Gangliosides and their cell density-dependent changes in control and chemically transformed C3H/10T1/2 cells

The cloned C3H/10T1/2 mouse embryo cells contained a complex pattern of gangliosides. Two cloned chemical transformants obtained from the C3H/10T1/2 cell line by treatment with 7,12-dimethylbenz(a) anthracene (DMBA-TCL1) and 3-methylcholanthrene (MCA-TCL15) also had complex ganglioside patterns; but the transformants had increased levels of the simplest ganglioside, N-acetylneuraminylgalactosylglucosylceramide (GM₃), and reduced levels of more complex gangliosides. Incorporation of [¹⁴C]glucosamine into gangliosides, as cell-to-cell contact increased in C3H/10T1/2 cells, showed that GM₃ synthesis was decreased and that the synthesis of the more complex ganglioside N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosylglucosylceramide (GD_1a) was increased. In the two transformants the percentage each individual ganglioside was of total labeled gangliosides was only slightly altered with changing cell density. Turnover of [¹⁴C]glucosamine-labeled gangliosides, as cell density increased, was approximately equal in C3H/10T1/2 cells and MCA-TCL15 cells, but more rapid in the DMBA-TCL1 cells. Most individual gangliosides turned over at about the same rate in the respective cell lines. However, GD_1a increased slightly as a percentage of total labeled gangliosides with increasing cell density in both C3H/10T1/2 cells and transformed cells. The labeling data indicated that the majority of GD_1a synthesis was de novo and only a small part occurred by transfer of sialyl or glycosyl residues to simpler gangliosides or catabolism of more complex gangliosides already present in the outer membrane. Exogenous complex gangliosides added to the medium were more effective inhibitors of DMBA-TCL1 cell growth than of C3H/10T1/2 cell growth. Furthermore, gangliosides added to exponentially growing C3H/10T1/2 and DMBA-TCL1 cells caused both cell lines to incorporate a greater percentage of [¹⁴C]glucosamine into gangliosides more complex than GM₃.     

Response of plateau-phase C3H 10T1/2 cells to radiation and concurrent administration of bleomycin

The mode and extent of interaction between bleomycin and radiation were assessed in contact-inhibited cultures of C3H 10T1/2 cells, which in confluent monolayers display a low turnover rate and behave more like late-responding normal tissues in vivo with respect to response to fractionated radiotherapy (i.e., having a low alpha/beta value). Plateau-phase C3H 10T1/2 cultures were exposed to gamma rays delivered in 1, 2, 5, or 10 fractions. The radiation doses administered ranged from 2 Gy in one exposure to 26 Gy in 10 fractions. Half of the cultures were also treated with 1 micrograms/ml of bleomycin for 5 days during which radiation was also given. It was found that 1 micrograms/ml of bleomycin sterilized approximately 40% of the C3H 10T1/2 cells in the cultures. The radiation dose-survival curves of various fractionation schedules (1, 2, 5, and 10 fractions) plus bleomycin were displaced downward (i.e., to lower survival levels) but not modified in shape. The alpha/beta ratios, parameters of the linear-quadratic model of cell survival, were 2.6 (2.2-3.1) and 2.4 (1.8-3.1) Gy for radiation only and radiation plus bleomycin, respectively. This observation indicates that the effect of combining irradiation and bleomycin on C3H 10T1/2 cells in monolayers was additive.     

Differences in membrane electrical properties between C3H 10T1/2 mouse embryo fibroblasts and their ionizing radiation and chemically transformed counterparts

Membrane electrical properties of mouse embryo fibroblasts and their ionizing radiation and chemically transformed counterparts were investigated using dielectric relaxation measurements in the radio frequency range. This determination is possible because, in the radio frequency range, suspensions of cells in an electrolyte buffer show a conductivity dispersion due to interfacial polarization. An analysis of the experimental data based on a single-shell model showed that conductivity and permittivity of the membranes of both radiation and chemically transformed fibroblasts were lower than in normal cells. In addition, the conductivity of the cytoplasm was higher in both transformed cell types than in the normal mouse fibroblasts. We discuss the significance of these findings in view of the possible structural and functional modifications brought about by the process of neoplastic transformation.     

