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.     

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.     

Reduction in the formation of carcinogen-induced transformed foci by pencillin G sodium in the C3H/10T1/2 CL8 cell lines.

The C3H/10T1/2 CL8 cell line is being widely used to study mechanisms of malignant transformation in vitro. As currently employed, the standard assay system uses a combination of penicillin (100 I.U./ml) and streptomycin (50 micrograms/ml) to reduce the occurrence of bacterial contamination. The penicillin component of this mixture has been discovered to cause a reduction in the number of transformed foci which develop after exposure of cells to MCA, DMBA and X-rays. This reduction is dose dependent; 500 I.U./ml virtually eliminates transformation, while 100 I.U./ml causes an approximate 50% decrease in the number of foci. This effect does not appear to be due to overt toxicity and is largely reversible on removal of the antibiotic. Gentamicin (25 micrograms/ml) causes no reduction in the formation of transformed foci when compared to cultures maintained in antibiotic-free medium and offers the advantages of chemical stability, a wider spectrum of antibacterial activity in comparison with penicillin/streptomycin and, in addition, is active against many mycoplasma. It is suggested that future studies with this cell line should ideally be performed without antibiotics or should employ Gentamicin for antibacterial protection.     

In vitro reversion of transformed phenotype in mouse C3H/10T1/2 cells

Spontaneous tumor regression is still one of the most puzzling events in human cancer. A cell culture model of malignant transformation designed to permit the study of this phenomenon in vitro was applied to examine reversion and re-expression of the transformed phenotype in two X-ray transformed mouse 10T1/2 cell clones. By alternating cell passages at low and high seeding density, the expression of cell contact inhibition and tumorigenic capacity were both reverted and restored. Growth of non-transformed wild-type cells was not affected by seeding density. This reversion of the transformed phenotype was associated with a modification in genomic 5-methylcytosine content. Initially, the transformed clones were hypomethylated, as occurs in most human tumors. After only four passages at low seeding density, the phenotype was reverted to that of non-transformed 10T1/2 cells and genomic 5-methylcytosine content was significantly increased to levels measured in non-transformed C3H/10T1/2 mouse cells. Thus, hypomethylation induced by ionizing radiation was not a permanent feature of malignantly transformed 10T1/2 cells. Although genomic 5-methylcytosine content returned to normal levels during low density passaging, the methylation pattern of the c-myc gene specifically was not associated with cell passages either at low or high seeding density. In an attempt to identify genes involved in this process, expression of the tumor suppressor gene p53 was measured. Western blot analysis failed to detect any correlation between expression of p53 protein and reversion of the transformed phenotype. The results of this study indicate that the transformed phenotype is not permanently associated with the malignant transformation of C3H/1OT1/2 cells, and can be modulated by growth conditions in vitro. We propose that modulation of genomic 5-methylcytosine levels may be involved in this process.     

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.     

Mutagenicity of 5-azacytidine and related nucleosides in C3H/10T 1/2 clone 8 and V79 cells

To determine whether 5-azacytidine (5-AzaCR)-induced transformation and/or differentiation of C3H/10T 1/2 clone B (10T 1/2) cells might have a mutational basis, we studied whether 5-AzaCR and structurally related nucleoside analogs could mutate 10T 1/2 and Chinese hamster V79 cells. In an assay for mutation to ouabain resistance in 10T 1/2 cells, which detects base substitution mutations but not frameshift mutations, 5-AzaCR and 6-azacytidine were not significantly mutagenic. 5-Aza-2'-deoxycytidine, 5-fluoro-2'-deoxycytidine, 5,6-dihydro-5-azacytidine, 5-fluoro-2'-deoxyuridine (FUdR), 5-bromo-2'-deoxyuridine (BUdR), and 1-beta-D-arabinofuranosylcytosine (ara-C) were only weakly mutagenic. In an assay for mutation to ouabain resistance in V79 cells, which also detects base substitution mutations but not frameshift mutations, 5-AzaCR, 5-aza-2'-deoxycytidine, FUdR, and ara-C were not detectably mutagenic, and BUdR was moderately mutagenic at highly cytotoxic concentrations. In an assay for mutation to 8-azaguanine resistance in V79 cells, which detects base substitution and frameshift mutations, 5-fluoro-2'-deoxycytidine and ara-C were weakly mutagenic, BUdR was moderately mutagenic at very cytotoxic concentrations, and 5-AzaCR, 5-aza-2'-deoxycytidine, FUdR, 6-azacytidine, and 5,6-dihydro-5-azacytidine were not significantly mutagenic. Therefore, 5-AzaCR and related cytosine analogs can be considered as negligibly mutagenic. This study does not provide support for a mutational basis for 5-AzaCR-induced differentiation in 10T 1/2 cells. Further, there was no correlation between the mutagenicity of the nucleosides 5-AzaCR, ara-C, BUdR, and FUdR studied here and their previously reported abilities to transform 10T 1/2 cells. The mutagenicities of 5-AzaCR and FUdR were so low that the biological significance of these effects is uncertain. Hence, it is not clear to what extent, if any, mutation contributes to the transformation caused by these two compounds, and other possible mechanisms of transformation should also be investigated.     

