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C R Geard

Publications and source records attributed to C R Geard.

At least 37 records · Page 2Linked to original sources
PubMed1995

Chromosome end associations, telomeres and telomerase activity in ataxia telangiectasia cells.

Cells derived from individuals with ataxia telangiectasia (AT) show enhanced spontaneous levels of chromosomal abnormalities and are sensitive to ionizing radiations and radiomimetic drugs, as evidenced by decreased survival and increased chromosome aberration frequencies at mitosis when compared with normal cell lines. The higher base line frequencies of chromosome aberrations in part involve chromosome end-to-end associations as seen at metaphase. Since telomeres of tumor cells and aging tissues are often reduced in length, chromosome end associations may be due to loss of telomeric repeats. We studied the chromosome behavior and telomeres of two ataxia telangiectasia lymphoblastoid cell lines compared to two normal control cell lines. The ataxia telangiectasia cell lines showed higher frequencies of chromosome end associations both at metaphase and in interphase, determined in prematurely condensed chromosomes of G1 and G2 cells. They also showed higher frequencies of chromosomal breaks at metaphase and fewer telomeric signals determined using fluorescent in situ hybridization with a (TTAGGG)n probe. The frequency of telomeric repeats was variable in the ataxia telangiectasia cell lines (4.3 and 8.2 kb) compared to the normal cell lines (9.6 and 12 kb) and an inverse correlation between telomere length and chromosome end associations was observed. Both ataxia telangiectasia cell lines showed more robust telomerase activity than the normal cell lines, precluding defective enzymatic capacity as the basis for the chromosome end associations. It is possible that chromatin structure in the form of telomere-nuclear matrix interactions are variant in ataxia telangiectasia cells negatively influencing telomerase function and contributing to telomere associations.

Ataxia Telangiectasia↗
PubMed1995

Neutron-induced cell cycle-dependent oncogenic transformation of C3H 10T1/2 cells.

Exposure of synchronized populations of mouse C3H 10T1/2 cells to a single dose (0.6 Gy) of 5.9 MeV neutrons at intervals after mitotic shake-off results in a distinctive variation in the oncogenic transformation frequency through the cell cycle. Previous findings show a sensitive window for X-ray-induced oncogenic transformants at late times after mitotic shake-off (14-16 h). Optimal sensitivity to neutrons was observed for cell populations irradiated soon after mitotic shake-off (4-6 h), where the majority of cells would be in the G1 phase of the cell cycle. Additionally, enhanced sensitivity was also found for that period after shake-off (14-16 h) which was maximally sensitive to X rays corresponding to cell populations with a high proportion of G2-phase cells. That is, low-LET radiation (250 kVp X rays) largely appears to produce oncogenic transformants in G2-phase cells, while intermediate-LET radiation (5.9 MeV neutrons) is effective principally on G1- and, to a somewhat lesser extent, G2-phase cells. Cells irradiated with neutrons showed less variation for lethality through the cell cycle than those irradiated with X rays, in agreement with previous findings. The mechanistic basis for the difference in the response of cells in the different phases of the cell cycle to radiations of different quality is unknown but is suggestive of distinct ("signature") molecular changes leading to the observed oncogenic transformation response.

3T3 Cells↗
PubMed1995

The biological effectiveness of radon-progeny alpha particles. IV. Morphological transformation of Syrian hamster embryo cells at low doses.

