Role of repair processes in neoplastic transformation induced by ionizing radiation in C3H/10T1/2 cells.
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Biomedical subjects
M M Elkind
Publications and source records attributed to M M Elkind.
Neoplastic transformation is enhanced by multiple low doses of fission-spectrum neutrons.
The neoplastic transformation of C3H mouse 10T1/2 cells was measured induced by fission-spectrum neutrons delivered at a high dose rate in five fractions over 4 days. The transformation frequency was significantly enhanced over that due to single equivalent total doses. These new data, in the low dose region, demonstrate an increased transformation frequency by fractionated versus single exposures of high-dose-rate fission-spectrum neutrons; an increase equal to that observed with low-dose-rate fission-spectrum neutrons (i.e., 0.086 rad/min). Estimates of the dose modifying factor (DMF), based upon the ratio of the initial linear portions of the induction curves for high and for low dose rates, suggest the same DMF (approximately 7.8) for both five daily fractions of high-dose-rate neutrons and for low-dose-rate neutrons. However, when these results are compared to those following high-dose-rate 60Co gamma rays (100 rad/min), the relative biological effectiveness (RBE) for low-dose-rate fission-spectrum neutrons based upon slope ratios is 19.6; similarly, the RBE relative to five daily fractions of 60Co gamma rays is 78.8.
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Radiobiology in radiotherapy: a personal history.
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Poly(ADP-ribose) metabolism in X-irradiated Chinese hamster cells: its relation to repair of potentially lethal damage.
Nicotinamide-adenine dinucleotide (NAD+) is the substrate used by cells in poly(ADP-ribose) synthesis. X-irradiation of log-phase Chinese hamster cells caused a rapid decrease in NAD+ levels which was linearly dependent on radiation dose. The activity of ADP-ribosyl transferase ( ADPRT ) also increased linearly with radiation dose. The decrease of NAD+ was slower, and the increase in ADPRT activity was less pronounced, in a radiation sensitive line, V79- AL162 /S-10. An inhibitor of ADPRT , m-aminobenzamide, largely prevented the depletion of cellular NAD+ and reduced the rate at which ADPRT activity disappeared during post-irradiation incubation. Post-irradiation treatment with hypertonic buffer or with medium containing D2O--which inhibit repair of radiation-induced potentially lethal damage--enhanced the depletion of NAD+ and prevented the reduction in ADPRT activity following irradiation. The characteristics of the effects of treatment with hypertonic buffer on NAD+ metabolism were qualitatively similar to the effects that such treatment has on radiation-induced cell killing. These results suggest that poly(ADP-ribose) synthesis after irradiation plays a role in the repair of potentially lethal damage.
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Fission-spectrum neutrons at a low dose rate enhance neoplastic transformation in the linear, low dose region (0-10 cGy).
The neoplastic transformation of C3H 10T1/2 cells induced by fission-spectrum neutrons delivered at a high dose rate is linear up to 40 cGy. Reducing the dose rate increases the frequency of transformation in the low dose region. At a dose rate of 0.086 cGy min-1, the initial part of the induction curve remains linear but it has a slope 9-fold greater than the initial part of the curve at a high dose rate.
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Multifractionation of 60Co gamma-rays reduces neoplastic transformation in vitro.
Incidences of neoplastic transformation of 10T 1/2 cells, derived from a C3H mouse embryo, were measured following irradiation of cells with 60Co gamma-rays delivered in single total doses, from 25 to 300 rads, or in different numbers of fractions and with different time intervals between fractions. Throughout the range of total doses that was examined (25-300 rads) exposure of asynchronous, actively growing 10T 1/2 cells to a dose of gamma-rays in five equal daily fractions, rather than in a single exposure, results in a significant reduction in transformation frequency. The initial part of the induction curve fits a straight line quite well. The slope of the line for five daily fractions is approximately 3-fold less steep than that for single exposures. A further increase in the interfraction interval, to 48 h, or further reductions in the dose per fraction, led to additional decreases in the incidence of neoplastic transformation. Our observations are consistent with a net 'error-free' repair of subtransformation damage as a result of protracted irradiation with 60Co gamma-rays.
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Inhibitors of poly (ADP-ribose) synthesis enhance radiation response by differentially affecting repair of potentially lethal versus sublethal damage.
