this is that for cohesive-ended dsb (caused by BamHI and EcoRI) the rate at which these breaks are rejoined matches or exceeds the rate of enzymatic incision and hence no dsb were observed. In the case of PvuII, the possibly slower rate of repair of blunt-ended termini would on this hypothesis result in the observed net accumulation of dsb.
Animals↗
Induction of mutations at the thymidine kinase locus in CHO cells by restriction endonucleases.
Induced mutation frequencies were measured at the tk locus (encoding for the enzyme thymidine kinase) following treatment of Chinese hamster ovary cells (CHO KI) with two restriction endonucleases (REs), PvuII and EcoRI, which generate 'blunt-ended' and 'cohesive-ended' DNA double-strand breaks (dsb), respectively. Electroporation was used to introduce these enzymes into the cells. Restriction endonucleases generating blunt-ended dsb have been shown to mimic the action of ionising radiation in causing chromosome aberrations, cell killing, mutations and oncogenic transformation. Here we show that the tk locus, PvuII induced an approximately 11-fold higher mutation frequency than EcoRI at the same enzyme concentrations. There are four PvuII and six EcoRI restriction sites in the Chinese hamster thymidine kinase gene. Hence the higher mutation induction by PvuII, despite the lower number of restriction sites than EcoRI in the tk gene, suggests that blunt-ended dsb represent more effective and critical mutagenic DNA lesions than the cohesive-ended type. In this respect, our results are similar to those we obtained previously for chromosomal aberrations and for cell killing. Results from the present study suggest that mutations could arise from unrepaired or misrepaired dsb possibly via induction of chromosomal deletions or stable exchanges between chromosomes.
Animals↗
Cytogenetic responses of human uroepithelial cell lines and a malignant bladder carcinoma cell line to X-rays.
The responses to X-rays of three simian virus 40 (SV40) immortalized but non-tumourigenic human bladder epithelial cell lines have been compared with a malignant bladder epithelial line using the micronucleus assay. A linear increase in induced micronuclei (MN) was observed for all four cell lines with increasing X-ray dose. The three SV40 immortalized lines were found to be significantly less sensitive than the malignant cell line. Spontaneous levels of MN indicate that certain cell lines within the SV40 immortalized lines have a higher genetic instability. These cell lines may have a predisposition towards the generation of a fully transformed phenotype when treated with carcinogenic agents.
Cell Line, Transformed↗
Enhanced mutability at the tk locus in the radiosensitive double-strand break repair mutant xrs5.
The thymidine kinase locus (tk) has been utilised as the target locus to measure the induced mutation frequency following X-irradiation in the X-ray-sensitive xrs5 mutant and its parent CHO K1 line of Chinese hamster cells. Mutations to tk- cells were measured by plating cells in selective medium containing trifluorothymidine after a post-irradiation expression time of 4 days. Our results show that the mutation frequency was 3-4 times higher in the xrs5 mutant than in the CHO K1 cell line. This enhanced mutation frequency in xrs5 is though to result from the deficiency in DNA double-strand break repair in this cell line which also results in the enhanced cell killing and higher frequencies of chromosomal aberrations in response to X-irradiation. The findings of the present study suggest that DNA double-strand break is a critical lesion leading to mutations in irradiated cells.
Animals↗
Effects of trypsin on X-ray-induced cell killing, chromosome abnormalities and kinetics of DNA repair in mammalian cells.
When cells are trypsinized before irradiation a potentiation of X-ray damage may occur. This is known as the 'trypsin effect'. Potentiation of X-ray damage on cell killing was seen in V79 Chinese hamster cells but was marginal in Chinese hamster ovary (CHO K1) cells and not evident in murine Ehrlich ascites tumour (EAT) cells. Trypsinization did however increase the number of X-ray-induced chromosomal abnormalities in all 3 lines. To investigate the possibility that trypsin acts by digestion of proteins in chromatin, further experiments were performed to monitor DNA damage and repair. Induction of DNA breaks by X-rays was unaffected by trypsin but trypsinized EAT (suspension) cells repaired single-strand breaks (ssb) less rapidly than controls indicating an inhibitory effect of trypsin on ssb repair. However double-strand break (dsb) repair was unaffected by trypsin. It was also found that the EDTA solution in which the trypsin was dissolved also contributes to the inhibition of dsb repair. The results show that trypsinization can enhance X-ray-induced cell killing, chromosomal damage and DNA repair, the effect varying between cell lines.
Animals↗
Neutral filter elution detects only limited inhibition of double-strand break repair by 9-beta-D-arabinofuranosyladenine.
Bradley and Kohn (1979) showed that the neutral filter elution technique detects DNA double-strand breaks (dsb), yet there is still some uncertainty regarding the interpretation of results obtained with this technique (viz. the curvilinear dose-response curve and the rapid repair kinetics). In this report we have investigated the effect of the nucleoside analogue 9-beta-D-arabinofuranosyladenine (ara A), a known inhibitor of dsb repair, on the DNA repair in X-irradiated Ehrlich ascites tumour cells as measured by this technique. We have compared the effect of ara A on repair in these cells with results previously obtained with the same cell line and using the neutral velocity sedimentation and DNA unwinding techniques (Blöcher, 1982; Bryant and Blöcher, 1982). Our results suggest that the lesions measured by the neutral elution technique are different from those measured by neutral velocity sedimentation or long-term repair measured by DNA unwinding.
Animals↗
The induction of DNA double-strand breaks in CHO cells by Pvu II: kinetics using neutral filter elution (pH 9.6).