Primary explants of Syrian hamster embryo (SHE) cells were exposed to either low-LET 250 kVp X rays or graded single doses of defined high-LET alpha particles (90, 100, 120, 150, 180 and 200 keV/microns), simulating those produced by radon progeny, and monitored for cell inactivation and oncogenic transformation. For the alpha particles the doses delivered ranged from 1 cGy to 1 Gy with an emphasis on doses less than 20 cGy, while for the X rays the doses ranged from 20 cGy to 4 Gy. The dose-response curves for cell killing by alpha particles approximated an exponential function of dose, whereas the X rays produced a curve with a shoulder characteristic of linear-quadratic relationships seen for low-LET radiations. The RBE at 10% survival varied between 3.6-7.0 depending on the LET of the alpha particles, with the RBEm ranging between 7-12. The most effective alpha particles were those with an LET of 120 keV/microns. All radiations produced initial increases in the frequency of morphological transformants, as a function of dose, with a rise to a maximum followed by a plateau in the response which was relatively constant at approximately 2-6 x 10(-3) transformants frequency, expressed per initial cell at risk, had a tendency to decline to parallel the cell survival response. Both the dose at which the maximum frequency of transformants was expressed and the initial slope of the dose-response relationship differed substantially between the different radiation qualities. Maximal transformation per initial cell at risk occurred at doses as low as 1-4 cGy for the 90 and 100 keV/microns particles with the maximum occurring at higher doses (to 16 cGy) as the LET increased toward 200 keV/microns. In contrast, the maximal transformation for 250 kVp X rays was at 50 cGy. The 90 and 100 keV/microns particles, with an RBEm of 60 and 37, respectively, based on the ratios of the initial slopes of the dose-response curves, were the most effective LETs in terms of the ability to induce morphological transformation of SHE cells. When expressed in terms of particle fluence, it appears that in the LET range of radon progeny approximately two to four particle traversals per nucleus are required per killing event, whereas it is at doses corresponding to less than one particle per nucleus that maximal oncogenic transformation is expressed.(ABSTRACT TRUNCATED AT 400 WORDS)

Alpha Particles↗
PubMed1994

Radiation and taxol effects on synchronized human cervical carcinoma cells.

PURPOSE: To evaluate the effectiveness of the plant derived chemotherapeutic agent taxol alone and in combination with ionizing radiation on synchronous and asynchronous human cervical carcinoma cells and to define the mechanistic basis for this cytotoxic response. METHODS AND MATERIALS: Asynchronous and synchronous cells (obtained by modified mitotic shake-off) derived from carcinomas of the human uterine cervix were treated with a range of concentrations of taxol (0, 1.0, 2.5, 5.0, 10.0 and 20.0 nM) for either 8, 24 or 48 h. Synchronized cell cycling was evaluated by counting mitotic indices and by uptake of bromodeoxyuridine (BrdUrd). Cells were irradiated (137Cs gamma rays at 1.12 Gy/min) alone and after taxol treatment and plating efficiencies and radiosensitivity determined. RESULTS: Taxol treatment resulted in a dose time dependent loss of colony forming ability with 10 nM for 24 h producing about 10% cell survival. Irradiating taxol treated cells resulted in a strictly additive response in contrast to previous supra-additive results with astrocytoma and melanoma cells. Mitotically synchronized cells rapidly moved into G1 phase with a second mitotic peak at 28 h (total cycle time). Taxol treatment resulted in a continued accumulation of mitoses, and a failure and/or delay of entry of a fraction of cells into S phase after a G1 phase of at least 10 h. That is, taxol effects cell cycling at a stage other than G2/M. Irradiating (3 Gy) synchronized cells showed a 10-fold variation in sensitivity, with mitosis as the most sensitive phase with taxol alone resulting in some cytotoxicity and combined effects additive or less than additive. CONCLUSION: Taxol effects these cervical carcinoma cells at other stages of the cell cycle than G2/M. This may explain the failure to obtain taxol radiosensitization with these cells and it may indicate that taxol has a multiplicity of actions with differences in effectiveness likely between cells of different origins.

Cell Cycle↗
PubMed1994

Taxol and ionizing radiation: interaction and mechanisms.

