In systems used to express transformation using focus formation as the end point, nontransformed cells generally express a down-regulation of cell growth and division made evident by the formation of a monolayer of cells that completely covers the growth surface. In C3H 10T1/2 cells, down-regulation is thought to be progressively effected principally by cell-to-cell communication via gap junctions. Starting with a sparse population in asynchronous growth-e.g. containing cells in all phases of the growth cycle-as the area density increases, cells are progressively lost from the distribution in the order M phase, G 2 phase, S phase and G 1 phase, leading to the accumulation of viable cells out of cycle in so-called G 0 phase. We have measured the progressive phase transitions as a function of inoculum size and time. The influence of a promoter and an antipromoter was also examined as well as the expression of the cyclin/cyclin-dependent kinase inhibitors p21 Waf1/ Cip1 and p27 Kip1 as the cells grew into confluence. Using cells synchronized in mitosis, we found that with increasing cell density the expression of p27 increased and concomitantly p21 decreased.

To date, few studies have evaluated the magnitude of the risks of somatic effects in humans from low-dose or protracted radiation exposure to neutrons using in vitro or in vivo techniques (A. Kronenberg, Radiat. Res. 128, S87-S93, 1991). In an earlier study a strong energy dependence was shown for neutron-induced mutations at both the hprt and the tk loci in a rodent fibroblast cell line (Zhu and Hill, Radiat. Res. 139, 300-306, 1994). Using fast neutrons produced by impinging protons on a beryllium target at the UCLA/VA cyclotron, we have been examining the energy dependence of mutation induction at the HPRT locus in a human epithelial cell line derived from solid tumor tissue. In the present study, human epithelial teratocarcinoma cells were exposed to neutron beams produced from protons with 46, 30, 20 and 14 MeV energy. We found that cytotoxicity increased by 50% as the neutron energy decreased from 46 MeV to 14 MeV, confirming many earlier reports. But as with the Chinese hamster cells, the mutation frequency at the HPRT locus increased 2.5-4-fold with decreasing neutron energy. Additionally, although there was a strong energy dependence for mutation induction, we noted that the shape of the induction curves was curvilinear for the human cells compared to the linearity of the curves obtained for the Chinese hamster cells and some other non-solid tissue human cell lines. Calculations of the RBE, using γ rays as the standard, reflected these differences. The RBE for mutation at the HPRT locus was dependent not only upon energy but also on dose, giving rise to RBEs that were in some cases distinctly different from those found in the Chinese hamster cell line. In the low-dose region (doses below 75 cGy) the maximum RBE of about 5 resulted from irradiation by the lowest-energy neutron beam (14 MeV protons on beryllium).

There has been a keen interest in the past decade in both elucidating the mutation frequency for different energy radiations and determining if mutation frequencies vary from one gene to another. This interest is driven in part by the strong link between mutational events and subsequent development of the carcinogenic state. Using fast neutrons produced by impinging protons on a beryllium target at the UCLA/VA cyclotron, we have examined the energy dependence of the induction of mutants at the hprt and tk loci. In this paper, we present studies using V79 Chinese hamster cells exposed to beams of neutrons produced from protons with 46, 30, 20 and 14 MeV energy. There is a gradually increasing cytotoxic effect of the neutrons as the energy decreases. In a similar fashion, the mutation frequency also shows a strong energy dependence with the frequency increasing as the energy decreases. The results also show that the frequency of induced mutants at the tk gene is higher than at the hprt gene. Calculations of RBE using γ rays as the standard radiation showed a maximum for 14 MeV neutrons of 5.4 for the hprt locus and 36.6 for TK normal-growth mutants (TKng). Most of the curves for induction are best fitted with a linear function in the low-dose region with a few becoming curvilinear at higher doses.

Since our original observation that low-dose, low-dose-rate JANUS neutrons produced more transformation in C3H 10T1/2 cells than equivalent doses at high dose rate (Hill, Buonaguro, Myers, Han, and Elkind, Nature 298, 67-69, 1982), there have been many reports on the dose-rate dependence of a variety of high-LET radiations. Some of these have qualitatively supported our original and subsequent observations, and some have found no evidence for a dose-rate effect. Thus there remain questions about the generality, size, tissue specificity, and reproducibility of these observations. Furthermore, there are no well-established mechanistic descriptions or observations to account for such effects. In this report studies are presented using a range of neutron energies produced by the UCLA cyclotron from 12- to 46-MeV protons on beryllium. In particular, emphasis is placed on comparing results between energies for neoplastic transformation and mutation end points, and preliminary data are presented on the molecular and mechanistic aspects of neutron-induced damage.

