Sarcomas routinely produced from putatively nontumorigenic Balb/3T3 and C3H/10T1/2 cells by subcutaneous inoculation attached to plastic platelets

The Balb/3T3 and C3H/10T1/2 lines, noted for their marked postconfluence inhibition of proliferation and anchorage dependence, and frequently studied as nontumorigenic lines that are compared with tumorigenic sublines transformed with various agents, produced tumors within two to four months at low-cell dosage (3 × 10⁴ cells) when implanted subcutaneously attached to 1 × 5 × 10 mm polycarbonate platelets. Platelets alone did not produce tumors. The cultured Balb/3T3 tumor cells showed loss of both postconfluence inhibition of proliferation and anchorage dependence. Tumors arising form attached Balb/3T3 cells in (BALB/c × C57B1/6)F1 hybrids were shown to be transplantable to BALB/c but not to C57B1/6 mice, proving that the tumors were derived form Balb/3T3 and not from host cells. The tumors exhibited unique transplantation rejection antigens that did not cross-react with each other. Scanning electronmicroscopy of Balb/3T3 cells and derive tumor cells on Teflon

1 Teflon: Registered trademark of DuPont Plastics.

substrates (on which only the tumor cells and not the parent Balb/3T3 cells could grow) revealed that the two cell types were remarkably similar in appearance, except that the tumor cells were larger and showed many more microvilli that tended to concentrate over the nucleus. We conclude that Balb/3T3 cells and C3H/10T1/2 cells are preneoplastic and give rise to spontaneously transformed clones when implanted in vivo attached to a solid substrate.     

In vitro transformation by bromodeoxyuridine and X irradiation in C3H 10T1/2 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.     

Control of cell multiplication in vitro. IV. Isolation and characteristics of C3H10T1/2-line cells with decreased dependence for multiplication on blood serum

Mouse embryo fibroblasts of C3H10T1/2 cell line were selected in the medium with a low serum content. The frequency of clone occurrence was about 1.10(-5) (for 0.5% serum). The treatment of cells by N-methyl-N'-nitrosoguanidine significantly increased the frequency of occurrence of these clones. The obtained spontaneous variants were assayed for stability of growth characteristics. The phenotypic analysis of clones with low serum growth dependence discovered at least two cell types differing in some morphological and growth characteristics. The former did not differ from the parental line C3H10T1/2 in any phenotype characteristics but their serum growth dependence; whereas, the latter were poorly spread over the substrate and showed an enhanced saturation density and a decreased population doubling time. These cells also differed in their growth dependence on the homologous conditioned medium.     

Lactate-mediated changes in growth morphology and transformation frequency of irradiated C3H 10T1/2 cells

Treatment of mammalian cells with lactate or inhibitors of glycolysis alters their radiation response, particularly in the low dose region of the dose response curve. The occurrence of both high lactate levels and high glycolytic metabolism in tumours is well known and therefore the effect of lactate on a cell line sensitive to radiation induced transformation was examined using a single exposure to Cobalt 60 gamma rays as the carcinogen challenge. The results indicate that cells treated with 5mM lactate before irradiation exhibit changes in morphology and growth rate and that the transformation frequency is increased by three to ten fold following 24 hours lactate treatment just prior to irradiation. Examination of radiation survival curves showed a positive correlation between transformation frequency and size of the shoulder, but increasing transformation frequency was associated with a decrease in Do. A mechanism involving altered Redox potential in lactate treated cells is suggested. The results are discussed in terms of their possible significance for radiotherapy.     

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.     

Colony transformation in C3H 10T1/2 cells: stepwise development of neoplastic change in vitro

This report demonstrates that low passage C3H 10T1/2 cells treated with the carcinogens benzo(a)pyrene or diepoxybutane are transformed morphologically as colonies in as little as 14 d after carcinogen treatment. A transforming dose-response curve is achieved but the frequency of transformation is less than half the expected for 38 d foci, compared on the basis of percent transformants per cell plated. Anchorage-independent cell growth, plating efficiency, doubling time, cell density, and modal chromosomal number were examined from transformed colonies and foci. The data from colony transformants show progressive alteration of these in vitro expressions of neoplastic character with continued subcultivation, consistent with the multistep hypothesis of carcinogenesis. Early in vivo data obtained from one colony-derived transformed cell line show tumorigenesis in irradiated mice within 13 wk of implantation. With continued in vivo passage, tumors were observed in 4 to 6 wk.     

