Biomedical subjects

B Marples

Publications and source records attributed to B Marples.

At least 37 records · Page 2Linked to original sources

PubMed1998

DNA damage in human and mouse spermatozoa after in vitro-irradiation assessed by the comet assay.

The comet assay is widely employed as a method to measure DNA damage in a wide variety of cell types following genotoxic insult. We have used this method in order to characterise DNA damage in spermatozoa following in vitro irradiation with 137Cs gamma rays. In contrast to somatic cells, the DNA of mammalian spermatozoa is bound by protamine molecules allowing a sixfold more highly compact structure and thus rendering conventional cell lysis protocols ineffective. Therefore, this new method uses an extensive lysis step to ensure effective removal of DNA-associated proteins allowing DNA damage to be scored reproducibly in both murine and human spermatozoa. Mouse spermatozoa collected from the vas deferens at post-mortem or human spermatozoa provided by donors were irradiated with doses of gamma-rays from 0-100 Gy using a 137Cs source and then processed for both alkaline and neutral comet assays. Under neutral electrophoresis conditions, which permits the measurement of double-stranded DNA breaks, a linear increase in the amount of DNA damage measured was observed with increasing radiation dose for both murine and human spermatozoa. Similarly, using alkaline electrophoresis conditions to examine DNA single-strand breaks and alkali-labile sites, a linear relationship was also observed for murine sperm but in contrast no such relationship was apparent for human spermatozoa subjected to the same radiation treatments. Interestingly, unirradiated sperm (both human and mouse) showed extensive DNA migration from the nucleus after alkaline assay. Since it is unlikely that the DNA of normal spermatozoa contains high numbers of single-strand breaks and damage was not detected for unirradiated sperm in the neutral assay, it is more likely that this DNA migration is due to the presence of high numbers of alkali labile sites within sperm DNA and that these may be related to the highly condensed structure of spermatozoal DNA. The large radiation doses used in these experiments to produce measurable amounts of DNA damage reflects the high radioresistance of spermatozoa compared to somatic cells and this may also be related to the differences in DNA packaging and conformation. In conclusion, this work shows that the comet assay represents a new method for examining DNA damage in spermatozoa and should be evaluated for use in reproductive toxicity testing.

Animals↗

PubMed1998

The radiosensitivity of human fibroblast cell lines correlates with residual levels of DNA double-strand breaks.

PURPOSE: To study the correlation of residual DNA double-strand breakage after irradiation and cellular radiosensitivity in cells showing marked differences in radiosensitivity. MATERIALS AND METHODS: The levels of DNA double-strand breaks remaining at 4 h after irradiation were measured by graded-voltage gel electrophoresis in fibroblast cell strains derived from seven individuals either with normal radiosensitivity (n = 2), or with genetic abnormalities known to show increased (two ataxia telangiectasia, one scid) or possibly decreased (two Li-Fraumeni family members) sensitivity. RESULTS: The slope of the dose-response curve for DNA breaks remaining unrepaired at 4 h showed a highly significant correlation with cellular radiosensitivity characterized by SF2, alpha, or D (r > or = 0.91, P < 0.001). Hence, this measure of genotoxic damage was predictive of radiation sensitivity for cells affected by a variety of mutations in different damage signalling/repair components. DISCUSSION: This correlation confirms another published study and extends it to cell lines with other genetic defects. The technique may be useful in the development of rapid assays to predict the sensitivity of normal tissues in patients receiving radiotherapy.

Blotting, Southern↗

PubMed1998

The ratio of initial/residual DNA damage predicts intrinsic radiosensitivity in seven cervix carcinoma cell lines.