Disposition of endogenous homocysteine by mouse fibroblast C3H/10T1/2 Cl 8 and the chemically transformed C3H/10T1/2 MCA Cl 16 cells following methotrexate exposure

The tumorigenic cell line termed "MCA Cl 16" was derived from C3H/10T1/2 clone (Cl) 8 cells by chemical transformation in the presence of 3-methylcholanthrene [(MCA) CAS: 56-49-5]. Transformed (Cl 16) cells were more sensitive toward the cytotoxic effect of methotrexate (MTX) than their normal counterpart Cl 8 cells. The disposition of endogenous L-homocysteine (Hcy) was investigated in these two cell lines after MTX exposure. Both nonmalignant and transformed cells exported Hcy into the extracellular medium, and only small amounts were retained within the cells. The Hcy efflux from the malignant cells was markedly increased after MTX exposure (0.5-10 microM), and this effect was almost completely prevented by 5-formyl-tetrahydrofolate (THF), whereas treatment with thymidine plus hypoxanthine did not inhibit the MTX-dependent Hcy efflux. Cytotoxic concentration of MCA reduced rather than increased the Hcy efflux from these cells. High concentrations of MTX (greater than 10 microM) were required to increase the release of Hcy from nonmalignant cells. The enhancement of Hcy export from the malignant cells in the presence of MTX was not associated with cellular build-up of S-adenosyl-L-homocysteine (AdoHcy), indicating that the amount of intracellular Hcy was kept below the level required for inhibition or reversion of the AdoHcy hydrolase reaction. MTX-dependent Hcy efflux probably reflects cellular deficiency of 5-methyl-THF required for the salvage of Hcy to methionine and may therefore be a measure of lack of this reduced folate relative to the metabolic demand.     

Induction of foci of morphologically transformed cells in synchronized populations of 10T1/2 cells by N-methyl-N'-nitro-N-nitrosoguanidine and the effect of spontaneous transformation on calculated transformation frequency

Exposure of synchronized C3H10T1/2 (clone 8) cell populations of various sizes to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) at a concentration of 2 micrograms/ml for 30 min at 24 h after release from confluence-induced arrest of proliferation produced neoplastic transformation (formation of foci of morphologically altered cells) by a random but episodic process in a small fraction of the cells at risk soon after treatment. The fraction of dishes that contained type II or type III foci increased as the number of cells at risk increased. In contrast, the development of spontaneous foci is a stochastic process that depends on the number of new cells that form during population growth and is independent of the number of cells that are plated (J. W. Grisham et al., Cancer Res., 48: 5969-5976,1988). When there were small numbers of cells at risk, spontaneous formation of foci was a source of considerable error in evaluating MNNG-induced transformation frequency. In surviving cell populations of less than 1000-3000 cells/100-mm dish, the frequency of induction of foci by MNNG could not be distinguished statistically from the frequency with which foci were expected to form spontaneously. When the fraction of MNNG-treated dishes that contained foci was adjusted for the fraction of pooled control dishes that contained foci, the number of foci induced by a uniform dose of MNNG was found to vary with the number of surviving cells. However, the MNNG-induced transformation frequencies calculated by the Poisson method were independent of the size of the population of cells at risk, provided the population of cells at risk was of sufficient size to allow spontaneous and induced transformation to be distinguished statistically. The results of this study show that the frequency of MNNG-induced transformation can be quantitated in cultures of 10T1/2 cells that contain varying but sufficient numbers of cells at risk when spontaneous transformation is considered. Furthermore, these observations suggest that MNNG-induced transformation of 10T1/2 cells occurs with the frequency and characteristics of a mutation-like change involving a single gene.     