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.     

Simultaneous determination of ascorbic acid and dehydroascorbic acid in cultures of C3H/10T1/2 cells

Summary A reproducible method is described for the separation and quantification of ascorbic acid and dehydroascorbic acid by ion-pairing reverse-phase high performance liquid chromatography and detection by absorbance at 232 nm. Lowest detectable concentrations with a linear response of detection were 5 nmol for ascorbic acid and 50 nmol for dehydroascorbic acid. This method was applied to the analysis of C3H/10T1/2 cells and culture medium after influx or efflux experiments and single or multiple treatments with ascorbic acid. Subsequent measurement of the radioactivity in the eluted fractions increased the detectability of both ascorbic acid and dehydroascorbic acid to 10 to 20 pmol. This research was supported by grant CA 09320 and CA 31574 from the National Cancer Institute, Bethesda, MD, and grant BC441 from The American Cancer Society.     

Activation of the Ha-ras gene in C3H 10T1/2 cells transformed by exposure to N-methyl-N'-nitro-N-nitrosoguanidine

A transfectable, presumably mutationally activated, c-Ha-ras gene was identified in a clonal population of 10T1/2 cells established from a Type II focus induced by exposure of a parental, wild-type population to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).     

Molecular phenotyping of non-transformed and chemically transformed C3H10T1/2 mouse fibroblasts

A systematic molecular phenotyping approach based on two-dimensional gel electrophoresis is being applied in an attempt to identify protein changes associated with malignant transformation. Using the C3H10T1/2 mouse cell line, two-dimensional polypeptide maps of the non-transformed cell line, several chemically transformed lines and a tumour cell line were compared. Although there is a large degree of similarity between the protein profiles of all cell lines, clear differences are evident. Initial results are consistent with the view that many of the protein changes are incidental to malignant transformation. Changes induced by 3-methylcholanthrene are retained after transplantation of the cells into nude mice.     

Possible importance of PLD repair in the modulation of BrdUrd and IdUrd-mediated radiosensitization in plateau-phase C3H10T1/2 mouse embryo cells

C3H10T1/2 mouse embryo cells exhibiting strong contact inhibition of growth at confluency were grown in the presence of 5-bromodeoxyuridine (BrdUrd) or 5-iododeoxyuridine (IdUrd) (0-1.2 microM) with daily refeeding and exposed to gamma-rays (6 Gy) either in the logarithmic or the plateau phase of growth. Sensitization to radiation was observed in both growth states with increasing concentration of BrdUrd or IdUrd but the degree of sensitization achieved was lower for plateau-phase cells. Because the degree of [H3]BrdUrd incorporation was found to be similar in exponentially growing and plateau-phase cells, it is hypothesized that the radiosensitization caused by pyrimidine analogues may be affected by the physiological state of the cells at the time of irradiation. Delayed plating of plateau-phase cells (6 h) caused an increase in survival, indicating repair of potentially lethal damage (PLD). A greater increase in cell survival was observed in cells that had been grown in the presence of BrdUrd and IdUrd and it was found to increase with increasing concentrations. This analogue-concentration dependent PLD repair activity resulted in an almost complete loss of the radiosensitizing effect in delayed plated plateau-phase cells up to a concentration of about 0.6 microM of BrdUrd and IdUrd. Both compounds, but especially BrdUrd, caused a relaxation in the mechanism of contact inhibition and led to higher cell densities in the plateau phase. The results suggest that repair and/or expression of PLD might be involved in the mechanism underlying BrdUrd and IdUrd-mediated radiosensitization and point out the potential importance of PLD repair in the modulation of the radiosensitizing effect of these compounds in their clinical application.     

