Neutron-energy-dependent oncogenic transformation of C3H 10T1/2 mouse cells

The relative biological effectiveness (RBE) of a range of neutron energies relative to 250-kVp X rays has been determined for oncogenic transformation and cell survival in the mouse C3H 10T 1/2 cell line. Monoenergetic neutrons at 0.23, 0.35, 0.45, 0.70, 0.96, 1.96, 5.90, and 13.7 MeV were generated at the Radiological Research Accelerator Facility of the Radiological Research Laboratories, Columbia University, and were used to irradiate asynchronous cells at low absorbed doses from 0.05 to 1.47 Gy. X irradiations covered the range 0.5 to 8 Gy. Over the more than 2-year period of this study, the 31 experiments provided comprehensive information, indicating minimal variability in control material, assuring the validity of comparisons over time. For both survival and transformation, a curvilinear dose response for X rays was contrasted with linear or nearly linear dose responses for the various neutron energies. RBE increased as dose decreased for both end points. Maximal RBE values for transformation ranged from 13 for cells exposed to 5.9-MeV neutrons to 35 for 0.35-MeV neutrons. This study clearly shows that over the range of neutron energies typically seen by nuclear power plant workers and individuals exposed to the atomic bombs in Japan, a wide range of RBE values needs to be considered when evaluating the neutron component of the effective dose. These results are in concordance with the recent proposals in ICRU 40 both to change upward and to vary the quality factor for neutron irradiations.     

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.     

Effects of split-dose irradiation on survival and oncogenic transformation induced by 31 MeV protons in C3H10T1/2 cells

Survival and oncogenic transformation were studied in C3H10T1/2 cells exposed to 31 MeV protons. Total doses of 0.5, 1 and 7 Gy were delivered as single and two equal fractions with various time intervals up to 10 h between doses. With split doses as compared with single doses to a total dose of 7 Gy, survival increased by a factor of 2.5 +/- 0.2, whereas the frequency of transformation per surviving cell declined by a factor of 3.1 +/- 0.5. Maximal split-dose recovery occurred within the first 5 h for both endpoints. Further, the transformation frequency decreased by factors of 3.1 +/- 0.6 and 1.5 +/- 0.3 respectively for total doses of 0.5 and 1.0 Gy split into two equal fractions. The data for 1 and 7 Gy are compatible with data in the literature for other low LET radiations.     

Morphological transformation of C3H/10T1/2 cells subcultured at low cell densities

Morphological transformation in $\text{C3H/10T}\text{1}\text{2}$ cells treated with varied concentrations of benzo (α) pyrene (BP) was measured following subculture at low cell densities. Subconfluent cultures exposed to BP were allowed to grow to confluence, trypsinized, and reseeded at cell densities ranging from 5 to 2,300 surviving cells/cm². These secondary cultures were incubated for 8 to 9 weeks, stained, and examined for evidence of morphological transformation. BP-treated cells reseeded in virtual isolation in microwells (approx. 5 surviving cells/cm²) transformed at frequencies up to 14.5%. At these low initial cell densities, transformation frequency did not demonstrate a significant dependence on BP concentration. However, BP-treated cells reseeded at higher densities (11 to 2,300 surviving cells/cm²) showed both density-dependent transformation frequencies and BP-concentration dependence of transformation. As reported previously (Haber et al., Cancer Res. 37 1644, 1977), the subculturing of treated cells did not affect the BP-concentration dependence of focus formation in the $\text{C3H/10T}\text{1}\text{2}$ transformation assay. Cell density-dependent suppression of morphological transformation has now been observed over a wide range of BP concentration. We suggest that this phenomenon is associated with colony interactions and consider various possible mechanisms of BP involvement.     

Modification of transformation of C3H/10T1/2 cells by a differentiation-inducing substance

Transformation of C3H/10T1/2 cells was induced by 3-methylcholanthrene. Treatment with hexamethylene bisacetamide (HMBA), a differentiation inducing and poly (ADP-ribose)-synthesis modifying substance, influences expression of multilayered foci in a treatment schedule-dependent manner. Inhibition of transformation occurred only if HMBA was present after the genotoxic damage. After HMBA treatment most of transformed cells showed an end-differentiation like form.     