Chinese hamster CHO K1 cells were treated with the restriction endonuclease Pvu II during electroporation and assayed for DNA double-strand breaks (dsb). Dsb were measured by the non-denaturing filter elution technique (pH 9.6) at various times up to 24 h after restriction endonuclease (RE) treatment. The frequency of dsb following electroporation in the presence of 200 units/ml Pvu II increased over the post-treatment incubation period. This was found not to be due to cell or DNA degradation, indicating that Pvu II remains active for at least 24 h inside the cell. We suggest that these kinetics of dsb result from a competition between incision (by Pvu II) and dsb repair.
Animals↗
Repair kinetics in CHO cells of X-ray induced DNA damage and chromatid aberrations during a cell cycle extended by transient hypothermia.
Transient hypothermia was employed to extend the G2 phase of CHO K1 cells in order to facilitate study of the repair of X-ray induced chromatid and DNA damage. Thus G2 + 1/2M at 37 degrees C of 2.9 h was lengthened to 5.7 h at 33 degrees C and 7.3 h at 29 degrees C. While chromatid break kinetics remained essentially unaltered at 33 degrees C, at 29 degrees C there was an initial shoulder followed by a decrease in breaks similar to that at 37 and 33 degrees C. Although fewer exchanges were observed at 33 and 29 degrees C than at 37 degrees C, a similar kinetic involving a sharp initial rise followed by a plateau was observed at 33 and 29 degrees C, and, as far as could be judged, also at 37 degrees C. The failure of G2 prolongation to influence the rate of break disappearance was taken as evidence in support of the view that the disappearance of chromatid breaks represented a repair process rather than the decline of chromosomal radiosensitivity throughout this phase, though the possibility of a reduced sensitivity close to the G2/M border remained open. This hypothesis was supported by the mainly flat kinetics of exchanges. The data were taken as further evidence that chromatid rejoining and misjoining (exchanges) are essentially different processes. The rates of repair of DNA double-strand breaks as measured by neutral filter elution were similar at 37 and 33 degrees C, while there was evidence of inhibition at 29 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Similar kinetics of chromatid aberrations in X-irradiated xrs 5 and wild-type Chinese hamster ovary cells.
We have studied the kinetics of chromatid aberrations in cells of the Chinese hamster ovary (CHO-K1) derived, X-ray sensitive cell line xrs 5 irradiated in the G2 phase at 37 degrees C, as well as during a cell cycle extended by transient hypothermia at 33 degrees C. While a given X-ray dose was estimated to produce about 4 times as many chromatid break and twice the frequency of exchanges in xrs 5 cells as in the parent line, there was no difference between the lines in the rates of disappearance of chromatid breaks during G2 at either temperature; and similar patterns of chromatid exchange kinetics were observed in the two lines. Both the frequencies and distributions of chromatid breaks at different times after irradiation are consistent with the view that the disappearance of these during incubation represents a repair process. These results imply that the G2 chromosomal radiosensitivity of the xrs 5 mutant residues at the level of initial chromatid damage.
Animals↗
Clastogenicity of PvuII and EcoRI in electroporated CHO cells assayed by metaphase chromosomal aberrations and by micronuclei using the cytokinesis-block technique.
The clastogenicity of two restriction endonucleases with almost equal cutting frequencies: PvuII, generating blunt-ended DNA double-strand breaks (dsb) and EcoRI, generating cohesive-ended dsb, has been measured after treatment of electroporated CHO cells with these enzymes. Chromosome damage was assessed by the micronucleus cytokinesis-block technique, and for certain electroporation voltages by analysis of metaphase preparations. As has been found in previous studies, PvuII was found to be more effective in causing chromosomal damage than EcoRI, indicating the greater importance of blunt-ended dsb in chromosome aberration induction. These findings also validate the use of the micronucleus cytokinesis-block technique for evaluating chromosomal damage from restriction endonucleases. The results show a biphasic induction of micronuclei in binucleate cells as a function of time after treatment. This pattern is interpreted as indicating variable sensitivity of cells to restriction endonucleases at different stages of the cell cycle. The micronucleus data show that late collection times (40-48 h after treatment) give higher frequencies than short times. Both micronucleus and metaphase aberration data indicate that voltages in excess of 260 V are more efficient in porating cells than lower voltages and, as a result, lower restriction endonuclease concentrations could be used.
Animals↗
Induction of chromosomal aberrations in CHO cells by restriction endonucleases: effects of blunt- and cohesive-ended double-strand breaks in cells treated by 'pellet' methods.
In recent reports it has been suggested that restriction endonucleases (RE) producing cohesive-ended double-strand breaks (dsb), are of comparable effectiveness to those producing blunt-ended dsb in causing chromosomal aberrations (CA) in mammalian cells. In several of these reports, trypsinized cells or suspension cultures were treated as cell 'pellets' in small volumes containing RE and storage buffers. In this study we have examined this by comparing 2 'pellet' methods in which trypsinized Chinese hamster cells were treated with RE in small volumes, after cells were centrifuged to a pellet. In the first method, cells were treated with RE in storage buffer as previously reported (e.g. Obe et al., 1985). In the second method, cells were treated as pellets with Sendai virus and purified RE. For both methods we show that the frequency of chromosomal aberrations was higher in cells treated with RE causing blunt-ended dsb than those causing cohesive-ended dsb. The first method however was found to lead to substantial loss in cell viability. The results strengthen the conclusion drawn from our earlier work, using treatment of attached V79 or CHO-K1 cells with Sendai virus, that cohesive-ended dsb are less effective than blunt-ended dsb in causing chromosomal aberrations.
Animals↗