C3H 10T Research Articles

DNA synthesis in the heterokaryons of non-dividing differentiated cells and culture cells with various proliferative potentials

Resident peritoneal mouse macrophages (non-dividing differentiated cells) were fused with mouse embryo fibroblasts (cells with a limited lifespan), NIH 3T3 and C3H 10T 1 2 cells (‘immortal’ cell lines) and SV 3T3 cells (a malignant cell line). DNA synthesis was investigated in the resultant heterokaryons. No inhibitory effect upon the transition of NIH 3T3 and mouse embryo fibroblasts nuclei to the S-phase was observed. C3H 10T 1 2 , NIH 3T3 and SV 3T3 cells induced the reactivation of DNA synthesis in the macrophage nuclei in the heterokaryons. At the same time, no replication was detected in the macrophage nuclei after fusion with mouse embryo fibroblasts.

Loss of Repair Capacity in Density-Inhibited Cultures of C3H 10T 1/2 Cells during Multifraction Irradiation

Multifraction survival curves for slowly cycling, density-inhibited C3H 10T1/2 cells were shown previously to bend toward lower survival levels with increasing total dose, even for doses per fraction as small as about 2.0 Gy. In an attempt to explain this, we tested the capacity of cells to repair potentially lethal damage (PLD) as fractionation progressed. Plateau-phase cultures were exposed to repeated doses of 4.0 Gy of 137Cs gamma rays delivered at 12-hr intervals. After zero, three, five, and seven fractions, some cultures were put aside, incubated for 12 hr at 37 degrees C, irradiated with a single dose of 9.0 Gy, and subsequently returned to a 37 degrees C incubator. At 0, 2, 4, 6, and 12 hr after the 9.0 Gy dose, cultures were trypsinized and plated for a survival assay. Following three fractions of 4.0 Gy, cells were able to repair PLD as well as those receiving a single dose of 9.0 Gy without prior fractionation. Following five fractions, cells were less able to repair PLD, and after seven fractions, only a very small amount of PLD repair was detectable using this method of measurement.

Patterns of cell loss and repopulation in irradiated cultures of plateau phase C3H 10T 1/2 cells

Patterns of cell loss and repopulation were studied in plateau phase cultures of slowly-cycling, contact-inhibited C3H 10T1/2 mouse fibroblasts following large single, and multiple small doses of 137Cs-gamma rays. A progressive, dose-independent cell loss was apparent within days after irradiation with large single doses, and similar patterns of loss were observed following the start of multifraction irradiations. This progressive cell loss culminated in the loss of integrity of the monolayer of cells, a loss of contact-inhibition, and therefore, an increased rate of cell division. The time of onset of measurable repopulation, and the time required to completely repopulate a culture varied with the number of clonogenic cells present at the time of breakdown of the monolayer. For cultures receiving multiple irradiations over time periods greater than those required for breakdown of the monolayer, repopulation began to occur during treatment, and its rate varied with the average dose per fraction being delivered. Thus, repopulation did not start immediately after the start of irradiation, but needed a triggering event, in this case, a decrease to a critical level in the cell density. Once initiated, repopulation was able to decrease or even eliminate the effectiveness of subsequent doses in reducing the number of viable cells per culture. To the extent that the responses of slowly-cycling, contact-inhibited cells in vitro can be applied to interpret the radiation responses of cell populations in vivo, these results further support the notion that it may be necessary, in some cases, to account for an increasing contribution from repopulation with increasing overall treatment time in dose fractionation isoeffect formulae used for predicting tissue tolerances or tumor control.