The effect of nicotinamide and m-aminobenzamide, representative inhibitors of poly(ADP-ribose) synthesis, on the survival of V79 Chinese hamster cells following ionizing radiation was studied in plateau- and log-phase cultures. In plateau-phase, after an acute dose of X-rays, nicotinamide partially inhibited the repair of potentially lethal damage that occurs when the plating of such cells is delayed. The incompleteness of the inhibition was attributed to the slowness of the uptake of nicotinamide by unfed plateau-phase cells. Postirradiation incubation of log-phase cells with either of these inhibitors caused a pronounced enhancement of the radiation response which was reflected mainly by a reduction of the shoulder on the survival curve. However, the ability of such treated cells to repair sublethal damage was hardly affected as demonstrated by the reappearance of the shouldered survival curve during a fractionation interval. NAD levels in irradiated cells were rapidly reduced. This reduction was somewhat slower in a radiation sensitive mutant and was almost completely blocked in the presence of m-aminobenzamide. Another treatment that enhances the radiation response, i.e., incubation in D2O medium, facilitated the rate of NAD depletion in irradiated cells. We conclude that inhibitors of poly(ADP-ribose) synthesis inhibit the repair of potentially lethal damage in plateau-phase as well as in log-phase cells with little if any effect on the repair of sublethal damage. Poly(ADP-ribose) appears, therefore, to have a role in the repair of radiation damage that is potentially lethal in mammalian cells.
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Inhibitors of poly(ADP-ribose) synthesis enhance X-ray killing of log-phase Chinese hamster cells.
Postirradiation incubation of V79 Chinese hamster cells with inhibitors of poly(ADP-ribose) synthesis was found to potentiate the killing of cells by X rays. Potentiation increased with incubation time and with concentration of the inhibitor. Preirradiation incubation had only a small effect. The enhanced response correlated well with the known extent of the inhibition of poly(ADP-ribose) synthesis. A radiation-sensitive line, V79- AL162 /S-10, was affected to a lesser extent than the normal cells. Cells repaired the radiation damage with which the inhibitors interacted within 1 hr, a process that has similar kinetics to what is observed when a postirradiation treatment with hypertonic buffer is used [H. Utsumi and M. M. Elkind , Radiat . Res. 77, 346-360 (1979)]. However, the sectors of damage affected by inhibitors of poly(ADP-ribose) synthesis and hypertonic buffer do not entirely overlap. The inhibitor nicotinamide enhanced the killing mainly of late S-phase cells and did not affect cells at the G1/S border. It is concluded that the repair process(es) involving poly(ADP-ribose) synthesis is important for cell survival in repair-competent cells and that the radiation-sensitive cells that were examined are partially deficient in a repair pathway in which poly(ADP-ribose) participates.
4-Aminobenzoic Acid↗
Repair processes in radiation biology.
Three dualities underscore the role of repair in radiobiology: the discrete and random nature of energy deposition; the coupled processes of damage and repair; and the harmful and beneficial properties of radiation in connection with issues of public health and the radiation therapy of cancer, respectively. Starting with the survival curve of HeLa S3 cells, published by T. T. Puck and P. I. Marcus [J. Exp. Med. 103, 653-666 (1956)], the survival response of repair-competent mammalian cells is discussed and is related to clinical experience. This is followed by an examination of DNA repair in surviving cells from which it is inferred that even in repair-deficient cells, very likely large numbers of single- and double-strand DNA breaks are repaired because the progeny of surviving cells are essentially free of such damage. These observations support the idea that even when single-hit dose-effect relationships are involved (e.g., survival of ataxia telangiectasia cells), repair processes may play a role. Lastly, single-hit kinetics connected with the induction of neoplastic transformation are discussed. The data demonstrate that linear dose-response dependencies due to ionizing radiation (i.e., gamma rays and fission-spectrum neutrons) can be modulated by dose protraction, but that neither the direction nor the extent of such modulations can be predicted on theoretical grounds. To be applied more effectively for the benefit of mankind, therefore, it remains necessary to understand fully the biological mechanisms of the action of ionizing radiation.
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Repair processes and radiation quality in neoplastic transformation of mammalian cells.
