Morphological correlates of transformation in cultured C3H/10T1/2 mouse embryo cells

In efforts to determine common and consistent morphological parameters of transformation in C3H/10T1/2 cells, 15 cell lines transformed by different carcinogens were examined with the scanning electron microscope (SEM) and compared to non-transformed cells. Cell lines were studied at different passages, at different cell densities, and after growth as anchorage dependent or independent cultures. The transformed cell lines could be distinguished in the SEM from non-transformed cultures by the expression of one or more of the following morphological characteristics: formation of mini- or macro-foci (random piling of cells on top of each other), pleiomorphism in cell size and shape, and cell surface complexity. The extent to which these characteristics were expressed varied widely in the different transformed cell lines. Light microscopic scoring for different types of foci also revealed broad variability among the different transformed lines. At the SEM level, cell lines could not be characterized as transformed on an individual cell basis. However, all transformed cell lines could be definitively characterized as transformed on a population basis due to the presence of mini-foci. The various transformed cell lines were classified semi-quantitatively into categories based on the extent of expression of the different morphological characteristics. There was broad correspondence between the morphological classification and the relative plating efficiencies of the cell lines in soft agarose.     

Vanadate enhances transformation of bovine papillomavirus DNA-transfected C3H/10T1/2 cells.

Bovine papillomavirus (BPV) DNA-transfected C3H/10T1/2 cells respond to tumor promoters by enhanced production of transformed foci. Vanadate, a suspected carcinogen, is a mitogen, generates active oxygen species and alters phosphorylation of proteins. We investigated whether vanadate would enhance transformation of BPV DNA-transfected C3H/10T1/2 cells. Transformed foci in BPV DNA transfected C3H/10T1/2 cells exposed continuously to vanadate for 21 days increased in a dose-dependent manner to 50-fold at 4 microM vanadate. This increase was not due to enhanced uptake of BPV DNA post transfection. Neither catalase nor superoxide dismutase inhibited the vanadate-mediated increase in transformed foci but this does not necessarily rule out the involvement of intracellular active oxygen species. At vanadate concentrations greater than 6 microM, cells lost adherence to the Petri plates. We conclude that vanadate is capable of enhancing BPV DNA-mediated cell transformation. Possible mechanisms may involve active oxygen species or altered patterns of protein phosphorylation.     

In vitro transformation of C3H/10T1/2 and NIH/3T3 cells by acrylonitrile and acrylamide

Acrylonitrile (AN) and acrylamide (AM) are carcinogenic in a number of rodent organs and AN is a suspected human carcinogen. We sought to determine whether AN and/or AM could produce morphological transformation in vitro in C3H/10T1/2 and NIH/3T3 mouse fibroblast cells. Both AN and AM induced a dose-dependent cytotoxic effect in C3H/10T1/2 and NIH/3T3 cells and readily transformed both cell lines. Our conclusions are based on the appearance of cells exhibiting a transformed phenotype and growth in soft agar. AN and AM transformed NIH/3T3 cells to a greater extent than C3H/10T1/2 cells. This is the first reported transformation of cells in vitro by AM.     

Inhibition of radiation-induced transformation of C3H/10T1/2 cells by specific protease substrates

In the current studies, we have examined the effect of two specific protease substrates, the thrombin substrate Boc-Val-Pro-Arg-MCA and the chymotrypsin substrate Suc-Ala-Ala-Pro-Phe-MCA, on the oncogenic transformation of C3H/10T1/2 cells induced with: (i) 6 Gy of X-radiation and (ii) 4 Gy of X-radiation followed by promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). Both substrates reduced radiation transformation while only Suc-Ala-Ala-Pro-Phe-MCA suppressed the TPA enhancement of radiation transformation. We have previously reported that C3H/10T1/2 cells contain at least two proteolytic activities which will cleave these substrates. Our results therefore suggest that: (i) these substrates may inhibit oncogenic transformation due to the fact that they are competitive substrates for these enzymes; and (ii) two or more proteases play an important role in the malignant transformation of these cells.     