A calcium requirement for electric field-induced cell shape changes and preferential orientation

C3H 10T 1 2 mouse embryo fibroblasts stimulated by a steady electric field (10 V/cm) for 30 min exhibited lamellar retraction on the sides facing the electrodes. Some cells elongated and preferentially oriented with their long axis perpendicular to the field direction. Depletion of external calcium or blockage of calcium influx with lanthanum or the calcium channel blocker D-600 resulted in a reduction of the field-induced response. When external calcium was elevated stepwise from 0 to 10 mM, the field-induced response increased correspondingly. Electric stimulation in the presence of the calcium ionophore A23187 resulted in an increase of spindle-shaped cells with no preferential orientation. This response was blocked by calcium depletion and lanthanum, but not by D-600. The anticalmodulin drug W-13 inhibited the field-induced responses observed in normal buffer as well as in the presence of A23187. Some cell death resulted from prolonged electric field exposure, and the mortality was reduced by calcium depletion, lanthanum or D-600, but was not affected by W-13. We postulate that local calcium influx through channels opened by the electric field produces areas of high intracellular calcium which stimulate the cytoskeletal network to induce lamellar retraction. Prolonged field-induced calcium influx may eventually overcome the cell's mitochondrial calciumbuffer system, leading to necrotic calcification.

In Vitro Transformation by Bromodeoxyuridine and X Irradiation in C3H 10T 1/2 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.

Ultraviolet radiation stimulates the release of arachidonic acid from mammalian cells in culture.

Abstract— C3H 10T½ cells in culture were prelabelled with [3H]arachidonic acid and exposed to UVB radiation. Almost immediately after irradiation cells released labelled arachidonate metabolites into media in a dose dependent manner. This release was inhibited by removing calcium ions from the system and by the addition of dexamethasone and parabromophenacyl bromide to the system. This suggests that the UVB stimulated release of arachidonic acid from membrane phospholipids is, in part, mediated by a phospholipase A2 enzyme system. Thin layer chromatographic examination of media extracts revealed a dose dependent UVB stimulation of prostaglandin production by cultured cells.

Dose-rate effects in mammalian cells: V. dose fractionation effects in noncycling c3h [formula omitted] cells

Multiple gamma ray dose fractionation effects were studied using contact-inhibited C3H 10T 1 2 murine fibroblast cultures in an attempt to simulate conditions in tissues with low rates of cell turnover. Multifraction survival curves were determined for different doses per fraction using 12 hour interfraction intervals 7 days per week. Isoeffect curves were generated from the multifraction survival curves. For doses per fraction greater than approximately 1.0 Gy, these isoeffect curves were similar to those derived from tissue reactions in humans and experimental animals published previously. For doses per fraction below 1.0 Gy, the isoeffect curves became essentially flat, thus deviating appreciably from the total dose which would be predicted by extrapolating the NSD equation to achieve an isoeffect. The existence of a non-repairable component of gamma ray damage can be inferred from this finding, which has implications both for basic radiobiology, and radiotherapy.

Timing of the steps in transformation of C3H 10T 1/2 cells by X-irradiation.

Transformation of cells in culture by chemical carcinogens or X rays seems to require at least two steps. The initial step is a frequent event; for example, after transient exposure to either methylcholanthrene or X rays, almost every cell of established lines of mouse embryo fibroblasts proved capable of yielding transformed, tumorigenic descendants. Although results were interpreted as indicating that 100% of the progeny of methylcholanthrene-treated cells were potentially transformed, later experiments showed that only a very small minority of the progeny of cells initiated by X rays or methylcholanthrene actually produced transformed colonies. We thus concluded that there must be a second step in transformation that is a very rare event. We assumed that this event occurred after the cultures became confluent, a time when transformed cells have a selective growth advantage. Since then, however, others have shown that transformation can occur soon after initiation and that clones of transformed cells may already be present by the time initiated cultures become confluent. It has been hypothesized that the second step behaves like a spontaneous mutation in having a constant but small probability of occurring each time an initiated cell divides. We show here that the clone size distribution of transformed cells in growing cultures initiated by X rays is, indeed, exactly what would be expected on that hypothesis.

Epidermal Growth Factor and tumor promoters prevent DNA fragmentation by different mechanisms

Serum deprivation of C3H 10T 1 2 fibroblasts resulted in DNA fragmentation which was prevented by growth factors such as Epidermal Growth Factor or the tumor promoters, 12-0-tetradecanoyl-13-0-phorbol acetate and Dihydroteleocidin B. Palmityl carnitine, an inhibitor of Ca2+-phospholipid-dependent protein kinase C, reversed the effects of the tumor promoters, but not the effect of Epidermal Growth Factor.

