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L Metzger

Publications and source records attributed to L Metzger.

11 recordsLinked to original sources
PubMed1992

Mechanism of radiosensitization by halogenated pyrimidines: effect of BrdU on repair of DNA breaks, interphase chromatin breaks, and potentially lethal damage in plateau-phase CHO cells.

There is evidence suggesting that radiosensitization induced in mammalian cells by substitution in the DNA of thymidine with BrdU has a component that relies on inhibition of repair and/or fixation of radiation damage. Here, experiments designed to study the mechanism of this phenomenon are described. The effect of BrdU incorporation into DNA was studied on cellular repair capability, rejoining of interphase chromosome breaks, as well as induction and rejoining of DNA double- and single-stranded breaks (DSBs and SSBs) in plateau-phase CHO cells exposed to X rays. Repair of potentially lethal damage (PLD), as measured by delayed plating of plateau-phase cells, was used to assay cellular repair capacity. Rejoining of interphase chromosome breaks was assayed by means of premature chromosome condensation (PCC); induction and rejoining of DNA DSBs were assayed by pulsed-field gel electrophoresis and induction and rejoining of DNA SSBs by DNA unwinding. A decrease was observed in the rate of repair of PLD in cells grown in the presence of BrdU, the magnitude of which depended upon the degree of thymidine replacement. The relative increase in survival caused by PLD repair was larger in cells substituted with BrdU and led to a partial loss of the radiosensitizing effect compared to cells tested immediately after irradiation. A decrease was also observed in the rate of rejoining of interphase chromosome breaks as well as in the rate of rejoining of the slow component of DNA DSBs in cells substituted with BrdU. The time constants measured for the rejoining of the slow component of DNA DSBs and of interphase chromosome breaks were similar both in the presence and in the absence of BrdU, suggesting a correlation between this subset of DNA lesions and interphase chromosome breaks. It is proposed that a larger proportion of radiation-induced potentially lethal lesions becomes lethal in cells grown in the presence of BrdU. Potentially lethal lesions are fixed via interaction with processes associated with cell cycle progression in cells plated immediately after irradiation, but can be partly repaired in cells kept in the plateau-phase. It is hypothesized that fixation of PLD is caused by alterations in chromatin conformation that occur during normal progression of cells throughout the cell cycle.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Detection of DNA double-strand breaks in synchronous cultures of CHO cells by means of asymmetric field inversion gel electrophoresis.

A pulsed field gel electrophoresis technique, asymmetric field inversion gel electrophoresis (AFIGE), was used to evaluate induction by X-rays of DNA damage in CHO cells. The fraction of DNA activity released from the plug (FAR) was used as a measure for the amount of radiation-induced DNA damage, predominantly DNA double-strand breaks (dsb) (Stamato and Denko 1990), and was determined at various stages of growth and phases of the cell cycle in a range of doses between zero and 70 Gy. The FAR per unit dose fluctuated throughout the cell cycle and was high for cells irradiated in G1; it decreased as cells entered S and reached a minimum in the middle of this phase. The FAR per unit dose increased again as cells progressed towards the end of S, and reached values in G2 similar to those measured in G1. When damage was introduced into DNA by means of 125I decay similar fluctuations in the FAR per decay were observed throughout the cell cycle, suggesting that the variations in the FAR per unit of radiation dose observed throughout the cell cycle do not derive from alterations in the induction of dsb. The fluctuations in the FAR per unit dose throughout the cell cycle were quantitatively similar to the fluctuations in the fraction of activity eluted in irradiated cells assayed by the non-unwinding filter elution assay throughout the cycle (Okayasu et al. 1988), and suggest that both techniques respond to similar DNA replication-associated alterations of the biophysical and/or biochemical properties of the DNA molecule. It is concluded that caution needs to be exercised before differences observed in the FAR between different cell lines or between various phases of the cell cycle after exposure to a given dose of radiation are interpreted as suggesting differences in the induction of DNA dsb.

Animals↗
PubMed1991

Measurement of DNA double-strand breaks in CHO cells at various stages of the cell cycle using pulsed field gel electrophoresis: calibration by means of 125I decay.