The single-cell gel electrophoresis (comet) assay was used to measure radiation-produced DNA double-strand breaks (dsbs) in a series of seven cervical tumour cell lines (ME180, HT3, C33A, C41, SiHa, MS751 and CaSki). The proportion of DNA dsbs was measured immediately after radiation treatment (initial damage) and 16 h later after incubation at 37 degrees C (residual damage). Linear dose-response curves were seen for initial (slopes 0.23-0.66) and residual (slopes 0.16-0.87) DNA dsbs. Neither of the slopes of the linear regression analysis on the initial and on the residual DNA dsbs dose-response curves (range 0-80 Gy) correlated with SF2 (surviving fraction at 2 Gy) measured after high- (HDR) or low-dose-rate (LDR) irradiation. An association was evident between SF2 after HDR and LDR irradiation and the ratio of the absolute level of initial and residual damage after a single dose of 60 Gy. However, a significant correlation was found between HDR (r= -0.78, P = 0.04) and LDR (r = -0.86, P = 0.03) SF2 values and the ratio of the slopes of the initial and residual DNA dsbs dose-response curves (range 0.47-0.99), representing the fraction of DNA damage remaining. These results indicate that the neutral comet assay can be used to predict radiosensitivity of cervical tumour cell lines by assessing the ratio of initial and residual DNA dsbs.

Carcinoma↗

PubMed1998

Repair of radiation-induced DNA double-strand breaks in human fibroblasts is consistent with a continuous spectrum of repair probability.

PURPOSE: To propose a novel interpretation of DNA double-strand break (dsb) repair based on the distribution of energy micro-deposition. MATERIALS AND METHODS: Double-strand break repair curves were studied either after irradiation at 4 degrees C or at 37 degrees C (low dose rate). Two human fibroblast cell lines were used: a control line, HF19, and an ataxia telangiectasia repair-deficient line, AT5BI. Irradiations were made with gamma-rays or alpha-particles (241Am). Repair data were fitted by the variable repair half-time (VRHT) model. Assuming that each dsb has its own inherent repair half-time (IRHT) and that the VRHT is the average of the IRHT at any time during repair, the distribution of the IRHT was calculated. RESULTS: At the end of the irradiation, the distribution was a continuous asymmetric curve with a maximum of dsb having a short IRHT. After 1 h of repair, the curve became bell-shaped. There is a striking similarity between the distribution of dsb repair half-times and that of energy micro-deposition described by Goodhead et al. (1993). CONCLUSION: This similarity suggests a possible causal relationship between the energy density deposition and the repair rate or the probability of dsb repair.

Americium↗

PubMed1997

A correlation between residual DNA double-strand breaks and clonogenic measurements of radiosensitivity in fibroblasts from preradiotherapy cervix cancer patients.

PURPOSE: To study the relationship between residual DNA damage and clonogenic measurements of radiosensitivity in fibroblasts from pretreatment cervix cancer patients. METHODS AND MATERIALS: Early passage vaginal fibroblasts from nine preradiotherapy cervix cancer patients and two radiosensitive skin fibroblast cell strains were studied. Cell survival was measured by clonogenic assay following both high and low dose rate irradiation. Residual DNA damage was measured using pulsed-field gel electrophoresis (PFGE) after irradiating radiolabeled, plateau-phase cells at 37 degrees C and allowing 24 h for repair. DNA damage was expressed both in terms of the residual damage slope (fitted to data from 60 to 150 Gy) and the fraction of activity released (FAR) following 150 Gy. RESULTS: The surviving fraction at 2 Gy (SF2) values after high dose rate irradiation for the vaginal fibroblasts ranged from 0.15 to 0.32 (a 2.2-fold difference). When the two radiosensitive cell strains were included, residual damage, expressed as the residual damage slope, correlated with alpha (r = 0.82, p = 0.002), D bar (r = -0.91, p < 0.001) and SF2 (p = -0.79, p = 0.004), and when the vaginal fibroblasts alone were studied, the residual damage slope again correlated with clonogenic survival, although less strongly [alpha (r = 0.66, p = 0.053), D bar (r = -0.83, p = 0.006), and SF2 (r = -0.63, p = 0.07)]. Within the group of vaginal fibroblasts there was a 4.0-fold difference in residual DNA damage slope. When residual damage was expressed as FAR at 150 Gy, then for all cell strains the correlations were alpha: r = 0.78, p = 0.004, D bar: r = -0.86, p = 0.001, and SF2: r = -0.78, p = 0.004, and for the vaginal fibroblast strains alone the correlations were alpha: r = 0.60, p = 0.088, D bar: r = -0.75, p = 0.02, and SF2: r = 0.62, p = 0.077. CONCLUSION: This study confirms previous findings that residual DNA damage correlates with clonogenic survival in fibroblasts. In addition, it demonstrates a correlation for fibroblasts from pretreatment cervix cancer patients demonstrating a relatively small range of SF2 values.