Loss of repair capacity in density-inhibited cultures of C3H 10T1/2 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.     

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.     

Evidence for reduced capacity for damage accumulation and repair in plateau-phase C3H 10T1/2 cells following multiple-dose irradiation with gamma rays

The effects of multiple-dose gamma irradiation on the shape of survival curves were studied with mouse C3H 10T1/2 cells maintained in contact-inhibited plateau phase. The dose-fractionation intervals included 3, 6, and 24 h. Following three fractionated doses (5 Gy per dose) of exposures, cells responded to further irradiation by displaying a survival curve with a much reduced shoulder width (Dq) compared to that of the survival curve measured in cells irradiated with single-graded doses alone. The effect on the mean lethal dose (D0) was small and appeared to be significant. The effect on reduction of Dq could not be completely overcome by lengthening the fractionation intervals from 3 to 6 h or 24 h, times in which repair of sublethal damage (SLD) measured by simple split-dose scheme and potentially lethal damage (PLD) measured by postirradiation incubation was completed. Other experiments showed that pretreatments of cells with fractionated irradiation appeared to slow down the cellular repair processes of SLD and PLD. Therefore, the observed change in the shape of survival curves after fractionation treatments may be attributed to a reduction of the cells' capacity for damage accumulation by an enhancement of the lethal expression of SLD and PLD. Although the molecular mechanism(s) is not known, the results of this study indicate that the acute graded dose-survival curve cannot be used a priori to extrapolate and reliably predict results of hyperfractionation. It is probable that for a nondividing or slowly dividing cell population, such an extrapolation may lead to an underestimation of cell killing. Furthermore, the findings of this investigation appear to support an interpretation, alternative to the high-linear energy transfer (LET) track-end postulate, for the effects on cell survival seen at low doses or low dose rates.     

Oxygen concentration regulates 5-azacytidine-induced myogenesis in C3H/10T1/2 cultures.

This study reports that changing the oxygen concentration within a physiologic range has a striking effect on myogenesis induced by the cytidine analog 5-azacytidine. Reducing oxygen from 20% to 2.5% increases 7-fold the number of myocytes that appear in cultures of C3H/10T1/2 mouse embryo cells 10 days after they receive a 24-h exposure to 5-azacytidine. Reducing oxygen does not alter the extent to which a 24-h exposure to 5-azacytidine inhibits cytosine methylation in newly synthesized DNA. Instead, the oxygen-sensitive step in myogenesis occurs after 5-azacytidine is removed from the culture medium. Reducing oxygen increases the rate of logarithmic growth in C3H/10T1/2 cultures after 5-azacytidine exposure, suggesting that survival and proliferation of myocyte stem cells (morphologically indistinguishable from uncommitted C3H/10T1/2 cells) may be the oxygen-sensitive steps in myogenesis.     

Ammonia effects in cultures of normal and transformed 3T3 cells

3T3 and SV-40 transformed 3T3 mouse fibroblasts were cultured in media with serum and antibiotics plus ammonia (NH₃ z NH₄⁺) added as NH₄C1. Both cell lines cultured without added ammonia showed normal morphology and multiplication even though ammonia in the medium at the end of the culture period ranged from 35 to 48 μg/ml. Ammonia concentrations being significantly higher in media removed from cells at the end of the culture period than in media incubated identically without cells, verified that cells released substantial quantities of ammonia in addition to components of the medium which underwent spontaneous breakdown. Both cell lines showed changes in morphology and highly significant reductions in cell multiplication which increased progressively as the concentration of added ammonia on the initial day of culture was increased to 35μg/ml. Control 3T3 cultures released significantly greater quantities of ammonia per cell than control cultures of transformed cells but their multiplication was more adversely affected by added ammonia. There were downward shifts in pH of the culturing medium for both cell lines as culture age increased at all concentrations of added ammonia, However, significant reductions in cell multiplication resulted from additions of ammonia that did not produce significant changes in extracellular pH. The data show that studies upon the effects of pH of the medium on cultured cells require control of ammonia concentrations.     