A continuum of transformed phenotypes in C3H/10T1/2 derivatives

Two-stage transformants have been isolated from C3H/10T1/2 cellsexposed to UV-irradiation followed by treatment with TPA. TheseUV-TDTx cells form foci in co-culture with C3H/10T1/2 cellsonly in the presence of TPA. In the absence of TPA, UV-TDTxcells are indistinguishable from control C3H/10T1/2 cells inco-cultures. Single-step transformants of C3H/10T1/2 cells isolatedafter exposure to high doses of chemical carcinogens, however,show TPA-independent focus formation in co-culture with C3H/10T1/2cells. We now show that three independently isolated two-stageUV + TPA transformants as well as a single-step, high dose,methylcholanthrene (MCA) transformant (MCATxle) isolated inour laboratory are anchorage-dependent and non-tumorigenic.In contrast, another single-step, high dose MCA transformant(MCAC1 # 16/39) known to contain an activated c-Ki-ras geneshows TPA-independent focus formation in mixed culture withC3H/10T1/2 cells, anchorage independence and tumorigenicity.Analysis of UV-TDTx: C3H/10T1/2 and MCAC1 # 16/39:C3H/10T1/2somatic cell hybrids indicates that a similar percentage ofhybrids of each cell type are able to form foci in co-culturewith C3H/10T1/2 cells. However, focus-forming UV-TDTx:C3H/10T1/2hybrids remain dependent on TPA for focus formation in mixedculture.     

Prostaglandin endoperoxide synthetase-dependent cooxidation of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene in C3H/10T 1/2 clone 8 cells

(+/-)trans-7,8-Dihydroxy-7,8-dihydrobenzo(a)pyrene (BP-7,8-diol), the proximate form of the carcinogen benzo(a)pyrene, is cooxidized during the oxidation of arachidonic acid to prostaglandins by prostaglandin endoperoxide synthetase (PES). This enzyme can oxidize BP-7,8-diol to the reactive intermediate (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, which binds covalently to macromolecules, is mutagenic in bacterial test systems, and forms 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BP-tetrol) isomers. We have examined the cooxidation of BP-7,8-diol in an intact cell culture system of C3H/10T 1/2 clone 8 mouse embryo fibroblasts, in which both the mixed-function oxidase and PES systems are present. When BP-7,8-diol is incubated for 72 hr with approximately 10(6) confluent cells, high-performance liquid chromatography analysis of the organic extractable products reveals all four pairs of BP-tetrols, with those from (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene predominating. The addition of arachidonic acid (100 microM) produced a 2- to 3-fold increase in the formation of BP-tetrols from (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, while the metabolism of BP-tetrols from (+/-)-7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10 tetrahydrobenzo(a)pyrene was unchanged. The addition of the PES inhibitor indomethacin (100 microM) completely eliminated this stimulation. Cell transformation assays were carried out under the same conditions. The addition of arachidonic acid resulted in a 10-fold increase in foci formation, while indomethacin inhibited the increase in foci formation by 70%. These results suggest that cooxidation of BP-7,8-diol to reactive intermediates by PES can occur in an intact cell system if stimulated with arachidonic acid. In addition to mixed-function oxidase-dependent activation of carcinogens, the cooxidation of chemicals to reactive metabolites during prostaglandin biosynthesis may also play a role in carcinogenesis.     

Isolation of mutants temperature-sensitive for expression of the transformed state from chemically transformed C3H/10T1/2 cells