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.     

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.     

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.     

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).     

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.     

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.     

Control of alkaline phosphatase activity in C3H10T1/2 cells: role of retinoic acid and cell density.

The enzyme alkaline phosphatase (AP) has been shown to be lost or inappropriately expressed during carcinogenesis in some tissues. Because retinoic acid (RA) appears to play a role in the normal regulation of the enzyme (RA up-regulates AP in a variety of cell types) we have suggested that altered AP expression in some cancers may be caused by a defect in the ability of the cells to respond normally to retinoid. We have begun to use the chemically transformable mouse embryo fibroblast cell, C3H10T1/2, to investigate this possibility. In this initial study we characterized AP regulation in normal C3H10T1/2 cells and show that: (1) 10(-7) M RA increases AP activity within 3-4 h in serum-free medium; (2) serum inhibits short-term induction (0-8 h) in a concentration-dependent manner (10% serum causes complete inhibition); (3) during long-term RA exposure (24 h and 48 h), induction can be detected in serum-containing medium; (4) AP induction is dose related at RA concentrations from 10(-10) M to 10(-6) M in serum-free medium; (5) 10(-5) M RA is ineffective at inducing AP in serum-free medium during 8 h but is the most effective concentration in serum-containing medium during 24 h and 48 h exposures; (6) AP inducibility by RA requires near-confluent cell densities; and (7) when cultures become confluent, cells become constitutive for AP and no longer require RA for enzyme expression. The effects of serum and cell density on AP inducibility by RA and implications of the RA up-regulation of AP for teratogenesis are discussed.     

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.     

Glutaraldehyde-fixed transformed and non-transformed cells induce contact-dependent inhibition of growth in non-transformed C3H/10T1/2 mouse fibroblasts, but not in 3-methylcholanthrene-transformed cells

C3H/10T1/2 mouse fibroblasts showed a pronounced inhibition of growth when reaching a critical cell density. The situation of high cell density could be mimicked by the addition of glutaraldehyde-fixed cells to sparsely seeded proliferating cells. Treatment of the C3H/10T1/2 cells with 3-methylcholanthrene led to a high frequency of piled up foci (118 type II and type III foci in 78 cultures). Cells of a type III focus of a treated culture were cloned. These cells grew in soft-agar and reached 10 times higher cell densities when grown in culture dishes, than did their non-transformed counterparts. Glutaraldehyde-fixed transformed cells did not differ from fixed non-transformed cells in the ability to inhibit the growth of sparsely seeded non-transformed cells. On the other hand, both the addition of fixed normal or transformed C3H/10T1/2 cells did not affect the growth rate of transformed cells. In a concept explaining the density-dependent inhibition of growth of non-transformed cells by a specific interaction of plasma membrane-localized effectors with plasma membrane-localized receptors, the present findings would indicate that the transformed cells used express active effectors but are functionally defective in the receptors or in the signal transmission.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin reduces high-affinity binding of epidermal growth factor to cell surface receptors in C3H 10T1/2 cells

The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on epidermal growth factor (EGF)-binding characteristics was studied in a cultured embryonic fibroblast cell line, C3H 10T1/2. At very low concentrations, TCDD was found to cause a persistent decline in EFG binding, the median effective concentration (EC-50) being 10(-12) M. This particular effect was most conspicuous when TCDD was added at the time of medium change with fresh Dulbecco's modified Eagle's medium. Cells at an early stage of confluency were more responsive to TCDD than those at a later stage. Although most reported TCDD-evoked biological changes are recognized to occur slowly during the course of a few days to weeks, the response of C3H 10T1/2 cells to TCDD was swift, showing a sign of decline of EGF binding as early as three hours after TCDD addition. C3H 10T1/2 cells appear to be an excellent in vitro model to study TCDD's biochemical action mechanisms.     

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.