Cell Survival↗

PubMed1997

A comet assay of DNA damage and repair in K562 cells after photodynamic therapy using haematoporphyrin derivative, methylene blue and meso-tetrahydroxyphenylchlorin.

Single-cell electrophoresis (comet assay) has been used to evaluate DNA damage and repair in the human myeloid leukaemia cell line K562 after low-dose (predominantly sub-lethal) treatments of hyperthermia and photodynamic therapy (PDT). Three different photosensitizers were examined: haematoporphyrin derivative (HpD), methylene blue (MB) and meso-tetrahydroxyphenylchlorin (mTHPC). None of the drugs in the absence of light, nor in light alone, resulted in detectable DNA damage. However, a significant amount of DNA damage was detected immediately after treatment with haematoporphyrin derivative or methylene blue PDT compared with drug-only or light-only treatments; no residual level of DNA damage was evident for either drug following a 4-h post-treatment incubation at 37 degrees C. No significant DNA damage was detected after meso-tetrahydroxyphenylchlorin PDT or hyperthermia either immediately or 4 h after treatment. We conclude that the alkaline comet assay can be applied as an effective screening assay for DNA damage induced by a range of laser therapies.

DNA Damage↗

PubMed1997

Low dose hyper-radiosensitivity and increased radioresistance in mammalian cells.

This manuscript reviews the low-dose survival work using the DMIPS cell analyser that has been carried out at the Gray Laboratory in the U.K. and the British Columbia Cancer Research Centre in Canada. It describes low dose hyper-radiosensitivity (HRS) detected after single doses of X-rays less than approximately 0.3 Gy and the subsequent increased radioresistant response (IRR) seen as the dose increases up to 1 Gy. Work is summarized from studies in V79 cells, normal human and human tumour cell lines and mutant cell lines deficient in DNA repair. The data are considered in light of the hypothesis that hyper-radiosensitivity and increased radioresistance reflect the existence of an inducible protective mechanism, possibly triggered by DNA damage.

Animals↗

PubMed1996

Hypersensitivity to very-low single radiation doses: its relationship to the adaptive response and induced radioresistance.

There is now little doubt of the existence of radioprotective mechanisms, or stress responses, that are upregulated in response to exposure to small doses of ionizing radiation and other DNA-damaging agents. Phenomenologically, there are two ways in which these induced mechanisms operate. First, a small conditioning dose (generally below 30 cGy) may protect against a subsequent, separate, exposure to radiation that may be substantially larger than the initial dose. This has been termed the adaptive response. Second, the response to single doses may itself be dose-dependent so that small acute radiation exposures, or exposures at very low dose rates, are more effective per unit dose than larger exposures above the threshold where the induced radioprotection is triggered. This combination has been termed low-dose hypersensitivity (HRS) and induced radioresistance (IRR) as the dose increases. Both the adaptive response and HRS/IRR have been well documented in studies with yeast, bacteria, protozoa, algae, higher plant cells, insect cells, mammalian and human cells in vitro, and in studies on animal models in vivo. There is indirect evidence that the HRS/IRR phenomenon in response to single doses is a manifestation of the same underlying mechanism that determines the adaptive response in the two-dose case and that it can be triggered by high and low LET radiations as well as a variety of other stress-inducing agents such as hydrogen peroxide and chemotherapeutic agents although exact homology remains to be tested. Little is currently known about the precise nature of this underlying mechanism, but there is evidence that it operates by increasing the amount and rate of DNA repair, rather than by indirect mechanisms such as modulation of cell-cycle progression or apoptosis. Changed expression of some genes, only in response to low and not high doses, may occur within a few hours of irradiation and this would be rapid enough to explain the phenomenon of induced radioresistance although its specific molecular components have yet to be identified.