Experiments were performed to calibrate a recently developed pulsed field gel electrophoresis assay, the asymmetric field inversion gel electrophoresis (AFIGE), for the measurement of double-strand breaks (dsb) in the DNA of mammalian cells. Calibration was carried out by means of 125I decay accumulation under conditions preventing repair, and is based on the observation that each 125I decay in the DNA produces approximately one dsb. Iodine was incorporated into DNA in the form of 5'-iododeoxyuridine and decay accumulation was allowed in cells kept frozen at -80 degrees C. Since widely different DNA damage dose-response curves were obtained in cells exposed to X-rays in various phases of the cell cycle, calibration was performed using synchronized populations of cells that were allowed to accumulate DNA damage in G1, G1/S, mid-S, and G2 + M. For this purpose the fraction of activity (in DNA) released from the plug (FAR) was measured and correlated to the number of 125I decays accumulated during the elapsed period of time. Fluctuations in the FAR per 125I decay were observed throughout the cell cycle that were similar to those previously reported for the FAR per Gy of X-rays. Comparison of the FAR per 125I decay with the FAR per Gy gave an induction of 21 +/- 3, 31 +/- 3, 21 +/- 3 and 26 +/- 8 dsb per Gy per diploid DNA complement for G1, G1/S, S, and G2 + M cells, respectively. The results suggest that the observed fluctuations in the FAR per Gy throughout the cycle reflect cell-cycle-associated differences in the physicochemical properties of the DNA molecules that alter their electrophoretic mobility, rather than variations in the induction of dsb per Gy, i.e. the sensitivity of the assay fluctuates throughout the cycle. We propose that similar phenomena underlie the observed fluctuations throughout the cell cycle in the fraction of activity eluted per Gy in the non-unwinding filter elution assay. 125I decays accumulated at 4 degrees C in partly purified DNA from cells embedded in agarose plugs and lysed immediately, gave FAR identical to those obtained with cells kept frozen. This finding suggests that indirect effects do not significantly contribute to DNA damage induction by 125I decay, and indicate that calibration of electrophoresis techniques for dsb measurements can be carried out using this simplified protocol.

Animals↗
PubMed1991

Comparison of DNA double-strand break rejoining as measured by pulsed field gel electrophoresis, neutral sucrose gradient centrifugation and non-unwinding filter elution in irradiated plateau-phase CHO cells.

The initial (up to 30 min) rate of DNA double-strand break (dsb) rejoining was measured in irradiated plateau-phase CHO cells, in a set of parallel experiments using the same cell suspension, by means of non-unwinding filter elution, neutral sucrose gradient centrifugation, and two pulsed-field gel electrophoresis assays: asymmetric field inversion gel electrophoresis (AFIGE) and clamped homogeneous electric field (CHEF) gel electrophoresis. The rate of DNA dsb rejoining was compared to the rate of rejoining of chromatin breaks measured, also in the same cell population, using the technique of premature chromosome condensation (PCC). Two radiation exposures, 25 Gy and/or 50 Gy, were used and applied to the individual parts of the experiments according to the sensitivity of the assay under investigation. Similar values for the initial rate of DNA dsb rejoining were obtained with all assays used, with t 1/2 ranging between 10 and 12 min after exposure to 25 Gy and between 15 and 20 min after exposure to 50 Gy. The initial rate of rejoining of chromatin breaks was slower than that of DNA dsb and occurred with t 1/2 of 87 min. The results suggest that all major techniques currently used for assaying rejoining of DNA dsb give similar results despite their widely different biophysical basis, and indicate that more information is required before a direct correlation between rejoining of DNA dsb and rejoining of chromatin breaks can be established.

Animals↗
PubMed1991

Kinetics of DNA double-strand break repair throughout the cell cycle as assayed by pulsed field gel electrophoresis in CHO cells.