Inhibition of malignant transformation in vitro by inhibitors of poly(ADP-ribose) synthesis.

Malignant transformation in vitro of hamster embryo cells and mouse C3H 10T 1/2 cells by x-rays, ultraviolet light, and chemical carcinogens was inhibited by benzamide and by 3-aminobenzamide at concentrations that are specific for inhibition of poly(ADP-ribose) formation. These compounds slow the ligation stage of repair of x-ray and alkylation damage but not of ultraviolet light damage. At high concentrations they also inhibited de novo synthesis of DNA purines and DNA methylation by S-adenosylmethionine. The suppression of transformation by the benzamides is in striking contrast to their reported effectiveness in enhancing sister chromatid exchange, mutagenesis, and killing in cells exposed to alkylating agents. Our results suggest that mechanisms regulating malignant transformation are different from those regulating DNA repair, sister chromatid exchange, and mutagenesis and may be associated with changes in gene regulation and expression caused by alterations in poly(ADP-ribosyl)ation.

Thermal radiosensitization in Chinese hamster (V79) and mouse C3H 10T 1/2 cells. The thermotolerance effect.

The sensitivity of V79 cells and normal or morphologically transformed C3H-10T 1/2 cells to X-rays, heat or heat plus X-rays was examined. The normal and transformed C3H-10T 1/2 cell lines were equally sensitive to heat at 42.0 degrees C and radiation. The V79 cells were more heat sensitive. Thermal radiosensitization occurred for all 3 cell lines for the combined heat and radiation treatments and was greatest for simultaneous treatment. Recovery occurred when the treatments were separated by an incubation interval at 37 degrees C. For the V79 cells, recovery was much greater for X-rays preceding heat compared to X-rays following heat. This difference was not as great in the C3H-10T 1/2 cell lines. The transformed C3H-10T 1/2 cells were more sensitive compared to the normal for the simultaneous treatment or for heating followed by irradiation. For prolonged heating at 42.0 degrees C, after which thermotolerance occurred in all 3 cell lines, the radiosensitivity still increased as a function of heating time even though no additional cell killing occurred from the heat treatment alone. For heating V79 cells at 41.0 degrees C no further increase in radiosensitivity occurred, as cells became thermotolerant during prolonged heating. Also for the development of thermotolerance during incubation at 37 degrees C between two heat treatments, thermal radiosensitization decreased demonstrating that thermotolerance can affect radiosensitization by hyperthermia.

DNA binding of aflatoxin B 1 by co-oxygenation in mouse embryo fibroblasts [formula omitted

The mechanism of activation of aflatoxin B 1 to ultimate metabolites capable of DNA binding was investigated in mouse embryo fibroblasts C3H 10T 1 2 . The contribution of co-oxygenation reactions which are coupled to arachidonic acid metabolism was assessed by the use of inhibitors of prostaglandin endoperoxide synthetase and lipoxygenase. Indomethacin and 5,8,11,14-icosatetraynoic acid inhibited AFB 1-binding to maximally 60%. The antioxidant glutathoone was also inhibitory while CuZn superoxide dismutase had no effect or slightly stimulated binding at high concentrations. These results indicate that co-oxygenation plays a major role in AFB 1-metabolism in 10T 1 2 cells. The observation that the phospholipase A 2 inhibitor p-bromophenacylbromide diminished AFB 1-DNA binding supports the notion that AFB 1, because it is membrane-active, may enhance its own co-oxidative metabolism by stimulating the arachidonic acid cascade.