Adaptation, Physiological↗

PubMed1996

Response of V79 cells to low doses of X-rays and negative pi-mesons: clonogenic survival and DNA strand breaks.

Mammalian cells are hypersensitive to very low doses of X-rays (< 0.2 Gy), a response which is followed by increased radioresistance up to 1 Gy. Increased radioresistance is postulated to be a response to DNA damage, possibly single-strand breaks, and it appears to be a characteristic of low linear energy transfer (LET) radiation. Here we demonstrate a correspondence between the extent of the increased radioresistance and linear energy transfer of 250 kVp X-rays and plateau and Bragg peak negative pi-mesons. The results support our hypothesis since the size of the increased radioresistant response appears to correspond to the number of radiation induced single-strand breaks. Furthermore, since survival prior to the increased radioresistant response (< 0.2 Gy) was LET-independent, these data support the notion that the increased radioresistant response may dictate the overall survival response to higher doses. However, while these data provide further circumstantial evidence for the involvement of DNA strand breaks in the triggering of increased radioresistance, more direct conclusions cannot be made. The data are not accurate enough to detect structure in the single-strand break profiles, the production of single-strand breaks being apparently linear with dose.

Animals↗

PubMed1996

Small doses of high-linear energy transfer radiation increase the radioresistance of Chinese hamster V79 cells to subsequent X irradiation.

Chinese hamster V79 cells show a complex X-ray survival response which is characterized by hypersensitivity followed by increased resistance as the dose increases to 1 Gy. This hypersensitivity can be eliminated by pretreating cells with X rays or hydrogen peroxide. Accordingly, the protective effect that results from the priming treatments could be considered analogous to the "adaptive response" induced by low-linear energy transfer (LET) radiation and some chemical agents in human lymphocytes. Indeed, no hyper-radiosensitive response after single treatments in V79 cells or adaptive response in human lymphocytes has been reported after exposure to high-LET radiation. To investigate this further, we measured the survival after X irradiation of V79-379A cells previously irradiated with small priming doses of high-LET radiation. After a 0.2-Gy priming dose of neutrons followed by a 1-Gy 250 kVp X-ray dose given 4 h later, survival was 1.08 +/- 0.04 compared to 0.73 +/- 0.03 when the doses were given concurrently. Increases in survival were also observed from 0.80 +/- 0.03 to 0.96 +/- 0.05 after a 0.2-Gy priming treatment with 250 kVp X rays and from 0.78 +/- 0.03 to 0.84 +/- 0.03 with a priming dose of Bragg-peak negative pi mesons. The results indicate that a protective effect, as measured by an increase in radioresistance, is induced by high-LET neutrons, as well as by Bragg-peak pi mesons and X rays, and that a threshold level of damage is required for adaptation to occur.

Adaptation, Physiological↗

PubMed1995

The elimination of low-dose hypersensitivity in Chinese hamster V79-379A cells by pretreatment with X rays or hydrogen peroxide.

To explain increased radioresistance over the X-ray dose range approximately 0.5-1 Gy an inducible radioprotective mechanism triggered by DNA damage was proposed; hypersensitivity to doses << 0.5 Gy reflected the response prior to the activation of this system (Marples and Joiner, Radiat. Res. 133, 41-51, 1993). To test this hypothesis, cells were pre-exposed to DNA-damaging agents in an attempt to induce the process prematurely. An increase in survival was evident at X-ray doses below 0.3 Gy after a priming treatment of X rays (0.05, 0.2, 1 Gy) given 6 h earlier. The protective effect was found to be transitory, requiring time for development and diminishing after two to three cell cycle times. Cycloheximide administered in the interval between the priming and challenge doses of X rays abolished the protection conferred by pretreatment, indicating the involvement of de novo protein synthesis. Oxidative damage by nontoxic doses of hydrogen peroxide (10(-4) M, but not 10(-6) M) also produced a protective effect against subsequent X irradiation. These experiments indicate survival in the hyper-radiosensitive region (<< 0.5 Gy) can be modified by pretreatment with agents known to affect DNA repair. In addition, the development of increased radioresistance after single doses of X rays was inhibited by cycloheximide treatment. These studies provide evidence to support the explanations proposed previously for the phenomena of increased radioresistance and hyper-radiosensitivity observed at very low X-ray doses.