58 MCA Cl 16 is an oncogenic methylcholanthrene-transformed variant of the non-transformed mouse embryo fibroblast cell line, C3H/10T1/2 Cl 8. Using two different protocols, we have isolated six temperature-sensitive mutants from N-methyl-N'-nitro-N-nitrosoguanidine treated cultures of 58 MCA Cl 16. C3H/10T1/2 Cl 8, 58 MCA Cl 16 and the six mutant lines were characterized with respect to several properties associated with the transformed state: morphology, saturation density, anchorage independence, cell surface morphology and growth in medium containing 1% fetal calf serum. In general, C3H/10T1/2 cells behaved as non-transformed, whether grown at 33 degrees C or 39.5 degrees C. The transformed parental line and all six mutants behaved as transformed cells at 33 degrees C. At 39.5 degrees C, only the parental transformed line retained the transformed phenotype. Three of the mutants revert towards non-transformed behavior at 39.5 degrees C for all of the properties tested. The remaining mutants are temperature-sensitive for some, but not all, transformed characteristics. Thus, while the expression of these transformed properties is sometimes coupled, we have been able to dissociate the expression of traits such as saturation density, anchorage independence and transformed morphology from each other. These mutants should prove to be valuable tools in the study of the mechanisms which underly the expression of the chemically-induced transformed state.     

Co-cultured transformed and untransformed C3H 10T1/2 cells: preferential killing of transformed cells by low dose rate irradiation

Co-cultured C3H 10T1/2 cells, in which transformed cells were grown as discrete colonies on top of density-inhibited monolayers of untransformed cells, were used to determine the potential usefulness of a short term assay system for the study of differential radiation effects as they may apply to cell populations with differing turnover rates, but in close physical contact. Mixed cultures were exposed to either an acute dose of 20 Gy of Cs-137 gamma rays, or a dose of 72 Gy delivered at a low dose rate of 0.34 Gy per hour. These treatments resulted in approximately equal levels of damage to the untransformed monolayers. At 10-day intervals after treatment, representative flasks from each dose group were examined for evidence of degeneration, and subsequent regrowth, if any, of the transformed colonies and untransformed monolayers. For comparable amounts of visible damage to the untransformed monolayers, the low dose rate irradiation was more effective at delaying regrowth of, or even eradicating, transformed colonies. These results are consistent with expectations based on previous results in which dose-rate or dose fractionation isoeffect curves were compared for these two cell types, grown independently in plateau phase cultures.     

Oncogene-mediated multistep transformation of C3H10T1/2 cells

We have examined the response of the mouse embryonic cell line C3H10T1/2 to transfection with the activated human c-H-ras oncogene and the gag-myc oncogene from avian myelocytomatosis virus 29. C3H10T1/2 cells are not morphologically transformed following transfection with the gag-myc oncogene. A low level of focus formation is observed following transfection of the c-H-ras oncogene. When C3H10T1/2 cells are cotransfected with the ras and myc oncogenes, focus formation is increased by an average of 13 fold. In addition, C3H10T1/2 ras/myc foci have a distinct, transformed morphology which correlates with an increased potential for anchorage-independent growth. Although morphological transformation in this system is largely a function of ras oncogene expression, our studies demonstrate that it is potentiated by the presence of a functional gag-myc protein. Oncogene-mediated multistep transformation, which was first described in primary embryo cultures, is not a general property of established cell lines. The C3H10T1/2 cell line is an exception and provides a model system in which partially transformed phenotypes, in a progression toward malignant transformation, can be isolated and studied.     

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.     

Induction of neoplastic transformation and DNA single-strand breaks in C3H/10T1/2 clone 8 cells by polycyclic hydrocarbons and alkylating agents

The standard C3H/10T1/2 clone 8 (C3H/10T1/2 CL8) cell transformation assay was tested for its ability to identify a variety of polycyclic hydrocarbons and alkylating agents. Dose-dependent morphologic transformation occurred with benzo[a]pyrene (BaP), 3-methylcholanthrene (MCA), 7,12-dimethylbenz[a]anthracene, BaP-7,8-dihydroxy-7,8-dihydrodiol (BaP-7,8-diol), as well as with the relatively weak in vivo carcinogen benzo[e]pyrene. Dibenz[a,h]anthracene yielded a relatively weak response, whereas anthracene and phenanthrene were negative. In contrast, treatment of C3H/10T1/2 CL8 cells with two directly acting alkylating agents, N-nitroso-N-methylnitroguanidine (MNNG) and styrene oxide, gave no transformation, whereas a third alkylating agent, ethyl methanesulfonate (EMS), gave a weak response. Treatment with MCA (2.5 micrograms/ml) yielded a reproducible positive response and, therefore, served as a positive control for routine use of the C3H/10T1/2 CL8 assay. When cells treated with the hydrocarbons BaP, BaP-7,8-diol, or MCA were analyzed for nonspecific DNA damage (single-strand breaks or alkaline-labile sites) by alkaline elution techniques, little if any DNA damage was observed. In contrast, the alkylating agents MNNG, styrene oxide, and EMS yielded substantial numbers of single-strand breaks.     

