Regulation of proliferation and migration in retinoic acid treated C3H10T1/2 cells by TGF-beta isoforms

Proliferation of mesenchymal precursors of osteogenic and chondrogenic cells and migration of these precursors to repair sites are important early steps in bone repair. Transforming growth factor-beta (TGF-beta) has been implicated in the promotion of bone repair and may have a role in these processes. Three isoforms of TGF-beta, TGF-beta1, -beta2, and -beta3, are expressed in fracture healing, however, their specific roles in the repair process are unknown. Differential actions of the TGF-beta isoforms on early events of bone repair were explored in the multipotent mesenchymal precursor cell line, C3H10T1/2. Cell migration was determined using a modified Boyden chamber in response to concentrations of each isoform ranging from 10(-12) to 10(-9) g/ml. All three isoforms demonstrated a dose-dependent chemotactic stimulation of untreated C3H10T1/2 cells. Checkerboard assays indicated that all three isoforms also stimulated chemokinesis of the untreated cells. C3H10T1/2 cells treated with all-trans-retinoic acid (ATRA) and expressing relatively higher levels of osteoblastic gene markers such as alkaline phosphatase and collagen type I, lower levels of chondrocytic gene markers collagen type II and aggrecan, and unchanged levels of the adipose marker adipsin did not demonstrate significant chemokinesis or chemotaxis in response to TGF-beta1 or -beta3 at concentrations ranging from 10(-12) to 10(-9) g/ml. In the ATRA-treated cells, TGF-beta2 stimulated a significant increase in chemotaxis only at the highest concentration tested. Cell proliferation was assessed by mitochondrial dehydrogenase activity and cell counts at TGF-beta concentrations from 10(-11) to 10(-8) g/ml. None of the TGF-beta isoforms stimulated cell proliferation in untreated or ATRA-treated C3H10T1/2 cells. Analysis of TGF-beta receptors (TGF-betaR1, -betaR2, and -betaR3) showed a 1.6- to 2.8-fold decrease in mRNA expression of these receptors in ATRA-treated cells. In conclusion: (1) while all three TGF-beta isoforms stimulate chemotaxis/chemokinesis of multipotent C3H10T1/2 cells, TGF-beta1 and -beta3 do not stimulate chemotaxis in C3H10T1/2 cells treated with ATRA while TGF-beta2 stimulated chemotaxis only at the highest concentration tested. (2) TGF-beta isoforms do not appear to stimulate cell proliferation in C3H10T1/2 cells in either a multipotent state or after ATRA treatment when expressing higher levels of alkaline phosphatase and collagen type I gene markers. (3) Decrease in mRNA expression for TGF-betaR1, -betaR2, and -betaR3 upon ATRA treatment could potentially explain the lack of chemotaxis/chemokinesis in these cells expressing higher levels of alkaline phosphatase and collagen type I.     

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.     

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.     

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.     

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.     

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.     

Rapid induction of alkaline phosphatase activity by retinoic acid

Retinoic acid (vitamin A acid) increased alkaline phosphatase activity in cultured cells derived from both normal rat prostate and the Dunning R-3327 transplantable prostatic adenocarcinoma. Retinoic acid was found to be 3–4-fold more effective as an inducer of enzyme activity than retinol or retinal. In one rapidly-growing cell line (UMS-1541Q) which has a barely-detectable level of enzyme activity in the uninduced state, increased activity could be detected as early as 3–4 hours after the addition of 10μM retinoic acid. This increase was totally blocked by actinomycin D and cycloheximide. The demonstrated rapid inducibility of alkaline phosphatase activity provides a specific marker for the action of retinoic acid at the molecular level.     

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.     

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.     

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.     

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.     

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.     

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.     

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.