The induction of potentially lethal damage and potentially mutagenic damage expressed by hypertonic salt treatment in late S-phase V79 cells after exposure to 46-MeV fast neutrons and 50-kVp X rays has been studied. Resistance to 6-thioguanine was used as the mutagenic end point. When cells in late S phase were treated with hypertonic salt solution immediately after fast neutrons or X irradiation, both cell killing and mutation induction were enhanced compared to fast neutrons or X irradiation alone. These results suggest that neutron as well as X irradiation of cells in late S phase induces both potentially lethal damage and potentially mutagenic damage. However, some differences in mutation frequency were observed between the two radiations after salt treatments. These results are discussed in terms of repair of mutagenic damage by an error-free or errorprone process.

We have measured γ-ray-induced neoplastic transformation in C3H10T1/2 mouse embryo cells irradiated at an average 10 cGy/day throughout the useful life span of these cells for transformation studies. At cumulative total doses of 50, 150, 300, and 450 cGy, samples of cells were assayed for cell survival and neoplastic transformation with or without the administration of 0.1 μg/ml of 12-O-tetradecanoylphorbol-13-acetate (TPA) starting 24 h after the irradiation. The results indicate that, at a dose rate of 10 cGy/day, the rate of induction of neoplastic transformation is reduced by a factor of thirteen compared to that at 100 cGy/min. Still, frequencies above the background level are observed. These results are consistent with previous data which, at 144 cGy/day (0.1 cGy/min), showed that radiation-induced initiation events could be repaired during exposure, thus reducing the frequency of transformation from that observed at 100 cGy/min [A. Han et al., Cancer Res. 40, 3328-3332 (1980)]. Although the addition of TPA after the delivery of a particular dose at 10 cGy/day produced a significant increase in the frequency of neoplastic transformation, the degree of enhancement was less than after higher-dose-rate exposures [C. K. Hill et al., Radiat. Res. 109, 347-351 (1987)]. These results indicate that during 7 weeks of exposure, the repair of radiation-induced initiation was extensive but not complete, and suggest that a significant part of the damage persists which can be promoted by TPA. These observations support the inference that initiation and promotion are not tightly coupled and are probably independent processes.

Chinese hamster V79 cells (subline M12G) were exposed repeatedly to fractionated doses of germicidal 254 nm light (far-uv) at <tex-math>$6\ {\rm J}\cdot {\rm m}^{-2}/\text{fraction}/\text{day}$</tex-math> or sunlight-simulating 290-330 nm (mid-uv) at <tex-math>$150\ {\rm J}\cdot {\rm m}^{-2}/\text{fraction}/\text{day}$</tex-math> and sensitivities to cell killing action and mutation of far-uv and mid-uv were examined. As the number of exposure fractions increased, the cell cultures became resistant to cell killing induced by both far-uv and mid-uv. Increases in both D 0 and <tex-math>$D_{{\rm q}}$</tex-math> were observed. Treatment with exposures of <tex-math>$6\ {\rm J}\cdot {\rm m}^{-2}$</tex-math> far-uv is more efficient in yielding cell cultures that are resistant than exposures of <tex-math>$150\ {\rm J}\cdot {\rm m}^{-2}$</tex-math> mid-uv. In contrast to the cells exposed to repeated far-uv, the cells exposed to repeated mid-uv were relatively more resistant to cell killing effects of mid-uv than far-uv, suggesting a possible role of photolesions other than pyrimidine dimers. When mutants resistant to 6-thioguanine were assayed during repeated exposure to far- or mid-uv light, the yield was initially linear with accumulating dose. At high total accumulated doses, the frequency decreased gradually (<tex-math>$6\ {\rm J}\cdot {\rm m}^{-2}$</tex-math> mid-uv) or reached a plateau (<tex-math>$150\ {\rm J}\cdot {\rm m}^{-2}$</tex-math> mid-uv). The sensitivity of N80 cells (exposed to 80 fractions of mid-uv) to mutation induction by uv light is higher than that of the original M12G cells, whereas U81 cells (exposed to 81 fractions of far-uv) have a sensitivity similar to that of the original cells. Although an initial decrease in resistance to cell killing was observed, resistant cells retained their characteristics after 100 days in culture without further exposure. Cross-resistance to X rays was not shown. The data in this paper suggest that the capacity for repair of photolesions in DNA by repair processes was enhanced in cell cultures by repeated exposure to far-uv or mid-uv and that this altered the cells' ability to cope with lethal and mutagenic lesions. It remains to be seen if these changes in cell sensitivity were brought about by selective or inductive processes or a combination of both.