In vitro transformation by bromodeoxyuridine and X irradiation in C3H 10T1/2 cells

The effect of 10(-5) M bromodeoxyuridine (BrdUrd) substitution in C3H 10T1/2 cells was evaluated. Cellular toxicity increased rapidly for BrdUrd exposure times that were longer than the population doubling time. Radiosensitization by BrdUrd exposure was almost complete after one cell doubling time and was characterized by a decrease in D0 and the survival curve shoulder. Exposure to BrdUrd for one cell doubling time produced only very low transformation levels, but for prolonged BrdUrd exposure times, the transformation frequency per viable cell increased significantly. BrdUrd incorporation also enhanced radiation induction of transformation above the transformation levels resulting from the independent action of X rays or BrdUrd treatment. These results show that BrdUrd is a transforming agent in C3H 10T1/2 cells and thus may be a carcinogen and that BrdUrd can enhance radiation-induced transformation.     

Radiosensitivity parameters for neoplastic transformations in C3H10T1/2 cells

We have evaluated radiosensitivity parameters for cellular transformation from published experimental data on neoplastic transformations induced in C3H10T1/2 cells by BEVALAC ions. The measured RBE values are well reproduced by a track theory calculation using sets of m-target parameters with either m = 2 or m = 3, suggesting a quadratic or cubic extrapolation to low doses of gamma rays. Using track theory one is thus able to predict transformation frequencies in those cells after an arbitrary radiation field, under known or assumed conditions of exposure, in a manner shown earlier for cellular survival. Extension of these calculations to interpret cancer incidence in vivo is also discussed.     

Morphological transformation and chromosome damage by amsacrine in C3H/10T1/2 clone 8 cells

Morphological transformation, cell survival, chromosomal aberrations and micronuclei were measured in C3H/101/2CL8 cells after 24 h exposure to amsacrine. A weak but dose-related increase in the percentage of dishes containing transformed foci occurred. As previously reported for alkylating agents, this effect was increased by treating 5 days instead of 1 day after plating. There was no evidence for gene mutation at the Na/K ATPase locus, although amsacrine induced micronuclei in a large percentage of cells and chromosomal aberrations, including interchange events and double minute chromosomes, in dividing cells. In would appear that transformation and chromosomal events may be related in amsacrine-treated C3H/10T1/2CL8 cells. The results strongly suggest that amsacrine has carcinogenic potential, possibly related to its chromosome-breaking properties.     

Radiation-induced neoplastic transformation of C3H10T1/2 cells is suppressed by ascorbic acid

X-ray induced transformation of C3H10T1/2 cells was suppressed in a concentration-dependent manner by administration of ascorbic acid after irradiation (0.1-20 micrograms/ml for the first week) in the culture medium. The dose-response curve was shifted about 60% downward and was slightly steeper in the presence of ascorbic acid (5 micrograms/ml for the first week) than in its absence. The 1-week treatment procedure revealed that cells initiated by radiation remained susceptible to ascorbic acid until the time of morphological phenotype expression. The neoplastically transformed phenotype expressed after incubation for 8 weeks could no longer be suppressed by ascorbic acid even after culture transfer. Similarly, the neoplastically transformed phenotype suppressed for 8 weeks by ascorbic acid treatment was not subsequently expressed in the absence of ascorbic acid. On the basis of the oxygen-detoxifying nature of ascorbic acid, we postulated that expression of the neoplastically transformed phenotype is promoted by reactive oxygen species and peroxy radicals generated in cells during the whole assay period. The data may be useful as a guide for chemopreventive efforts against radiation carcinogenesis.     