Animals↗

PubMed1994

Recognition of platinum-induced DNA damage by nuclear proteins: screening for mechanisms.

The association of high mobility group (HMG) proteins with cisplatin-damaged DNA has attracted considerable interest because of a possible relationship with drug-acquired resistance and the repair of DNA damage caused by this important chemotherapeutic agent. We have further characterized the binding of HMG proteins to cisplatin-damaged DNA using a modification of the damaged DNA affinity precipitation assay (DDAP) and proteins isolated from the nuclei of V79 cells. HMG proteins recognized cisplatin adducts only in double-stranded DNA sequences. Pre-treatment of cells with cisplatin (1 microM) prior to protein extraction decreased the apparent yield of HMG proteins. However, the pre-treatment of isolated protein did not prevent recognition of the DNA adducts. To investigate the possible role of HMG proteins in toxicity and resistance, we have extended the DDAP method to study other platinum agents, some of which are active in cisplatin-resistant cells. A comparison of trans- and cis-[PtCl2(NH3)quinoline] is presented as an example. HMG proteins recognized DNA damage caused by the cis, but not the trans isomer. However, the trans isomer is known to be significantly more toxic and is highly active in cisplatin-resistant cells, suggesting a mechanism of action different from cisplatin. Therefore, the toxicity of trans-[PtCl2(NH3)quinoline] appears to be unrelated to the recognition of damage by HMG proteins. The DDAP assay may provide an additional screen for new mechanisms of cytotoxic platinum agents.

Animals↗

PubMed1994

The effect of radiosensitizers on the survival response of hypoxic mammalian cells: the low X-ray dose region, hypersensitivity and induced radioresistance.

It has been shown previously that the extent of chemical modification of the hypoxic radiation response is dependent on dose. Some types of sensitizer are more effective at low doses (to 4 Gy) than at higher doses. Since such drugs are possible adjuvants to radiotherapy, the mechanisms responsible for the variable response at clinical doses are of interest. Existing reports on sensitization at low doses are summarized, and the effects of cisplatin and buthionine sulfoximine on the purported induced response to radiation in hypoxic cells are presented. Cisplatin at a low, nontoxic concentration (1 microM) appears to abolish the increased radioresistant portion of the survival response. A role for high-mobility-group protein binding by platinum drugs is hypothesized to explain their interaction with radiation, and conversely, it is suggested that the heretofore unexplained different behavior of certain hypoxic sensitizers at low doses could be, at least in part, an effect on the induction of resistance.

Animals↗

PubMed1994

The effect of two topoisomerase inhibitors on low-dose hypersensitivity and increased radioresistance in Chinese hamster V79 cells.

A preliminary investigation of the effect of topoisomerase inhibitors on the structure of the survival curve at low doses has been carried out in Chinese hamster V79 cells, where there is a deviation from the predicted response to radiation (Marples and Joiner, Radiat. Res. 113, 41-51, 1993). Cells were treated with one representative drug for each enzyme (topoisomerase I or II) prior to X irradiation in air and assessed for cell survival using automated microscopic location of cells. VP-16 causes little or no effect, while camptothecin has a measurable effect up to 2 Gy. The results are discussed in terms of the role of DNA damage and cell cycle in increased radioresistance, which encourage further investigation of the effects of this class of drugs at low doses.

Animals↗

PubMed1994

A preliminary investigation into the extent of increased radioresistance or hyper-radiosensitivity in cells of hamster cell lines known to be deficient in DNA repair.