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.     

Increased labelling of polyphosphoinositide in chemically transformed cell line C3H10T1/2 CL8

The effect of malignant transformation of cells on phosphatidylinositol metabolism was investigated using C3H10T1/2 cells and its chemically transformed cell line, MCA CL-16 cells. We found that incorporation of [³²P]P_i into polyphosphoinositide was greatly increased in the transformed cells. A similar tendency was observed when myo-[2-³H]inositol was used as a labelling reagent. It is also observed that influx of labelled inorganic phosphate is enhanced 2-fold by the cell transformation. Therefore, promotion of polyphosphoinositide labelling in the transformed cell might be caused not only by the enhanced metabolism of phosphatidylinositol but also by the increased membrane permeability for radioactive labelling reagents.     

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.     

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.     

BMP-2 and sonic hedgehog have contrary effects on adipocyte-like differentiation of C3H10T1/2 cells

The signaling pathways of bone morphogenic protein 2 (BMP-2) and Sonic hedgehog (Shh) are related during embryogenesis. Both proteins have been implicated as important components during osteogenic differentiation; e.g., considering their in vitro effects in the pluripotent C3H10T/1/2 cell system. Also, BMP-2 has been frequently reported to stimulate adipogenesis as well as osteogenesis in these cells. We investigated the relative potencies of Shh and BMP-2 with regard to adipogenesis. We performed differentiation experiments by stimulating C3H10T1/2 cells with BMP-2, Shh, or a combination. We monitored adipocyte-like differentiation via gene expression analysis and cytologic staining. An adipocytic phenotype was observed in BMP-2-treated cells, as shown by upregulation of two adipocytic marker mRNAs, PPAR-gamma and aP2, and by staining of lipid-filled cell vesicles with Oil Red O. In contrast, no adipocyte-like differentiation could be detected either after treatment with Shh or after exposure to a combination of Shh and BMP-2. Our results demonstrate for the first time that Shh and BMP-2 have contrary effects on adipocyte-like differentiation. Whereas BMP-2 promotes the adipocytic lineage, Shh suppresses the expression of the BMP-2-induced fat-cell phenotype.     

Vanadate stimulates ornithine decarboxylase activity in C3H/10T1/2 cells.

Ornithine decarboxylase (ODC) activity of C3H/10T1/2 cells reflects their response to conflicting actions of many tumor promoters and tumor suppressors. In cultured C3H/10T1/2 cells, addition of vanadate (50 nM) increased ODC activity. Over the range 0.05-5 microM, vanadate increased ODC levels in a dose dependent manner to 11 times control levels. The presence of retinoic acid (5 microM) or the absence of fetal calf serum blocked the stimulation by vanadate.     

Galactosylceramide expression factor-1 induces myogenesis in MDCK and C3H10T1/2 cells

We previously reported that galactosylceramide expression factor-1 (GEF-1), a rat homolog of hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs/Hgs), induces galactosylceramide and/or sulfatide expression and morphological changes in epithelial cells. Here, we show that GEF-1 induces myogenesis in MDCK and C3H10T1/2 cells. GEF-1 overexpression in MDCK cells (MDCK/GEF-1) appeared to promote trans-differentiation to myoblasts that expressed MyoD and myosin heavy chain (MHC). MDCK/GEF-1 cells also expressed several DNA-binding proteins (MyoD and MEF-2) that are essential for myogenesis. These results suggest that GEF-1 induces MDCK cells to enter an early stage of myogenesis. Subsequently, we tested whether GEF-1 could induce myogenesis in C3H10T1/2 mouse fibroblasts, which have the potential to differentiate into myoblast-like cells. Indeed, GEF-1 induced morphological changes that were consistent with myoblast-like cells, and both MyoD and MHC were expressed. Our results suggest that GEF-1 may induce MDCK and C3H10T1/2 cells to trans-differentiate into myoblast-like cells.