Dose-Rate Effects in Mammalian Cells: IV. Repairable and Nonrepairable Damage in Noncycling C3H 10T 1/2 Cells

Repairable and nonrepairable components of gamma-ray damage leading to cell reproductive death were determined by measuring the range over which dose rate influenced the response of non-cycling C3H 10T 1/2 mouse cells. Cell proliferation and cell cycle redistribution were eliminated as factors influencing the dose-rate effect in the system by irradiating confluent monolayers of contact inhibited cells. The radiosensitivity of the cells did not change, and no selective loss of damaged cells occurred over the extended treatment times. A pronounced dose-rate effect was observed over the range between 55.6 and 0.29 Gy/hr, but a limit to the repair-dependent dose-rate effect was reached at 0.29 Gy/hr since no further reduction in effect per unit dose was observed when the dose rate was reduced to 0.17 or 0.06 Gy/hr. The survival curves, which were simple exponential functions of dose at dose rates of 0.29 Gy/hr and below, have a common Do of 7.32 Gy and represent an accurate measurement of the nonrepairable component of damage. Log-phase cultures showed remarkably different responses over the range of dose rates, due in large part to cell cycle redistribution and in some cases, cell proliferation during exposures. The results of these studies were compared with time-dose relationships used in clinical brachy-therapy and agree remarkably well with corrections in total dose suggested by R. Paterson [Br. J. Radiol. 25, 505-516 (1952)] and A.E.S. Green [cited in F. Ellis, Curr. Top. Radiat. Res. Q. 4, 357-397 (1968)] when the standard treatment time is changed. Comparison of our data with in vivo isoeffect curves of total dose vs dose per fraction for "early" and "late" tissue responses indicate that cell cycle redistribution should not be ignored as a factor influencing time-dose relationships in radiotherapy.

Differential effect of tumor promoters on phorbol-ester-receptor binding and membrane fluorescence anisotropy in C3H 10T 1/2 cells.

The presence of high‐affinity membrane‐associated receptor sites for phorbol ester tumor promoters has been demonstrated with [3H]phorbol dibutyrate ([3H]phorbol Bt2) in the mouse embryo fibroblast cell line C3H 10 T1/2. Binding of [3H]phorbol Bt2 to intact cells occurs rapidly at 37°C and is also rapidly reversed upon addition of excess unlabeled phorbol Bt2. The dissociation can be resolved into two phases at 23°C. Scatchard analysis reveals a biphasic curve which is consistent with two classes of binding sites: a high‐affinity class with a dissociation constant of 16 nM and a total concentration of binding sites of 1.9 pmol/106 cells, and a low‐affinity class with a dissociation constant of 1000 nM and a total concentration of binding sites of 29.4 pmol/106 cells. Unlabeled phorbol Bt2, 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (Myrphobol Ac), and the indole alkaloid tumor promoter teleocidin are potent inhibitors of [3H]phorbol Bt2 binding, producing 50% inhibition at 98 nM, 38 nM and 23nM, respectively. On the other hand, two compounds that lack tumor‐promoting activity, 4α‐phorbol‐12,13‐didecanoate (4α‐phorbol Dec2) and 4‐O‐methyl‐Myrphorbol Ac, only slightly inhibited phorbol Bt2 binding. The effects of these compounds on the physical state of the membrane of intact C3H 10T 1/2 cells was also assessed using two different fluorescence lipophilic probes. Phorbol Bt2. Myrphorbol Ac and teleocidin, but not 4α‐phorbol Dec2, induce significant decreases in the fluorescence anisotropy parameter obtained with both of the probes. However, the dose response curves for these effects are quite different from those obtained when the same compounds were tested for inhibition of [3H]phorbol Bt2 binding. This discrepancy suggests that although C3H 10T 1/2 cells contain an abundance of highaffinity receptor sites for phorbol ester and related tumor promoters, some of the membrane effects of these compounds may be mediated by direct binding of the tumor promoter to lipid domains in cell membrane.

Effects of fluence fractionation on UV-induced malignant transformation and cell killing in non-cycling plateau phase mouse cells.

AbstractPlateau phase C3H 10T 1/2 mouse cells were used to measure the response to split fluence UV light irradiation in the absence of any cell cycle effects. It was found that fluence fractionation with up to 24 h between the fluences resulted in no survival enhancement. The frequency of malignant transformation was potentiated 2.5‐fold when the time interval between the fluences was greater than 4h. This potentiation of transformation was attributed to plateau phase holding rather than to fluence fractionation per se.