Alterations of intercellular communication associated with the transformation of C3H/10T1/2 cells

Cultures of C3H/10T1/2 mouse embryo cells were treated in accordancewith several treatment regimens that induced the focal growthof morphologically transformed cells. Inter cellular communicationbetween focus cells, and between focus and monolayer cells,was examined in late stages of transformation experiments bymicroinjection of Lucifer yellow dye into cells and observationof dye transfer to surrounding cells. Transformed foci producedby treatment with 3-methylcholanthrene, by initiation with N-methyl-N'nitro-N-nitrosoguanidine (MNNG) and promotion with 12-O-tetradecanoylphorbol-13-acetate(TPA), or by initiation with MNNG and promotion with 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD) were studied. These included focus types thought to possesstumongenic potential (Types II and III) and those believed tolack such potential (Type I). Cells within all focus types exhibitedonly limited communication with each other or with surroundingmonolayer cells. In contrast, microinjection of monolayer cellstypically resulted in dye transfer to an average of {small tilde}50other monolayer cells. The presence of the tumor promoters TPAor TCDD did not alter intercellular communication between monolayercells. These studies demonstrate that alterations in intercellularcommunication are evident during the growth of transformed foci.These changes are relatively independent of both the treatmentregimen used to produce foci and the presumed oncogenic potentialof different focus types.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) promotes the transformation of C3H/10T1/2 cells

Continuous treatment of C3H/10T1/2 cells with low concentrations(>4pM) of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) enhancedfocus production in cultures pretreated with N-methyl-N'-nitro-N-nitrosoguanidine.Maximal enhancement occurred at 40 pM TCDD, a concentration10 000-fold lower than that required to produce an optimal responsewith 12-O-tetradecanoylphorboI-13-acetate. Single treatmentswith 0.06 nM-5 µM TCDD did not transform C3H/10T1/2 cellsor initiate the process of transformation in cultures subsequentlyexposed to the tumor promoter 12-Otetradecanoylphorbol-13-acetate.Promotion of transformation is thus the predominant effect ofTCDD in the C3H/101/2 cell transformation system.     

Inhibition of radiation-induced transformation of C3H/10T1/2 cells by chymotrypsin inhibitor 1 from potatoes

In this report, we have investigated whether a protease inhibitorobtained from potatoes (chymotrypsin inhibitor 1; CI-1) willinhibit carcinogen-induced transformation of C3H/10T cells.CI-1 was as effective as the soybean-derived Bowman Birk inhibitorat suppressing radiation-induced transformation of C3H/10T cells,at a concentration of 10 µg/ml. The inhibitor was nottoxic to the cells at concentrations of 0.1 –10 µg7sol;ml,the concentrations of CI-1 employed in the transformation experiments.To investigate the interaction of this inhibitor with the targetcells, binding studies were carried out. ¹²⁵I-labelled CI-1could not be displaced from C3H7sol;10T cells by co-incubationof the cells with a 5000-fold excess of unlabelled inhibitor.These results suggest that this inhibitor does not reversiblybind to specific receptor proteins on the surface of these cells.     

In vitro correlates of transformation in C3H/10T1/2 clone 8 mouse cells.

Various potential in vitro correlates of malignancy were studied in four chemically transformed C3H/10T1/2 Clone 8 mouse cell lines and were compared with controls cells. The degree of tumorigenicity was best predicted by the relative plating efficiencies of the morphologically transformed cells in soft agar. All transformed cells also showed an increase in extracellular fibrinolytic activity which may be an additional marker for transformation. Intracellular fibrinolytic activity and loss of 125I-labeled cell surface protein (M.W. 250,000) were not correlated with morphological transformation or tumorigenicity in these cells.     

Improved transformation of C3H10T1/2CL8 cells by direct- and indirect-acting carcinogens

Oncogenic transformation of C3H10T1/2CL8 cells was improvedby treating the cells 5 days after seeding. Benzo[a]pyrene-inducedtransformation was increased 3.5-fold by this method, comparedwith treating the cells 1 day after seeding. N-Methyl-N' -nitro-N-nitrosoguanidine,which does not transform asynchronous cultures of C3H10T1/2CL8cells when administered 1 day after seeding, produced an averageof 1 focus/dish, with 61% of the dishes exhibiting foci, whenadministered 5 days after seeding. Propane sultone and aflatoxinB₁ also produced marked transformation responses when administered5 days after seeding. However, 4-dimethylaminoazobenzene didnot induce transformation when administered either 1 day or5 days after seeding. With all chemicals examined, clonal cytotoxicitywas reduced when they were administered 5 days after seeding.These results indicate the utility of this new procedure forthe qualitative analysis of the transforming ability of chemicals.     