PURPOSE: Taxol has been shown to be clinically active against several types of human tumors. To assess the potential oncogenic effect of taxol, the in vitro cytotoxic and oncogenic transforming effects of taxol, either alone or in combination with gamma-irradiation, were examined. METHODS AND MATERIALS: Exponentially growing mouse C3H 10T1/2 cells were treated with taxol with or without concurrent gamma-irradiation. After treatment, cultures were replated for both clonogenic survival and transformation assays. To determine the effects of taxol on cell cycle kinetics, treated cells were concurrently labelled with bromodeoxyuridine coupled with fluorescein. Accumulated mitotic cells were isolated by the shake-off technique and their plating efficiency and radiosensitivity were determined. RESULTS: Taxol induced a dose dependent toxicity in 10T1/2 cells. In contrast to human tumor cells in culture, the mitotic block induced by a 100 nM dose of taxol in 10T1/2 cells was only partial. While taxol was ineffective in transformant induction, it enhanced the oncogenic transforming potential of gamma-rays in a supra-additive manner. The fact that approximately 15% of taxol-induced mitotic cells were clonogenically viable and at a cell cycle stage that was most radiosensitive suggests a mechanistic basis for the observed enhancement in transformation incidence by ionizing radiation. CONCLUSION: Taxol enhances the oncogenicity of radiation by partially blocking the 10T1/2 cells in G2/M phases of the cell cycle, phases that are most sensitive to radiation induced oncogenic transformation.

Animals↗
PubMed1994

Oncogenic transformation through the cell cycle and the LET dependent inverse dose rate effect.

Synchronised populations of mouse C3H/10T-1/2 cells were obtained by a stringent mitotic dislodgment procedure. Mitotic cells rapidly attach and progress sequentially through the cell cycle. Irradiation (3 Gy of X rays) was carried out at intervals from 0 to 18 h after initiating cell cycle progression of the mitotic cells. Oncogenic transformation was enhanced 10-fold over cells irradiated soon after replating (G1 and S phases) for cells in a near 2 h period corresponding to cells in G2 phase but not in mitosis. The cell surviving fraction had a 2-1/2-fold variation with resistant peaks corresponding to the late G1 and late S phases. These findings provide experimental support for the hypothesis initiated by Rossi and Kellerer and developed by Brenner and Hall to explain the LET dependent inverse dose rate effect for oncogenic transformation.

Animals↗
PubMed1994

Initial damage in human interphase chromosomes from alpha particles with linear energy transfers relevant to radon exposure.

To determine the efficiency at which alpha particles at LETs chosen to simulate exposure to radon progeny break chromosomes, the premature chromosome condensation technique was used to measure breaks soon after irradiation. Noncycling human fibroblasts were irradiated with graded doses of monoenergetic alpha particles accelerated to produce LETs of 90, 120, 150, 180 and 200 keV/microns at the midpoint of the cell nuclei. Premature chromosome condensation was initiated immediately after irradiation and cells were scored for the total number of prematurely condensed chromosomes and fragments per cell. Similar experiments were conducted with 250 kVp X rays for comparison. Irradiation with alpha particles produced 8.6 to 13.1 excess fragments per gray, while X rays produced 5.8 excess fragments, resulting in RBEs around 2. Calculations of the number of breaks produced on average by a single particle traversal of a cell nucleus indicated that at the LETs tested more than one break (1.5-2.8) was produced by each traversal, the maximum being that produced by 180 keV/microns alpha particles. When chromosome aberrations are scored at metaphase after high-LET irradiation, RBEs considerably greater than those recorded here (approximately 2) have been reported. These results showing relatively small differences in initial break levels for alpha particles in the LET range of the radon progeny relative to X rays indicate that the greater aberration frequencies are not due principally to an increase in breakage efficiency, but interactions between breaks along the same particle track are important.

Alpha Particles↗
PubMed1994

Kinetics of chromosome rejoining in normal human fibroblasts after exposure to low- and high-LET radiations.

To determine whether chromosome breaks produced by alpha particles are processed differently from those produced by X rays, the premature chromosome condensation technique was used to follow chromosome rejoining after irradiation. Doses of 90 and 200 keV/microns alpha particles (2.7 Gy) and 250 kVp X rays (6 Gy) were chosen to produce approximately the same number of initial chromosome breaks (about 30 excess fragments per cell). Frequencies of excess fragments were assessed at eight times to 24 h after irradiation with the final yields being about 2, 4 and 8 excess fragments per cell for 250 kVp X rays and 200 and 90 keV/microns alpha particles, respectively. For each radiation the time for the initial measured fragment frequency per cell to be halved (i.e. to about 15) was the same (about 100 min). The results were fitted to three models of kinetics of the rejoining, and the initial and residual number of excess chromosome fragments as well as the rate of rejoining were determined. Even with eight times, discrimination between the models of the kinetics was not possible, such that a single-component first-order reaction could not be rejected for either X-ray- or alpha-particle-induced breaks. Although rejoining proceeds at similar rates, the probability of "correct" rejoining is apparently reduced for alpha-particle-irradiated cells.

