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.     

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.     

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.     

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.     

Nuclear import of factors involved in signaling is inhibited in C3H/10T1/2 cells treated with tetradecylthioacetic acid

The non-beta-oxidisable tetradecylthioacetic acid (TTA) is incorporated into cellular membranes when C3H/10T1/2 cells are cultured in TTA-containing medium. We here demonstrate that this alteration in cellular membranes affect the nuclear translocation of proteins involved in signal transduction. Analysis of cellular fatty acid composition shows that TTA and TTA:1n-8 constitute approximately 40 mol% of total fatty acids in cellular/nuclear membranes. Activation of c-fos expression is significantly inhibited in TTA-treated cells but the enzymatic activation of mitogen activated protein kinase (ERK) is not affected. Immunofluorescence and confocal microscopy studies demonstrate that in mitogene-stimulated TTA-treated cells, the translocation of phosphorylated ERK1/2, protein kinase C alpha (PKC alpha), and PKC beta(1) from the cytoplasm into the nucleus is considerably decreased and delayed. Concomitant with a decreased nuclear import, ERK1/2 dephosphorylation is decreased in TTA-treated cells. There is no TTA-induced inhibition of nuclear import of proteins with a classical nuclear localization signal (NLS), as seen by in vitro nuclear import experiments of BSA fused to the NLS from SV40 large T, or in vivo studies of hnRNP A1 nuclear import. The expression levels of Importin alpha, Importin beta, Importin 7, and NTF2 are not altered in the TTA-treated cells. Taken together, our data indicate that TTA treatment causes changes in cellular fatty acid composition that negatively affect NLS-independent mechanisms of protein translocation through the nuclear pore complex.     

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.     

Repair time for oncogenic transformation in C3H/10T1/2 cells subjected to protracted X-irradiation

With exponential cultures of C3H/10T1/2 cells, we have investigated the effect of X-ray dose protraction on oncogenic cell transformation in the dose range 0.25-2 Gy. Within a particular experiment a constant exposure time was used. In different experiments exposure time varied between 1 and 5h. Cell transformation was analysed using the linear-quadratic relation, gamma (D) = alpha 1D + alpha 2D2, between transformation frequency per surviving cell and X-ray dose. Based on values of the linear coefficients, we developed an empirical formula for relating slopes of dose induction curves obtained at high or reduced dose rate condition. Our estimate of repair half-time for cell transformation with 95 per cent confidence limits is 2.4 (1.8, 3.0) h.     

Ether lipid studies in mouse C3H/10T1/2 cells and a 3-methylcholanthrene-transformed clone

C3H/10T1/2 mouse embryo cells and a transformed clone were used in these initial experiments to investigate the future application of this model culture system to studies of ether-linked lipids in cancer cells. Clone 8 cells are nontumorigenic, nontransformed, and maintain normal morphology to passages 15–20. Clone 16 cells were derived from morphologically transformed foci of clone 8 cells exposed to the chemical carcinogen, 3-methylcholanthrene, and are highly tumorigenic. The data presented here demonstrate that the high amounts of ether-linked lipids, characteristic of tumors, are likewise elevated in cells that have been oncogenically transformed in vitro. When incubated with labeled fatty alcohols, the transformed cells show a stimulated incorporation of radioactivity into alkyldiacylglycerols (>100% over clone 8), whereas radioactivity in the alkyl moiety of the phospholipids is not altered. Analysis of the lipids formed from [1-¹⁴C]hexadecanol indicates that the nontransformed cells have a greater capacity to oxidize hexadecanol and incorporate the resulting carboxylic acid into acyl groups. Quantitative analysis of cellular lipids shows that in the oncogenically transformed cells alkyldiacylglycerols are increased (123% over clone 8).     