Promotion of C3H/10T1/2 morphological transformation by polychlorinated dibenzo-p-dioxin isomers

Few of the seventy-five chlorinated dibenzo-p-dioxin isomerspresent in the environment have been adequately characterizedfor their carcinogenic potential. In previous studies we observedthat the carcinogenic dioxin isomer 2,3,7,8-tetra-chlorodibenzo-p-dioxinpromoted cell transformation when continuously applied to C3H/10T1/2cells initiated by treatment with N-methyl-N'-nitro-N-nitrosoguanidine(MNNG). The current study was undertaken to evaluate the responseof the C3H/10T1/2 cell transformation system to several otherdioxin isomers of known carcinogenic potential. 1,2,3,6,7,8-and 1,2,3,7,8,9-hexachlorodibenzo-p-dioxin (HCDD) (1–1000nM) and 2,7-dichlorodibenzo-p-dioxin (0.1–20 µM)failed to transform C3H/10T1/2 cells or to initiate transformationin cultures subsequently treated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate.Continuous exposure of MNNG-initiated cultures to 2,7-DCDD (1–5000nM) produced elevated but not statistically significant numbersof transformed foci at the highest dose tested. 1,2,3,6,7,8-and 1,2,3,7,8,9-HCDD were promoters of transformation when appliedat concentrations 12 and 40 pM, respectively, to C3H/10T1/2cultures initiated with MNNG. Maximum responses for both HCDDisomers were attained at concentrations between 120 and 400pM. These studies suggest that the C3H/10T1/2 cell transformationsystem may provide a relevant in vitro model for the identificationand study of carcinogenic dioxin isomers.     

Tiadenol-mediated induction of peroxisomal enzymes in cultured C3H/10T1/2CL8 cells and in chemically transformed C3H/10T1/2 MCA16 cells

The levels of peroxisomal enzyme activities in cultured C3H/10T1/2 CL8 cells and in chemically transformed C3H/10T1/2 MCA16 cells were studied after treatment with tiadenol and niadenate, two hypolipidemic drugs which are both carcinogenic and cause peroxisome proliferation in vivo. Administration of these peroxisome proliferators to the cells resulted in large increases in specific palmitoyl-CoA hydrolase, carnitine acetyl-transferase, and catalase activities. A reproducible induction of cyanide-insensitive palmitoyl-CoA oxidative activity was observed 5 and 9 days after initiation of tiadenol treatment. Basal activity was 0.16 nmole/min/mg protein compared to 0.95 nmole/min/mg protein in cells treated with 18 microM tiadenol (cytotoxicity of about 25%) for 9 days. The enzyme activities were more increased in the transformed MCA 16 cells than in the non-transformed cells and the order of increase in enzyme activities was: niadenate greater than tiadenol. In non-transformed cells, the specific activity of palmitoyl-CoA hydrolase was enhanced approximately 2.1-fold within 4 days after tiadenol treatment. During this time period the enzyme activity was constant in untreated cells, but decreased during longer incubation periods. The enhancement of palmitoyl-CoA hydrolase, carnitine acetyl-transferase and catalase activities was dose-related over a concentration range of 2 to 20 microM tiadenol, depending on the enzyme assayed. Tiadenol concentrations above 10 microM were increasingly cytotoxic, while 18 microM niadenate had no toxic effect on the C3H/10T1/2 C18 cells. Moreover, the stimulation of the 3 enzyme activities by the peroxisome proliferators were inhibited by cycloheximide. Neither of the two cell lines contained any appreciable urate oxidase activity. The responses of these cells to hypolipidemic drugs show that they constitute a useful system for studies on the role of peroxisomes in lipid metabolism and the relationship between hypolipidemic activity and carcinogenic potential of these drugs.     