Chromosomes↗
PubMed1993

Expression of immediate early genes after treatment of human astrocytoma cells with radiation and taxol.

PURPOSE: The promising chemotherapeutic agent, taxol, has been shown to sensitize the G18 line of human astrocytoma cells to ionizing radiation. The present studies were performed to identify specific changes in gene expression associated with this altered sensitivity. METHODS AND MATERIALS: The radioresistant, grade 3 human astrocytoma cell line, G18, was exposed for varying periods of time to treatment with taxol, tetradecanoyl phorbol acetate (TPA), serum, isoproterenol, dibutyryl cyclic adenosine monophosphate, or ionizing radiation alone or in combination with taxol pretreatment. Ribonucleic acid samples from the cells were monitored for the expression of a group of immediate early genes (IEGs), including c-fos, c-jun, TIS1, TIS7, TIS8, TIS11 and TIS21, by northern blot hybridization analysis. RESULTS: Transient immediate early gene induction was observed after treatment of G18 cells with tetradecanoyl phorbol acetate, serum, isoproterenol, or ionizing radiation, but not after treatment with taxol. Of the seven immediate early genes analyzed, all but TIS7 were found to be inducible by one or more of the treatments. Only TIS8 (also known as egr-1 or zif268) was significantly inducible by radiation, and this transient induction was decreased by at least four-fold by pretreatment for 24 hr with a dose of taxol that was previously shown to block 96.5% of the cells in G2/M and enhance radiosensitivity. CONCLUSION: The products of immediate early genes, which are induced transiently in cells in response to a variety of treatments, including growth factors, neurotransmitters, and irradiation with UV light or X rays, are thought to initiate a cascade of genetic responses to alterations in cellular environment. The present results demonstrate a dramatic attenuation in one immediate early gene response in association with a treatment that enhances radiosensitivity in a refractory human brain tumor line.

Astrocytoma↗
PubMed1993

Taxol and radiation.

The cytotoxic effects of Taxol and/or ionizing radiation were evaluated in four human tumor cell lines. The recognized antimicrotubular effects of the drug leading to transitory accumulations of cells in the G2/M phase of the cell cycle, the most radiosensitive phase of the cycle, prompted this assessment of the potential for Taxol to function as a cell-cycle, phase-specific radiosensitizer. Taxol alone was cytotoxic to all four cell lines at low (< 25 nM) concentrations. A Taxol concentration of 10 nM for 24 hours led to 48, 15, 8, and 4.4% of cells retaining clonogenic potential for melanoma, two cervical carcinomas, and astrocytoma, respectively. There were significant Taxol concentration-time-dependent differences in response between the cell lines. Cell lines also showed significant differences in their responses to ionizing radiation. Combined treatment resulted in a demonstration of radiation sensitization with the astrocytoma and melanoma cell lines but not with the cervical carcinoma cell lines. Sensitizer enhancement ratios at the 10% cell survival level were 1.8 for 10 nM Taxol for 24 hours with the astrocytoma cells and 1.2 for 40 nM Taxol for 24 hours with the melanoma cells. The cervical carcinoma cell lines showed an additive effect for radiation and Taxol at all drug concentrations; that is, combined treatments elicit an additive or supra-additive response with, however, no simple relationship between Taxol concentration, Taxol time of treatment, and radiation dose in optimizing cytotoxic effectiveness. Combined modality treatments using relatively low concentrations of Taxol and ionizing radiation can result in an enhanced response and, at the least, an additive response, which could be advantageous in a clinical setting.

Cell Survival↗
PubMed1993

Induction of sister chromatid exchange as a function of charged-particle linear energy transfer.