Screening of genes responsible for differentiation of mouse mesenchymal stromal cells by DNA micro-array analysis of C3H10T1/2 and C3H10T1/2-derived cell lines

BACKGROUND: The molecular mechanisms underlying the biologic effects or differentiation of mesenchymal stromal cells (MSC) have not been clarified. Screening for genes differentially expressed at different stages is an important step in determining these molecular mechanisms. METHODS: In this study, we analyzed the gene expression profiles of C3H10T1/2 (10T1/2) cells and two sublines, A54 (pre-adipocyte) and M1601 (myoblast), as a model of MSC and downstream committed progenitors. RESULTS: We found up-regulated expression of delta-like-1 (Dlk), Wnt-5a and IL-1 receptor-like-1 (ST2) in 10T1/2 cells; stem cell factor (SCF) and stromal derived factor-1 (SDF-1) in A54 cells; and cardiac muscle-specific gene in M1601 cells. Overexpression of Dlk in A54 cells did not induce any effects on their differentiation into adipocytes. After differentiation into adipocytes, A54 cells reduced the expression of SCF, SDF-1 and Ang-1 as well as the ability to support the formation of a cobblestone appearance. DISCUSSION: The results suggest that these three lines hae different gene profiles and are a useful system for analyzing the differentiation and function of MSC and progenitor cells.     

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.     

DNA methylation in 5-aza-2''-deoxycytidine-resistant variants of C3H 10T1/2 C18 cells.

A cell line (T17) was derived from C3H 10T1/2 C18 cells after 17 treatments with increasing concentrations of 5-aza-2'-deoxycytidine. The T17 cell line was very resistant to the cytotoxic effects of 5-aza-2'-deoxycytidine, and the 50% lethal dose for 5-aza-2'-deoxycytidine was ca. 3 microM, which was 30-fold greater than that of the parental C3H 10T1/2 C18 cells. Increased drug resistance was not due to a failure of the T17 cell line to incorporate 5-aza-2'-deoxycytidine into DNA. The cells were also slightly cross-resistant to 5-azacytidine. The percentage of cytosines modified to 5-methylcytosine in T17 cells was 0.7%, a 78% decrease from the level of 3.22% in C3H 10T1/2 C18 cells. The DNA cytosine methylation levels in several clones isolated from the treated lines were on the order of 0.7%, and clones with methylation levels lower than 0.45% were not obtained even after further drug treatments. These highly decreased methylation levels appeared to be unstable, and DNA modification increased as the cells divided in the absence of further drug treatment. The results suggest that it may not be possible to derive mouse cells with vanishingly low levels of 5-methylcytosine and that considerable de novo methylation can occur in cultured lines.     

Wnt-3A enhances bone morphogenetic protein-2-mediated chondrogenesis of murine C3H10T1/2 mesenchymal cells

We have recently reported the chondrogenic effect of bone morphogenetic protein-2 (BMP-2) in high density cultures of the mouse multipotent mesenchymal C3H10T1/2 cell line and have shown the functional requirement of the cell-cell adhesion molecule N-cadherin in BMP-2-induced chondrogenesis in vitro (Denker, A. E., Nicoll, S. B., and Tuan, R. S. (1995) Differentiation 59, 25-34; Haas, A. R., and Tuan, R. S. (1999) Differentiation 64, 77-89). Furthermore, BMP-2 treatment also results in an increased protein level of beta-catenin, a known N-cadherin-associated Wnt signal transducer (Fischer, L., Haas, A., and Tuan, R. S. (2001) Signal Transduction 2, 66-78), suggesting functional cross-talk between the BMP-2 and Wnt signaling pathways. We have observed previously that BMP-2 treatment up-regulates expression of Wnt-3A in high density cultures of C3H10T1/2 cells. To assess the contribution of Wnt-3A to BMP-2-mediated chondrogenesis, we have generated C3H10T1/2 cell lines overexpressing Wnt-3A and various forms of glycogen synthase kinase-3beta (GSK-3beta), an immediate cytosolic component of the Wnt signaling pathway, and examined their response to BMP-2. We show that overexpression of either Wnt-3A or kinase-dead GSK-3beta enhances BMP-2-mediated chondrogenesis. Furthermore, Wnt-3A overexpression results in decreases in both N-cadherin and GSK-3beta protein levels, whereas Wnt-3A as well as kinase-dead GSK-3beta overexpression increase total and nuclear levels of both beta-catenin and LEF-1. Direct cross-talk between Wnts and BMP-2 was also indicated by the up-regulated interaction between beta-catenin and SMAD-4 in response to BMP-2. These results suggest that Wnt-3A acts in a manner opposite to that of other Wnts, such as Wnt-7A, which were previously identified as inhibitory to chondrogenesis, and is the first BMP-2-regulated, chondrogenesis-enhancing member of the Wnt family.     