Suppression of x-ray induced transformation by Valium and aspirin in mouse C3H10T1/2 cells

Two commonly used drugs, Valium (diazepam) and Aspirin (acetylsalicyclic acid), were shown to suppress X-ray induced transformation in mouse C3H/10T1/2 cells. Valium was studied in an ethanol solution. Aspirin, which is soluble in both water and ethanol, was active only in the ethanol solution. Both drugs were effective only when present throughout the entire assay period.     

Increased thymidine uptake by methylcholanthrene-treated C3H/10T1/2 cells.

The uptake of 3H-thymidine in post-confluent cultures of methylcholanthrene-transformed C3H/10T1/I mouse embryo cells was markedly higher than in their non-transformed counterparts. In a reconstruction experiment as few as 2% transformed cells could be detected by increased thymidine uptake. Measurements made at various times up to 110 days in multi-chambered plates revealed that after 25 days methylcholanthrene-treated cultures incorporated significantly more thymidine than the acetone-treated controls. This increased uptake correlated with the appearance of Type III transformed foci.     

Oncogenic transformation of C3H/10T1/2 clone 8 mouse embryo cells by halogenated pyrimidine nucleosides.

Oncogenic transformation has been induced in vitro in the C3H/10T1/2 clone 8 line of mouse cells by exposure to 5-fluoro-2'-deoxyuridine (FUdR) or 5-fluorouracil. This transformation is both dose and time dependent and can be markedly decreased by simultaneous exposure of the cells to thymidine. The transformation induced by 5-fluorouracil is probably due to its intracellular conversion to FUdR or its monophosphate. Transformation by FUdR was found to be cell cycle dependent with maximum sensitivity to transformation occurring in early S phase. Cell lines that produced sarcomas in antithymocyte-treated syngeneic mice were isolated from FUdR-transformed cultures. Trifluorothymidine, 5-bromo-2'-deoxyuridine, and 5-iodo-2'-deoxyuridine induced no transformed foci in the C3H/10T1/2 clone 8 cell line. Thus, not all mutagens produce oncogenic transformation nor does the lack of mutagenicity, as classically measured, completely exclude the possibility that a given agent is oncogenic. Also, there was no evidence of the "switch on" of oncornaviral information in the FUdR-transformed cell lines.     

Inhibition of radiation-induced transformation of C3H/10T1/2 cells by carboxypeptidase inhibitor 1 and inhibitor II from potatoes

In the current study, the ability of four protease inhibitorsto suppress radiation-induced transformation of C3H/10T1/2 cellswas investigated. The inhibitors tested included: (i) aprotinin(a serine protease inhibitor), (ii) N-acetyl-L-tyrosine ethylester (a chymotrypsin substrate and competitive inhibitor ofprotein degradation), (iii) carboxypeptidase inhibitor (a metallo-exopeptidaseinhibitor) and (iv) Inhibitor II (a chymotrypsin/trypsin inhibitor).While none of the inhibitors were toxic to the cells at theconcentrations employed, only carboxypeptidase inhibitor andInhibitor II are internalized radiation-induced transformationin a statistically significant manner. Utilizing fluorescentlabeled inhibitors, we found that carboxypeptidase inhibitorsand Inhibitor II are internalized by the cells. Fluorescent-labeledinhibitor could be observed in the cells within 15 min of incubationand is present in distinct intercellular vacuoles within 1 h.These results indicate that carboxypeptidase inhibitor and InhibitorII are internalized by C3H/10T1/2 cells and thus would be ableto inhibit intracellular proteases (or other enzymes) involvedin the conversion of a cell to the malignant state.     