Frequencies of sister chromatid exchanges (SCEs) were evaluated in chromosome spreads of CHO-AA8 cells at the second mitosis after irradiation with charged particles of 10, 40, 80, and 120 keV/microns. At each LET there was a dose-dependent increase in the frequency of SCEs. In contrast to the majority of end points where relative biological effectiveness increases as LET increases up to an optimum and then declines, it was found that the most biologically effective particles were protons at 10 keV/microns, followed by deuterons at 40 keV/microns, then alpha particles at 80 and 120 keV/microns. Nuclear cross-sectional areas of these cells were log-normally distributed with a mean of 77 microns 2. When induced SCEs per chromosome were related to charged particles per cell nucleus (down to a mean of 0.5 particles per nucleus), results at low fluences favored a linear relationship between SCEs and particles which was relatively independent of LET. These observations are not compatible with the origin of radiation-induced SCEs being DNA double-strand breaks and favor the notion that they may be consistent with DNA single-strand breaks.

Alpha Particles↗
PubMed1992

Taxol sensitizes human astrocytoma cells to radiation.

Taxol is a chemotherapeutic drug which acts by stabilizing microtubules, preventing normal mitosis and resulting in a block of the cell cycle at G2 and M. The drug is isolated from the yew, Taxus sp. L., and is currently being evaluated in a series of Phase II and Phase III clinical trials. Taxol blocks cells in the most radiosensitive phases of the cell cycle and thus could act as a cell cycle-specific radiosensitizer. We report the results of combined taxol-radiation exposures in the human Grade III astrocytoma cell line, G18. Taxol is a potent inhibitor of G18 cell division; a concentration of 10 nM is cytostatic for a cell population observed for at least two doubling times. Cell survival curves for G18 cells showed a significant concentration-dependent interaction between taxol and radiation. Treatment of G18 cells with a fixed taxol concentration and radiation dose showed the interaction to be dependent on the duration of taxol exposure and consequently the fraction of cells in the G2 or M phase of the cell cycle. The sensitizer enhancement ratio for 10 nM taxol at 10% survival is 1.8 and, for 1 nM taxol, it is 1.2. These results suggest that appropriate combinations of taxol have a more than additive interaction in human tissue culture and may have a role in clinical protocols.

Alkaloids↗
PubMed1992

Correlation of sensitizer enhancement ratio with bromodeoxyuridine concentration and exposure time in human cervical carcinoma cells treated with low dose rate irradiation.

The effect of a range of bromodeoxyuridine concentrations and exposure times on the sensitizer enhancement ratio of two human cervical carcinoma cell lines treated with low rate irradiation was evaluated. Both cell lines show a linear, though different, dose response to low dose rate irradiation at dose rates up to 0.62 Gy/hr. Cells were pre-incubated with 0, 1, 5, or 10 microM bromodeoxyuridine for 0, 24, or 48 hr prior to low dose rate irradiation. Based on the survival of cells exposed to 4 Gy, delivered at 0.62 Gy/hr, a slope was determined for an exponential survival curve for each bromodeoxyuridine concentration and exposure time. Sensitizer enhancement ratios were calculated as the ratio of the slopes of the treated and untreated cells from a particular exposure time. Ratios increased linearly initially with a plateauing at higher levels with respect to the product of bromodeoxyuridine concentration and time of exposure for both cell lines with similar degrees of enhancement. A linear relationship has been noted previously for acute irradiation of hamster cells over a similar range of sensitizer concentrations and exposure times. At the highest dose-time points (540 microM-hr) ratios of greater than 2 were attained, which were comparable to results obtained following acute radiation exposures of V-79 cells. These results demonstrate that significant sensitizer enhancement ratios with low dose irradiation can be obtained in human tumor cells after exposures of 2 to 3 doubling times using relatively low concentrations of sensitizer.

Bromodeoxyuridine↗
PubMed1992

Taxol: a novel radiation sensitizer.