Inhibition of respiration by a phorbol ester tumor promoter in murine cultured cells

The tumor promoter 12-O-tetradecanoyl phorbol-13-acetate is a potent inhibitor of mitochondrial respiration in both normal and methylcholanthrene-transformed C3H 10T1 2 mouse fibroblasts. This inhibition is seen at concentrations of tumor promoter in the range of 10 −8M, occurs within a few minutes after exposure of the intact cells, and is not seen with a biologically inactive analog. The effect appears to be exerted through inhibition of the function of an oligomycin-sensitive ATPase. It is possible, therefore, that alterations in mitochondrial function are associated with the process of tumor promotion.

Hyperthermic Modulation of X-Ray-Induced Oncogenic Transformation in C3H 10T 1/2 Cells

The C3H 10T 1/2 mouse embryo cell line was used to determine the effect of hyperthermia on the in vitro oncogenic transforming potential of X irradiation. Heat exposures of 15 min at 45°C or 60 min at 43°C yielded no transformation (frequency < 0.3× 10-4). When either of these heat exposures preceded exposure to 200 rad of X rays, a four- to fivefold increase in the mean transformation frequency occurred over that resulting from X rays alone. These results may have important clinical implications.

Effects of dexamethasone and cortisone with x-ray irradiation on transformation of C3H 10T 1/2 cells.

Hormones can influence carcinogenesis in experimental animals1 and it has been proposed that sex hormones may act like tumour-promoting agents in their ability to enhance the incidence of cancer2. On the other hand, it has been observed that the glucocorticoid hormone, dexamethasone, suppresses the enhancement of chemical carcinogenesis in vivo2 and of transformation in vitro3 brought about by exposure to the tumour-promoting agent, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). We report here that the glucocorticoid hormones, cortisone and dexamethasone, do not suppress radiation transformation of C3H 10T½ cells in vitro. Indeed, cortisone induces transformation of cells by itself, and increases the yield of radiation-induced transformants in a synergistic fashion.

Mutagenesis and transformation of C3H 10T 1/2 cells treated with ethyl methanesulfonate.

Survival, mutagenesis and transformation were measured in mouse embryo C3H 10T 1/2 cells following treatment with ethyl methanesulfonate (EMS). Ouabain-resistant cells and transformed cells were isolated, and reconstruction experiments were carried out to determine the optimum conditions for the measurement of mutation and transformation frequencies. Survival was measured by plating efficiency; mutagenesis was measured in terms of the induction of cells able to form colonies in the presence of ouabain; and transformation was measured by the induction of cells forming either morphologically altered colonies on a monolayer of contact-inhibited cells or of cells capable of forming colonies in semi-solid media. When confluent monolayers were incubated for 4 h after treatment with EMS, to allow excision repair before the resumption of DNA synthesis, survival as well as the frequencies of both mutation and transformation increased. When this repair (or holding) period was extended to 24 h, the frequencies of mutation and transformation both decreased as compared to the 4-h holding period. Thus, the holding periods affect the frequencies of EMS-induced mutagenesis and transformation similarly.

Initiation of [formula omitted] cell transformation by formaldehyde

The effects of formaldehyde were evaluated in the C3H 10T 1 2 Cl 8 cell transformation system. Treatment of the cells with 0.1–2.5 μg/ml of formaldehyde alone did not result in significant rates of transformation. If formaldehyde treatment was followed by continuous treatment with 0.1 μg/ml of the tumor promoter 12- O-tetradecanoyl phorbol-13-acetate (TPA), transformed foci were produced. Methanol and formic acid lacked significant transforming activity under either treatment regimen. The results suggest that formaldehyde is an initiating agent for C3H 10T 1 2 Cl 8 transformation. The fact that some compounds may act solely as initiators should be considered when this transformation system is used to study chemicals which may interact with cells by mechanisms similar to that of formaldehyde.