Cell density dependence of focus formation in the C3H/10T1/2 transformation assay

Some of the dynamics of neoplastic transformation in vitro have been studied with the use of benzo(a)pyrene as the carcinogen in the C3H/10T1/2 morphological transformation assay. Experiments that involved the dsipersion of cells into new culture dishes at various times after carcinogen treatment have shown that no change in the fraction of potentially transformed cells occurs while cultures grow to form a confluent monolayer, that little or no change in the fraction of potentially transformed cells occurs for approximately 3 weeks after confluence is attained, and that this fraction increases rapidly some 7 weeks after BP treatment. When confluent benzo(a)pyrene-treated cultures are dispersed in new culture dishes prior to the onset of growth toward focus formation, the formation of transformed foci is suppresssed at high cell densities of seeding. This phenomenon is independent of the total number of divisions undergone by cells after treatment. We suggest that phenotypic expression of morphological transformation is dependent on colony interactions in the C3H/10T1/2 system, which we do not yet understand, but which are independent of time posttreatment either in cell generations or absolute time.     

Maintenance of adult rat hepatocytes on C3H/10T1/2 cells.

A procedure is described for maintaining primary cultures of adult rat hepatocytes on a layer of irradiated C3H/10T1/2 cells. These hepatocytes were capable of metabolizing the liver carcinogen N-2-acetylaminofluorene to water-soluble products and after 14 days in culture could still metabolize approximately 70% of the Day 1 level. Hepatocytes maintained on the C3H/10T1/2 cells were inducible for the liver-specific enzyme tyrosine aminotransferase, and exhibited approximately a 4-fold induction by hydrocortisone during a 10-day culture period. Morphologically, these hepatocytes retained many characteristics of hepatocytes in vivo. By contrast, hepatocytes maintained on plastic lost both N-2-acetylaminofluorene-metabolizing ability and tyrosine aminotransferase activity by Day 5. This was presumably due to degeneration of the hepatocytes and an overgrowth by fibroblasts. The maintenance of morphologically and biochemically functional hepatocytes in culture on feeder cells may provide a valuable approach for studying drug metabolism and liver cell transformation in vitro.     

Inhibition of benzo(a)pyrene-induced transformation of C3H/10T1/2 cells by allylisopropylacetamide and isopropylvaleramide

Allylisopropylacetamide (AIA) and isopropylvaleramide (IVA) have been demonstrated previously to protect in vivo against the acute toxicity and adrenal necrotic effect of 7,12-dimethylbenz(a)anthracene. In the present study, the influence of these two amides on the in vitro transforming ability of two potent carcinogens, benzo(a)pyrene [B(a)P] and 7,12-dimethylbenz(a)anthracene, on C3H10T1/2 cells was investigated. Both AIA and IVA showed a dose-dependent inhibition of B(a)P-induced transformation of C3H10T1/2 cells when added simultaneously for 24 hr with the carcinogen. While pretreatment, simultaneous treatment, and posttreatment of the cells with AIA or IVA inhibited transformation, the 24-hr posttreatment was somewhat more effective. The protective effect did not appear to results from alterations in B(a)P metabolism inasmuch as aryl hydrocarbon hydroxylase activity and the metabolic products of B(a)P detected by high-pressure liquid chromatography were not changed by AIA or IVA treatment. Furthermore, AIA and IVA did not selectively kill chemically transformed C3H10T1/2 cells, as indicated by the absence of their effect on an established, chemically transformed cell line. AIA and IVA also inhibited 7,12-dimethylbenz(a)anthracene-induced transformation of C3H10T1/2 cells. These data suggest that AIA and IVA may be useful protective agents and that they presumably exert their protective effect at some stage between the activation of the carcinogen and the expression of the transformed phenotype.     

A reassessment of methylcholanthrene transformation in the C3H10T1/2 cell culture system

We previously demonstrated that four tumorigenic methylcholanthrene (MCA) transformed cell lines derived from C3H10T1/2 cells each contain a common G34----T nucleotide alteration in the c-Ki-ras gene. In contrast, a non-tumorigenic MCA transformant does not contain this mutation. We have now examined 75 newly isolated MCA transformants of C3H10T1/2 cells for their degree of morphological transformation, the presence of the c-Ki-ras G34----T mutation, colony formation in soft agar, and tumorigenicity in nude mice. Although many of these new MCA transformants exhibit morphological characteristics indistinguishable from previously isolated tumorigenic MCA transformants, none contain the G34----T mutation in the c-Ki-ras gene. Only one newly isolated MCA transformant can grow in soft agar. Of 14 tested, none of the new MCA C3H10T1/2 transformants are tumorigenic in nude mice.