The investigational antineoplastic agent, taxol, a natural product from the yew, Taxus sp. L., is currently being evaluated in a series of Phase II clinical trials. To date, the drug has shown activity against ovarian cancer, lung cancer, and melanoma. Taxol is a potent microtubule stabilizing agent that selectively blocks cells in the G2 and M phases of the cell cycle and is cytotoxic in a time-concentration dependent manner. It is well known from radiobiological principles that G2 and M are the most radiosensitive phases of the cell cycle. On the rationale that taxol could function as a cell-cycle selective radiosensitizer, we examined the consequences of combined drug-radiation exposures on the human grade 3 astrocytoma cell line, G18. Survival curve analysis shows a dramatic interaction between taxol and ionizing radiation with the degree of enhanced cell killing dependent on taxol concentration and on the fraction of cells in the G2 or M phases of the cell cycle. The sensitizer enhancement ratio (SER) for 10 nM taxol at 10% survival is approximately 1.8. These results obtained with cycling aerated radioresistant brain tumor cells indicate that significant advantage may derive from appropriate time-concentration dependent interactions in combined modality protocols.

Alkaloids↗
PubMed1992

Cytogenetic assays for genotoxic agents.

The induction of genetic damage has clear and dramatic implications for human health, with teratogenic, mutagenic, cataractogenic and carcinogenic consequences resulting from cellular chromosomal alterations in appropriate tissues. When analysing the potential of an agent to initiate genetic damage or in evaluating possible incumbent genomic damage a variety of complementary assays may be employed. These apply to cells in vitro, to in vivo assessments involving small mammals and most importantly to derived human cells and tissues including those of ocular origin. Cytogenetic assays have the important advantage that they enumerate damage at the level of the individual cell. Assays involving the examination of chromosomal aberrations at mitosis, of cells prior to mitosis using the technique of premature chromosome condensation, of micronuclei in post-mitotic cells and of sister chromatid exchanges will be described. The development of human chromosome specific probes and fluorescent in situ hybridisation (FISH) techniques combine the resolution of molecular biology with classical cytogenetics in a powerful approach to defining genomic change and its consequences. These techniques and assays can be further augmented by in situ cytometry such that overall a number of parameters can be quantified involving cellular kinetics, clastogen and/or aneugen definition and ultimately the establishment of dose response relationships. A rational basis for avoidance or control, for intervention or for defining probable cause of the role of genotoxicants in the development of human disease can then be established.

Animals↗
PubMed1992

Cell-cycle-dependent radiation-induced oncogenic transformation of C3H 10T1/2 cells.

C3H 10T1/2 cells were synchronized by a modified mitotic shake-off procedure. X irradiation of cells at various intervals after mitotic harvest indicated a single narrow window (about 2 h) of sensitivity to the induction of oncogenic transformation. It is not possible to delineate precisely the time in the cycle at which this sensitivity is expressed. The most likely candidate is G2 phase, though we cannot eliminate the possibility that the sensitive period begins in late S phase. In the same synchronized cells, cell lethality showed the conventional pattern, i.e., sensitivity in mitosis and resistance in late S and in G1 phase.

Animals↗
PubMed1991

Low dose rate irradiation and halogenated pyrimidine effects on human cervical carcinoma cells.

The response of two human cervical carcinoma cell lines to the combination of bromodeoxyuridine (BrdUrd) incorporation and low dose rate irradiation (LDRI) was assessed. Survival curves were generated following both acute (67.2 Gy/hr) and low dose rate (0.11-0.62 Gy/hr) 137Cs gamma-ray irradiation. The two cell lines exhibited markedly different radiation responses, but for the chronic irradiation, there was no significant difference in response between the low dose rates for either cell line. In all cases, the low dose rate radiation response could be described using a single exponential y = e-alpha D. The sensitizer enhancement ratio (SER) at 10% survival was determined and for the first cell line the low dose rate slope (alpha) was approximately 0.7 Gy-1 with an SER of 1.3-1.5 and an SER of approximately 1.6 for the acute response. In contrast, the second cell line had a low dose rate slope (alpha) of approximately 0.4 Gy-1, an SER of 1.3-1.5, and an SER of approximately 1.7 for the acute response. The similarity of the SER's is notable in light of the marked differences between the intrinsic radiation response of the cells. As the concentration and/or time of exposure to BrdUrd increased, both cytotoxicity and the degree of sensitization also increased. This study demonstrates that SER's with BrdUrd and LDRI are comparable to those obtained for acute exposures in two cervical carcinoma cell lines. The results suggest that this combination of modalities may have clinical applications.

Bromodeoxyuridine↗