Effects of 5-azacytidine on expression of endogenous retrovirus-related sequences in C3H 10T1/2 cells.

In a previous study (22) we found that transient exposure of C3H 10T1/2 mouse embryo fibroblasts to 5-azacytidine (5-azaC) induced several changes in growth properties. The treated cells showed progressive changes in morphology, saturation density, growth rate, and serum dependence. By passage 5, the cells had acquired the ability to grow in 0.3% agarose, and by passage 30, they had given rise to fully transformed foci that grew in agarose, agar, and liquid suspension. This progression was rapidly accelerated if the cultures derived from 5-azaC-treated cells were exposed for 48 h to the carcinogen benzo[a]pyrene. The present studies demonstrate that both type C and type A, but not type B, retrovirus-related sequences were expressed in the 5-azaC-treated cells. There was negligible expression of these sequences in the control 10T1/2 cells. The level of expression of the related RNAs tended to correlate with loss of anchorage dependence and other markers of an increase in the transformed phenotype. These changes were associated with hypomethylation of the corresponding cellular DNA sequences, as revealed by differential digestion with the restriction enzymes HpaII and MspI. These studies provide evidence that aberrations in DNA methylation and induction of expression of certain endogenous retroviruses may be one of a series of critical events during the course of multistage carcinogenesis, thus enhancing the evolution of malignant tumor cells.     

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.     

Multiple new phenotypes induced in 10T1/2 and 3T3 cells treated with 5-azacytidine.

Three new mesenchymal phenotypes were expressed in cultures of Swiss 3T3 and C3H/10T1/2CL8 mouse cells treated with 5-azacytidine or 5-aza-2'-deoxycytidine. These phenotypes were characterized as contractile striated muscle cells, biochemically differentiated adipocytes and chondrocytes capable of the biosynthesis of cartilage-specific proteins. The number of muscle and fat cells which appeared in treated cultures was dependent upon the concentration of 5-azacytidine used, but the chondrocyte phenotype was not expressed frequently enough for quantitation. The differentiated cell types were only observed several days or weeks after treatment with the analog, implying that cell division was obligatory for the expression of the new phenotypes. Oncogenically transformed C3H/10T1/2CL8 cells also developed muscle cells after exposure to 5-azacytidine, but at a reduced rate when compared to the parent line. Five subclones of the 10T1/2 line which were the progeny of single cells all expressed both the muscle and fat phenotypes following 5-azacytidine treatment. The effects of the analog are therefore not due to the selection of preexisting myoblasts or adipocytes in the cell populations. Rather, it is possible that 5-azacytidine, after incorporation into DNA, causes a reversion to a more pluripotential state from which the new phenotypes subsequently differentiate.     

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.     

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.     

Hydroxylation of methylated DNA by TET1 in chondrocyte differentiation of C3H10T1/2 cells

DNA methylation is closely involved in the regulation of cellular differentiation, including chondrogenic differentiation of mesenchymal stem cells. Recent studies showed that Ten–eleven translocation (TET) family proteins converted 5-methylcytosine (5mC) to 5-hydroxymethylcytosine, 5-formylcytosine and 5carboxylcytosine by oxidation. These reactions constitute potential mechanisms for active demethylation of methylated DNA. However, the relationship between the DNA methylation patterns and the effects of TET family proteins in chondrocyte differentiation is still unclear. In this study, we showed that DNA hydroxylation of 5mC was increased during chondrocytic differentiation of C3H10T1/2 cells and that the expression of Tet1 was particularly enhanced. Moreover, knockdown experiments revealed that the downregulation of Tet1 expression caused decreases in chondrogenesis markers such as type 2 and type 10 collagens. Furthermore, we found that TET proteins had a site preference for hydroxylation of 5mC on the Insulin-like growth factor 1 (Igf1) promoter in chondrocytes. Taken together, we showed that the expression of Tet1 was specifically facilitated in chondrocyte differentiation and Tet1 can regulate chondrocyte marker gene expression presumably through its hydroxylation activity for DNA.     

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.     

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.     

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.     

Effects of protease inhibitors on nuclear binding of glucocorticoid hormones in C3H10T1/2 cells

We have measured incorporation of the glucocorticoid hormone cortisone into nuclear hormone-receptor complexes in the C3H10T1/2 cell line. As we had found cortisone to be capable of malignantly transforming these cells in vitro, and certain protease inhibitors have been shown to suppress transformation in this cell line, we investigated the effects of these protease inhibitors (antipain, chymostatin and the Bowman-Birk inhibitor) on the formation of nuclear cortisone-receptor complexes. All 3 inhibitors were found to suppress wholly or partially formation of nuclear cortisone-receptor complexes, suggesting that such complexes may be involved in the process of glucocorticoid-enhanced 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.     

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.     

Effect of 5-azacytidine on DNA methylation in Ehrlich's ascites tumor cells

5-Azacytidine inhibited in vivo DNA methylation in Ehrlich's ascites tumor cells depending upon the dose at which 5-azacytidine did not inhibit DNA synthesis significantly. This drug did not inhibit DNA methylation in vitro. The DNA methylase activity in ascitic cells decreased with the increasing dose of 5-azacytidine. Hypomethylated DNA was obtained from the 5-azacytidine treated ascitic cells.     

Comet assay evaluation of DNA single- and double-strand breaks induction and repair in C3H10T1/2 cells

Ionizing radiation is a potent inducer of DNA damage because it causes single- and double-strand breaks, alkali-labile sites, base damage, and crosslinks. The interest in ionizing radiation is due to its environmental and clinical implications. Single-strand breaks, which are the initial damage induced by a genotoxic agent, can be used as a biomarker of exposure, whereas the more biologically relevant double-strand breaks can be analyzed to quantify the extent of damage. In the present study the effects of ¹³⁷Cs γ-radiation at doses of 1, 5, and 10 Gray on DNA and subsequent repair by C3H10T1/2 cells (mouse embryo fibroblasts) were investigated. Two versions of the comet assay, a sensitive method for evaluating DNA damage, were implemented: the alkaline one to detect single-strand breaks, and the neutral one to identify double-strand breaks. The results show a good linear relation between DNA damage and radiation dose, for both single-strand and double-strand breaks. A statistically significant difference with respect to controls was found at the lowest dose of 1 Gy. Heterogeneity in DNA damage within the cell population was observed as a function of radiation dose. Repair kinetics showed that most of the damage was repaired within 2 h after irradiation, and that the highest rejoining rate occurred with the highest dose (10 Gy). Single-strand breaks were completely repaired 24 h after irradiation, whereas residual double-strand breaks were still present. This finding needs further investigation. This revised version was published online in July 2006 with corrections to the Cover Date.