Exposure to extremely low frequency magnetic fields suppresses x-ray-induced transformation in mouse C3H10T1/2 cells

We designed and manufactured equipment for exposure of cultured cells to extremely low frequency magnetic fields (ELFMF) at 5, 50, and 400 mT and examined the effect of ELFMF on cellular transformation in mouse C3H10T1/2 cells (clone 8). Transformed foci, Type II and Type III, were independently counted as transformants. The cells were exposed to ELFMF alone at 5, 50, and 400 mT for 24 h or X-irradiated with 3 Gy followed by the ELFMF exposure. No significant difference in the transformation was observed between sham-exposed control and the ELFMF exposure from 5 to 400 mT. The transformation frequency for X-rays plus ELFMF was decreasing compared with X-rays alone. When 12-O-tetra-decanoylphorbol-13-acetate (TPA) was contained in the medium throughout the experiment, the transformation frequency by X-rays alone was elevated more. In the combined treatment with X-rays followed by ELFMF, the transformation frequency was slightly decreased at 50 and 400 mT even in the medium containing TPA. The long-term exposure at 5 mT suppressed both spontaneous and X-ray-induced transformations significantly. It is well known that overexpressing protein kinase C (PKC) failed to yield identifiable transformation of foci induced by ionizing radiation. We demonstrated previously that exposure to high-density ELFMF induced expression of several genes through an increase in PKC activity. From these results, it is suggested that ELFMF might suppress X-ray-induced transformation through activation of PKC by ELFMF.     

Lactate-mediated changes in growth morphology and transformation frequency of irradiated C3H 10T1/2 cells

Treatment of mammalian cells with lactate or inhibitors of glycolysis alters their radiation response, particularly in the low dose region of the dose response curve. The occurrence of both high lactate levels and high glycolytic metabolism in tumours is well known and therefore the effect of lactate on a cell line sensitive to radiation induced transformation was examined using a single exposure to Cobalt 60 gamma rays as the carcinogen challenge. The results indicate that cells treated with 5mM lactate before irradiation exhibit changes in morphology and growth rate and that the transformation frequency is increased by three to ten fold following 24 hours lactate treatment just prior to irradiation. Examination of radiation survival curves showed a positive correlation between transformation frequency and size of the shoulder, but increasing transformation frequency was associated with a decrease in Do. A mechanism involving altered Redox potential in lactate treated cells is suggested. The results are discussed in terms of their possible significance for radiotherapy.     

Colony transformation in C3H 10T1/2 cells: stepwise development of neoplastic change in vitro

This report demonstrates that low passage C3H 10T1/2 cells treated with the carcinogens benzo(a)pyrene or diepoxybutane are transformed morphologically as colonies in as little as 14 d after carcinogen treatment. A transforming dose-response curve is achieved but the frequency of transformation is less than half the expected for 38 d foci, compared on the basis of percent transformants per cell plated. Anchorage-independent cell growth, plating efficiency, doubling time, cell density, and modal chromosomal number were examined from transformed colonies and foci. The data from colony transformants show progressive alteration of these in vitro expressions of neoplastic character with continued subcultivation, consistent with the multistep hypothesis of carcinogenesis. Early in vivo data obtained from one colony-derived transformed cell line show tumorigenesis in irradiated mice within 13 wk of implantation. With continued in vivo passage, tumors were observed in 4 to 6 wk.     

Morphological transformation and tumorigenicity in C3H/10T1/2 cells transformed with an inducible c-Ha-ras oncogene

A C3H/10T1/2 cell line containing an inducible metallothionein-ras hybrid oncogene was conditionally and reversibly transformed upon exposure to zinc ions. Interestingly, although the cell line was fully malignant when expressing only low levels ofras, complete morphological transformation required much higher levels.     

Absence of a dose-rate effect in the transformation of C3H 10T1/2 cells by alpha-particles

The findings of Hill et al. (1984) on the greatly enhanced transformation frequencies at very low dose rates of fission neutrons induced us to perform an analogous study with alpha-particles at comparable dose rates. Transformation frequencies were determined with gamma-rays at high dose rate (0.5 Gy/min), and with alpha-particles at high (0.2 Gy/min) and at low dose rates (0.83-2.5 mGy/min) in the C3H 10T1/2 cell system. alpha-particles were substantially more effective than gamma-rays, both for cell inactivation and for neoplastic transformation at high and low dose rates. The relative biological effectiveness (RBE) for cell inactivation and for neoplastic transformation was of similar magnitude, and ranged from about 3 at an alpha-particle dose of 2 Gy to values of the order of 10 at 0.25 Gy. In contrast to the experiments of Hill et al. (1984) with fission neutrons, no increased transformation frequencies were observed when the alpha-particle dose was protracted over several hours.     