The response to low doses of X rays was assessed in cells of three hamster cell lines which are defective in DNA repair and was compared with their parental lines. Cells of the V79-derived double-strand break repair-deficient line XR-V15B showed no radioresistance in the 0.5-Gy range compared with the V79B wild type, but instead showed an exponential response. Cells of the single-strand break repair-deficient line EM9 showed hyper-radiosensitivity and exhibited increased radioresistance. Most interestingly, cells of the UV-20 cell line appeared to respond exponentially, as a continuation of the hyper-radiosensitive portion of the curve, with no evidence of increased radioresistance. This line is defective in an incision step of excision repair and is sensitive to crosslinking agents. Further studies are warranted to address the possible role of single- and double-strand break repair and excision repair in hyper-radiosensitivity and increased radioresistance.

Animals↗

PubMed1994

The effect of oxygen on low-dose hypersensitivity and increased radioresistance in Chinese hamster V79-379A cells.

Chinese hamster V79 cells irradiated in air are hypersensitive to X-ray doses less than 0.5 Gy and show an increased radioresistance over the dose range 0.5-1 Gy (Marples and Joiner, Radiat. Res. 133, 41-51, 1993). Of considerable interest from both a mechanistic and clinical viewpoint is the response of hypoxic cells over this dose range. The data presented here indicate that hypoxic cells are also hypersensitive to low X-ray doses and exhibit an increased radioresistant response, albeit triggered at a somewhat higher dose (0.69 Gy, SEM +/- 0.18 Gy) than observed in oxygenated cells (0.5 Gy, SEM +/- 0.21 Gy). These data indicate that the triggering event for increased radioresistance may be independent of oxygen. As reported by others previously, the oxygen enhancement ratio was found to decrease with a decreasing X-ray dose.

Animals↗

PubMed1994

Can colony size be used to detect low-dose effects?

Many insults at low doses are defined as nontoxic, even though obvious effects do occur. Among these are changes in colony size when clonogenic survival is assessed. Early attempts to quantify radiation effects on colony size have been reported, but the time-consuming nature of these experiments did not encourage the use of this parameter as an end point. Recently, however, developments in image cytometry technology have provided alternative, less labor-intensive means of measuring colony size. These techniques have been used in our ongoing investigations of radiation effects at low doses. Data accumulated to date show a measurable dose dependence of colony size in clones classed as survivors. This dose dependence is characterized by fluctuations in the 0-1.5-Gy dose range, followed by a gradual decrease in colony size at higher doses. The fluctuations at low doses correspond qualitatively to the concavity, thought to be indicative of inducible repair phenomena, that has been observed in cell survival curves in the same dose range. This concavity was also seen in the current study, but its detection appeared to be dependent on the method used to score survivors.

Animals↗

PubMed1994

The response of Chinese hamster V79-379A cells exposed to negative pi-mesons: evidence that increased radioresistance is dependent on linear energy transfer.

Chinese hamster V79-379A cells exhibit low-dose hypersensitivity to 250 kVp X rays followed by an increased radioresistant response over the dose range 0.5-1 Gy. This phenomenon is not seen with neutrons (Marples and Joiner, Radiat. Res. 133, 41-51, 1993). It was therefore postulated the induction of radioresistance might develop as a response to a cellular event(s) which predominates after low- and not high-LET radiation. To test this hypothesis, we measured the survival response of V79-379A cells exposed to pions. Clonogenic survival was assessed for cells irradiated in the Bragg peak (35 keV/microns) and plateau region (10-20 keV/microns) of the beam, using an automated microscope (DMIPS cell analyzer). As expected, peak pions were found to be more effective per unit of dose at killing cells than plateau pions. The survival curve for cells irradiated in the plateau of the pion beam was found to incorporate a region of low-dose hypersensitivity and increased radioresistance, the effective D0 was dose-dependent, ranging from 3.5-5. This was not seen with peak pions, where the effective D0 was, on average, constant reflecting a single-exponential survival curve. Fitting the data with an induced repair model indicates that the phenomenon of increased radioresistance is almost certainly dependent on LET.

Animals↗