Survival parameters and immediate DNA damage induced by 60 Co γ rays, 50-kVp X rays, and Janus fission-spectrum neutrons in human epithelial P3 cells (derived from an embryonic teratocarcinoma) are compared with those for Chinese hamster lung V79 cells. DNA damage caused by X and γ irradiation, measured by alkaline elution methods, is the same in both cell types, whereas the P3 cells are about two times more sensitive (as measured by D 0 ratios of the final survival curve slope) to the lethal effects of these radiations than are the V79 cells. Human P3 cells are also more sensitive to the lethal effects of fission-spectrum neutrons than V79 cells. Survival experiments with split radiation doses and hypertonic salt treatment indicate that both P3 cells and V79 cells can recover from radiation-induced damage efficiently.

C3H10T1/2 cells were exposed to low doses of 60 Co γ rays at 100 or 0.10 cGy/min and the incidence of neoplastic transformation was assayed with or without the addition of tetradecanoylphorbol-13-acetate (TPA). As we reported earlier [A. Han, C. K. Hill, and M. M. Elkind, Cancer Res. 40, 3328-3332 (1980); C. K. Hill, A. Han, F. Buonaguro, and M. M. Elkind, Carcinogenesis 5, 193-197 (1984)], the frequency of neoplastic transformation per unit dose following low doses appears to be linear and is reduced 2.3-fold at 0.10 cGy/min compared to 100 cGy/min. We report now that the addition of TPA 24 h after irradiation appreciably enhances the frequency after both low- and high-dose-rate exposures. The enhancement indicates that TPA leads to the expression of potentially effective, preneoplastic damage due to γ rays. Our data suggest that the enhancement of transformation by TPA is essentially independent of dose rate. Also, our results suggest that the sector of preneoplastic damage which is repaired during protracted exposures becomes unavailable to enhancement by TPA.

The neoplastic transformation of C3H mouse 10T 1/2 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 (∼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 60 Co γ 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 60 Co γ rays is 78.8.

Appropriate in vitro mammalian cell systems facilitate the study of cellular mechanisms of radiation oncogenesis. C3H 10T 1/2 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 60 Co γ 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 γ 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.

Following mid to large doses of X rays, or of fission spectrum neutrons, the repair of potentially lethal damage in V79 Chinese hamster cells can be inhibited by anisotonic phosphate-buffered saline [H. Utsumi and M. M. Elkind, Radiat. Res. 77, 346-360 (1979); Int. J. Radiat. Biol. 35, 373-380 (1979)] or by medium containing 90% D 2 O [E. Ben-Hur, H. Utsumi, and M. M. Elkind, Radiat. Res. 84, 25-34 (1980)]. The foregoing post-treatments do not affect the viability of unirradiated cells. Using single synchronized cells irradiated in late S-phase, the most resistant phase of the cell cycle, repair of potentially lethal damage in the "single-hit," initially exponential, or small-dose part of the survival curve was examined. The use of synchronized cells avoids misinterpretations due to population heterogeneity. The slope of the small-dose, exponential region of the neutron survival curve is much steeper than that of the X-ray survival curve. Even so, it is demonstrated with post-treatments consisting of hypertonic phosphate-buffered saline, medium containing D 2 O, or medium containing caffeine that in the small-dose region cells ordinarily repair potentially lethal, "single-hit" neutron damage. Sensitive cells-i.e., cells not able to repair potentially lethal damage expressible by hypertonic buffer-appear not able to repair "single-hit" damage.