Repair of DNA double-strand breaks (dsb) was measured in exponentially growing, plateau-phase and synchronized G1, G1/S, early S, mid-S, late S, G2 + M and mitotic CHO cells. Cells were exposed to 50 Gy X-rays and allowed to repair (up to 4 h) in suspension at 37 degrees C. The dsb rejoining was measured by means of asymmetric field inversion gel electrophoresis (AFIGE), a pulsed-field gel electrophoresis technique. The fraction of DNA-associated [14C]thymidine activity released from the plug (FAR) during electrophoresis was used as a parameter to determine the number of dsb present in the DNA. The assay had been previously calibrated using 125I-decay and its sensitivity in detecting dsb throughout the cell cycle established. Biphasic kinetics of dsb rejoining with a fast and a slow component were obtained throughout the cell cycle, including mitosis, as well as in the various stages of growth. Repair kinetics were described mathematically as the sum of two exponential functions. The repair half-times calculated for the fast component by least-square fitting to the data were in the range of 7-14 min, with the shorter values reached in mid-S and late S and the longer ones during G1 and mitosis. The repair half-times of the slow component were in the range between 60 and 90 min and did not show consistent fluctuations throughout the cell cycle. The fraction of dsb repaired by the slow component ranged from 18% to 37% and did not show consistent variation throughout the cell cycle either. These results suggest that the state of chromatin condensation has only a limited impact on the ability of the cells to rejoin dsb, and indicate that the cell cycle-dependent fluctuations in radiosensitivity cannot be explained by alterations in the rate of rejoining of dsb. The repair half-times of the slow component of dsb rejoining were similar to the half-times of rejoining of chromosome breaks as visualized by the technique of premature chromosome condensation, suggesting a cause-effect relationship between rejoining of this subject of dsb and rejoining of chromosome breaks.

Animals↗
PubMed1990

Induction and repair of DNA double strand breaks in radiation-resistant cells obtained by transformation of primary rat embryo cells with the oncogenes H-ras and v-myc.

Rat embryo cells (REC) transformed by the H-ras oncogene plus the cooperating oncogene v-myc are highly resistant to ionizing radiation as compared with the nontransformed parent cells, REC, or immortalized REC. In an attempt to understand the potential mechanism of resistance in these cells, the induction and repair of double strand breaks (dsb) in DNA were measured in a H-ras plus v-myc transformed (3.7) and an immortalized REC (mycREC) line using pulsed field gel electrophoresis. Cells were irradiated in the exponential phase of growth, and the amount of DNA dsb present was quantified by measuring the fraction of DNA activity released from the agarose plugs in which cells were embedded. Similar values of the fraction of DNA activity released were measured for both cell lines at equal X-ray doses, after correction for differences in cell cycle distribution, suggesting a similar induction of DNA dsb per Gy. Repair of DNA dsb measured after exposure to 40 Gy of X-rays was similar in both cell lines and displayed a fast and a slow component. The fast component had a 50% repair time of approximately 12 min, and the slow component, 50% repair time of about 3 h. These results suggest that the relative radioresistance of 3.7 cells is not conferred by a decrease in the amount of DNA dsb induced per Gy per dalton or by alterations in the capacity of the cells to repair DNA dsb. It is hypothesized that alterations in the expression of potentially lethal damage underlie this phenomenon.

Animals↗
PubMed1989

Use of roots transformed by Agrobacterium rhizogenes in rhizosphere research: applications in studies of cadmium assimilation from sewage sludges.

The use of roots transformed by Agrobacterium rhizogenes in models for the rhizosphere is discussed. A list of species for which transformed root cultures have been obtained is provided and the example of studies of cadmium assimilation from sewage sludges is given to illustrate how transformed root cultures can be used in physiological tests under non-sterile conditions.

Biological Availability↗
PubMed1989

Lead poisoning in Wilmington, Delaware, 1974-1984.

This paper demonstrates the efficacy of screening for lead poisoning by determining FEP for a rapid, accurate identification of children at risk and in need of treatment. Early detection and prompt treatment significantly reduces morbidity and mortality. With improvements in childrens' environments, screening procedures and treatment regimes, lead poisoning should eventually become obsolete.

Child↗
PubMed1971

Rabies.

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Animals↗