Appropriate in vitro mammalian cell systems facilitate the study of cellular mechanisms of radiation oncogenesis. C3H 10T1/2 cells derived from mouse embryo have been used to study the effects of protracted irradiation on cell killing and incidence of neoplastic transformation. Neoplastic transformation in these cells results in the appearance of colonies of densely piled up, disorganized cells that grow on top of the contact inhibited monolayer of nontransformed cells. Protracted exposures of 60Co gamma rays or fission-spectrum neutrons (from the JANUS reactor at the Argonne National Laboratory) were given either at low dose rates or as multifractionated regimens using high dose rates. Dose protraction of gamma rays by multifractionation at a high dose rate (50 rad/min) results in appreciable reduction in cell killing and also significantly reduces the incidence of neoplastic transformation. Irradiation at a low dose rate has the same qualitative effect. In contrast, protracted exposures of fission-spectrum neutrons, given at a low dose rate (0.086 or 0.43 rad/min), result in significant enhancement of the frequency of transformation in the dose region of up to 80 rad. However, the survival of cells is essentially the same as that for the same single dose of neutrons given at a high dose rate. These observations are consistent with net "error-free" repair of transformational damage following protracted exposure of a low LET radiation and possibly a net "error-prone" repair of transformational damage after protracted irradiations with fission-spectrum neutrons.
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Repair of neoplastic transformation damage following protracted exposures to 60Co gamma-rays.
The incidences of neoplastic transformation induced by 60Co gamma-rays in exponentially growing mouse embryo 10T1/2 cells were measured following acute and protracted exposures. Delivery of 60Co gamma-rays at a low dose rate (0.1, 0.5, 2.5 cGy min-1) compared with a high dose rate (100 cGy min-1) results in appreciable, dose rate dependent reductions in cell killing and, independent of the effect on cell survival, reduces significantly the incidence of neoplastic transformation. Exposure of exponentially growing 10T 1/2 cells to a dose of gamma-rays in 5 equal daily fractions also significantly reduces transformation frequency, compared with delivery in a single dose, throughout the dose range examined (25-300 cGy). The initial parts of the induction curves are fitted quite well by a linear dose dependence. The slopes of the regression lines for multifractionation delivery or irradiation at 0.1 cGy min-1, are one-third and one-half, respectively, of those for single exposures at a high dose rate. Increasing the inter-fraction interval up to 48 h, or reduction of the dose per fraction further reduce incidence of neoplastic transformation. We conclude that protracted exposures of low LET radiation result in a net "error-free" repair of subtransformation damage.
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Possible error-prone repair of neoplastic transformation induced by fission-spectrum neutrons.
We have examined the effect of fission-spectrum neutrons from the JANUS reactor at Argonne National Laboratory, delivered either as acute or protracted irradiation, on the incidence of neoplastic transformation in the C3H 10T1/2 mouse embryo cell line. Acute exposures were delivered at 10-38 cGy min-1, protracted exposures at 0.086 or 0.43 cGy min-1. The total doses for both ranged from 2.4 to 350 cGy. In the low dose region (2.4-80 cGy), there was a large enhancement in transformation frequency when the neutrons were delivered at the low dose rates compared with the high dose rates, but the survival of the cells was not significantly different between the two exposures conditions. Analysis of the initial parts of the curves shows that the regression line for protracted doses is about 9 times steeper than that for single acute exposures. Finally, the possibility is discussed that an "error-prone" repair process may be causing the enhanced transformation frequency by protracted neutron exposures.
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Repair processes in radiation-induced transformation.
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Suppressive action of near-ultraviolet light on ouabain resistance induced by far-ultraviolet light in Chinese hamster cells.
The interaction between ultraviolet light (UV-C) from germicidal lamps (254 nm) and near-ultraviolet light (UV-B) from Westinghouse Sun Lamps (290-345 nm) was studied in Chinese hamster V79 cells by measuring the effectiveness of combined exposures to induce the resistance to 6-thioguanine or to ouabain. Exposure of cells to a conditioning dose of UV-B (approximately 70% survival) results in significant inhibition of the induction by UV-C of cells resistant to ouabain. The inhibition is lost, however, if cells are incubated for 12 h at 37 degrees C between exposures. Inhibition is also observed when cells are preirradiated with a dose of UV-B filtered with polystyrene (300-345 nm) which, in itself, has no effect on cell killing. Conditioning exposures of unfiltered or filtered UV-B light do not inhibit the induction of 6-thioguanine-resistant cells by UV-C light, and the effects of UV-B and UV-C light are largely independent.