Modulation by retinyl acetate of microfilament bundle formation in C3H/10T1/2 cells

Retinyl acetate has been previously shown to inhibit carcinogen-induced neoplastic transformation in 10T1/2 cells and to accentuate many aspects of the nontransformed phenotype. Scanning electron microscopy of logarithmic phase 10T1/2 cells treated for 3 days with 0.3 micrograms/ml retinyl acetate revealed that this treatment caused extensive flattening of cells to the plastic substrate. In contrast the tumor promoter tetradecanoyl phorbol acetate, which antagonizes the antineoplastic activity of retinyl acetate, caused cell rounding and completely inhibited the action of retinyl acetate on cell morphology. During this same time course, the formation of microfilament bundles was also found to be modulated by retinyl acetate. Transmission electron micrographs of unsectioned peripheral regions of flattened cells showed that while the unit density of microfilament bundles was not influenced, the thickness of bundles, particularly those with a diameter of 100 nm or more, was increased by retinyl acetate. Tetradecanoyl phorbol acetate had little effect on microfilament bundle diameters but did partially antagonize the action of retinyl acetate. To determine if this increase was associated with an increase in total actin/cell, total cell proteins, and proteins not extractable by glycerol-triton extraction, were subjected to sodium dodecylsulfate/ polyacrylamide gel electro-phoresis. It was found that while total cellular actin was not increased by retinyl acetate, the proportion of nonextractable actin (which includes microfilament bundles) increased from 65% to 88% of total actin. This increase was not inhibited by inhibitors of protein or RNA synthesis. These studies again demonstrate that retinyl acetate accentuates the nontransformed phenotype of 10T1/2 cells; it is hypothesized that these actions are related to the antineoplastic activity of retinoids.     

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.     

Sister chromatid exchange in methotrexate resistant and sensitive C3H10T1/2 mouse cells

Sister chromatid exchange (SCE) induction by methotrexate (MTX) was analyzed in C3H10T1/2 clone 8 mouse cells and in two MTX-resistant subclones with numerous double minute chromosomes (DM) present in the majority of cells. Significantly higher SCE levels were found, as expected, in sensitive cells after treatments with 10^-2 or 10^-5M MTX but not in resistant cells permanently growing in the presence of a high concentration of MTX (2×10^-3M) and characterized by a markedly lower cell cycle replication index (R.I.), i.e. in conditions that are known to otherwise favour SCE induction. These observations suggest, for the MTX-resistant cells under study, the existence of conditions limiting SCE formation.     

Differentiation and mineralization of murine mesenchymal C3H10T1/2 cells in micromass culture

The murine mesenchymal cell line, C3H10T1/2 in micromass culture undergoes chondrogenic differentiation with the addition of BMP-2. This study compares the use of BMP-2 vs. insulin, transferrin, and sodium selenite (ITS) to create a chondrogenic micromass cell culture system that models cartilage calcification in the presence of 4 mM inorganic phosphate. BMP-2 treated cultures showed more intense alcian blue staining for proteoglycans than ITS treated cultures at early time points. Both ITS and BMP-2 treated cultures showed similar mineral deposition in cultures treated with 4 mM phosphate via von Kossa staining, however FTIR spectroscopy of cultures showed different matrix properties. ITS treated cultures produced matrix that more closely resembled mouse calcified cartilage by FTIR analysis. ⁴⁵Ca uptake curves showed delayed onset of mineralization in cultures treated with BMP-2, however they had an increased rate of mineralization (initial slope of ⁴⁵Ca uptake curve) when compared to the cultures treated with ITS. Immunohistochemistry showed the presence of both collagens type I and type II in BMP-2 and ITS treated control (1 mM inorganic phosphate) and mineralizing cultures. BMP-2 treated mineralizing cultures displayed more intense staining for collagen type II than all other cultures. Collagen type X staining was detected at Day 9 only in mineralizing cultures treated with ITS. Western blotting of Day 9 cultures confirmed the presence of collagen type X in the mineralizing ITS cultures, and also showed very small amounts of collagen type X in BMP-2 treated cultures and control ITS cultures. By Day 16 all cultures stained positive for collagen type X. These data suggest that BMP-2 induces a more chondrogenic phenotype, while ITS treatment favors maturation and hypertrophy of the chondrocytes in the murine micromass cultures.