Induction of ouabain-resistance mutation and cycle-dependent transformation of C3H/10T1/2 cells by N-nitroso-2-acetylaminofluorene

Ouabain-resistance mutation and cell cycle-dependent transformation were studied concurrently in the C3H/10T1/2 cell line treated with N-nitroso-2-acetylaminofluorene (N-NO-AAF) or N-nitroso-N-2-fluorenylacetamide. N-NO-AAF is a new direct-acting mutagen that exhibits a very short half-life (34 min) in complete medium independent of cell number seeded. With 0.1-0.3 mM of N-NO-AAF, cytotoxicity was noted after exposure for 2 h, but another phase of cytotoxicity was observed between 8 and 24 h. N-NO-AAF was more toxic than its parent compound 2-AAF. Moreover, maximal mutation frequency at the Na+/K(+)-ATPase gene locus (ouar mutation) was attained within 30 or 40 min of exposure, dependent on dosage of N-NO-AAF. With 2-AAF, 2-AF and 2-nitrofluorene, however, no detectable mutants were found under the same conditions. In cell cycle-dependent transformation assays, cells were synchronized by release from confluence-induced arrest of proliferation, 2 concentrations of N-NO-AAF were added for 2 h at various intervals during the cell cycle. The results clearly revealed that cells in 2 specific time intervals were susceptible to malignant transformation, i.e., at 10 and 18 h (early S phase) after release from the block.     

Induction of neoplastic transformation in C3H/10T1/2 cells by 2.45-GHz microwaves and phorbol ester

C3H/10T1/2 cells were exposed to 2.45-GHz microwaves for 24 h and/or 1.5 Gy of 238-kVp X rays at 3.75 Gy/min. Transformation frequency and cell survival were measured with or without postirradiation addition of the tumor promoter tetradecanoyl-phorbol-13-acetate (TPA) at 0.1 microgram/ml. We previously reported (Carcinogenesis 6,859-864, 1985) an enhancement of transformation frequency when 10T1/2 cells exposed to a special sequence of microwaves and X rays were subsequently cultured in TPA. The same sequence of microwaves and X rays without promotion resulted in a transformation response similar to that induced by X rays alone. We now report statistically significant (at P greater than 0.999) enhancement of transformation response by TPA in cells exposed to 2.45-GHz microwaves (SAR = 4.4 W/kg). Microwaves alone had no effect on transformation. Plating efficiency and cell survival were not affected by TPA or microwave treatments.     

Cyclic hydrostatic compression stimulates chondroinduction of C3H/10T1/2 cells

While the potential for intermittent hydrostatic pressure to promote cartilaginous matrix synthesis is well established, its potential to influence chondroinduction remains poorly understood. This study examined the effects of relatively short- and long-duration cyclic hydrostatic compression on the chondroinduction of C3H/10T1/2 murine embryonic fibroblasts by recombinant human bone morphogenetic protein-2 (rhBMP-2). Cells were seeded at high density into round bottom wells of a 96-well plate and supplemented with 25 ng/ml rhBMP-2. Experimental cultures were subjected to either 1,800 cycles/day or 7,200 cycles/day of 1 Hz sinusoidal hydrostatic compression to 5 MPa (applied 10 min on/10 min off) for 3 days. Non-pressurized control and experimental cultures were maintained in static culture for an additional 5 days. Cultures were then analyzed for alcian blue staining intensity, DNA and sulfated glycosaminoglycan (sGAG) content, and for the rate of collagen synthesis. Whereas cultures subjected to 1,800 pressure cycles exhibited no significant differences (statistical or qualitative) compared to controls, those subjected to 7,200 cycles stained more intensely with alcian blue, contained nearly twice as much sGAG, and displayed twice the rate of collagen synthesis as non-pressurized controls. This study demonstrates the potential for cyclic hydrostatic compression to stimulate chondrogenic differentiation of the C3H/10T1/2 cell line in a duration-dependent manner.