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Caffeine-enhanced survival of radiation-sensitive, repair-deficient Chinese hamster cells.
A clone of V79 Chinese hamster cells (V79-AL162/S-10) with unique properties has been isolated after a challenge of parental cells (V79-AL162) with 1 mM ouabain. Compared with parental cells, or with other clones isolated after the ouabain challenge, these cells form smaller colonies, are more sensitive to both X rays and fission-spectrum neutrons, and respond atypically to a postirradiation treatment with caffeine. Their enhanced response to X rays results mainly from a large reduction in the shoulder of their survival curve, probably because in late S phase, the most resistant phase in the cell cycle, the survival curve of these cells has a reduced shoulder width. Caffeine, and to a lesser extent theophylline, added to the colony-forming medium immediately after exposure appreciably increases the width of the shoulder of these sensitive cells, whereas caffeine has the opposite effect on the response of normal V79 cells. Because dibutyryl-cAMP added immediately after exposure actually further sensitizes these sensitive cells, it would appear that the enhanced survival due to methylxanthines, which inhibit cAMP phosphodiesterase, does not result from increases in intracellular levels of cAMP. Thus the unique response of the V79-AL162/S-10 cells to a radiation posttreatment with caffeine (increased survival) results from a net increase in their ability to repair damage that is otherwise lethal; caffeine treatment ordinarily prevents normal V79 cells from repairing damage that is only potentially lethal.
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Fission-spectrum neutrons at reduced dose rates enhance neoplastic transformation.
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Modifiers of radiation response in tumor therapy: strategies and expectations.
The administration of two (or more) cytotoxic agents to widen the differential between the responses of tumor and normal tissues depends upon the biological properties of the agents in the cells and tissue, their interactive potential, and the strategy employed in their administration. Assuming that one agent is ionizing radiation and considering response modification in broad terms, the qualitative features of various strategies are developed for physical as well as chemical modifiers. The heterogeneity of human tumor cells and the compensatory mechanisms of normal tissues following injury are identified as topical areas requiring sustained research effort. Finally, estimates are developed for the degree of improvement required from a response modifier to effect significant improvements in tumor cure rates.
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Enhanced transformation of mouse 10T1/2 cells by 12-O-tetradecanoylphorbol-13-acetate following exposure to X-rays or to fission-spectrum neutrons.
Addition of the tumor promoter of 12-O-tetradecanoylphorbol-13-acetate (TPA) to C3H/10T1/2 cells after exposure to either X-rays or to fission-spectrum neutrons increases significantly the frequency of transformation without any effect on cell survival. However, treatment of unirradiated cells with 0.1 micrograms of TPA per ml alone results in a small increase in transformation frequency above background (i.e., from 1.1 x 10(-5) to 1.0 x 10(-4). Thus, besides being a promoter, TPA is also a weak initiator. Enhancement of radiation-induced transformation by TPA was relatively greater after low compared to high doses of either radiation. In addition, TPA causes the relative biological effectiveness of neutrons compared to X-rays to increase with increasing dose or with increasing frequency of transformation rather than to decrease, when TPA is not used. For X-ray doses from zero to approximately 120 rads, TPA raises transformation to frequencies approximately equal to those due to neutrons alone. Analysis of TPA enhancement in the context of the combined effect of two inducing agents, i.e., TPA plus a radiation, indicates that with either radiation TPA acts synergistically. Lastly, TPA was found to alter the dependence of transformation frequency on the density of viable cells. As opposed to a constant frequency of transformants per surviving (or viable) cell, which we observed after a fixed dose of X-rays or neutrons for a range of cell inocula, the addition of TPA increased the frequency of transformation for cell inocula (i.e., from approximately 20 to 6000 viable cells per 90-mm Petri dish). However, the frequency of transformation decreases with increasing size of the inoculum, a result that we interpret to indicate the combined effect of an interference with cell-to-cell communication by TPA plus the fading of initiation events due to the radiation.
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