Equivalent expression of endogenous murine leukemia virus-related genes in C3H/10T1/2 cells and chemically transformed derivative cells.

The possibility that chemical carcinogens may induce enhanced expression of endogenous C-type RNA tumor virus genes in the absence of intact virus particle production has been partially tested in a model system. Thie was accomplished by measuring the abundance and diversity of murine leukemia virus-related RNA sequences associated with the polyribosome fraction of nontransformed C3H/10T1/2 clone 8 cells and a 3-methylcholanthrene-transformed derivative clone. Although both clones are virus nonproducers, they were found to contain significant amounts of polyadenylate-containing murine leukemia virus-related RNA sequences; however, both the types and quantities of such sequences appear indistinguishable in both clones. These results suggest that expression of the corresponding gene sequences into RNA is not related to the maintenance of the transformed state in these chemically transformed cells.     

60 Hz magnetic field acts as co-promoter in focus formation of C3H/10T1/2 cells

Disruption of communication between transformed cells and normalcells is involved in tumor promotion. We have tested the hypothesisthat 60 Hz electromagnetic (EM) field exposures and a chemicaltumor promoter, 12-O-tetra-decanoylphorbol-13-acetate (TPA)are co-promoters that enhance focus formation of transformedcells in co-culture with normal cells. EM field exposures alonedid not affect the growth curves of parental C3H/10T1/2 fibroblastsor daughter mutant cells, UV-TDTx10e. Furthermore, EM fieldexposures alone did not promote focus formation of mutant cellsin co-culture with parental cells under the conditions tested.However, EM field exposures co-promoted with TPA by increasingfocus formation in co-culture. Cell cultures were exposed toan EM field in custom-built solenoidal incubators. The fieldexposures were 1.0 Gauss in a schedule of 1 h epochs four timesdaily for 28 days. Video image analysis of three independentexperiments showed that field-exposed cultures produced 1.9-foldmore foci than sham-exposed cultures when treated with TPA.The total area of foci per dish increased 2.2-fold and the numberof cells in stained foci increased 2.3-fold. In a TPA dose-response,focus formation began at 3 ng/ml with no difference betweenfield-exposed and sham-exposed co-cultures. However, at theTPA concentrations of 10, 20, 40, 50 and 100 ng/ml EM fieldexposures enhanced focus formation by an average of 150%. Thisstudy suggests that chronic intermittent exposures to a 60 HzEM field and a chemical tumor promoter influenced membrane-relatedevents by co-promoting focus formation.     

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.     

Modulation of cellular interactions between C3H/10T1/2 cells and their transformed counterparts by phosphodiesterase inhibitors.

It has been demonstrated previously that nontransformed C3H/10T1/2CL8 mouse embryo fibroblasts (10T1/2) can induce a state of reversible growth inhibition in cocultured malignantly transformed mouse fibroblasts and that this inhibition is modulated by serum concentration. The present study suggests that cyclic nucleotides may be implicated in this intercellular communication. The phosphodiesterase inhibitors theophylline, caffeine, and 3-isobutyl-1-methylxanthine (IBX) at concentrations of 10(-3) M, maintained continuously, were all found to inhibit the expression of 3-methylcholanthrene-induced malignant transformation when added 7 days after removal of carcinogen. IBX was the most potent, causing 100% inhibition at 10(-4) M and 70% inhibition at 10(-5) M. This inhibition was partially reversible in the former case and completely reversible in the latter case by removal of drug. Complete inhibition by 10(-4) M IBX was still observed when treatment was delayed 21 days postcarcinogen. In reconstruction experiments, utilizing confluent monolayers of 10T1/2 cells overlaid with transformed cells, IBX caused a dose-dependent inhibition of colony size of the transformed cells. Adenosine cyclic 2':3'-monophosphoric acid (cAMP) and N6,O2'-dibutyryladenosine cyclic 3':5'-monophophoric acid potentiated this response. The presence of non-transformed 10T1/2 cells was required for this effect, since a concentration of IBX (10(-4) M) inhibitory for the growth of transformed cells in mixed cultures was without effect on the growth rate, plating efficiency, or saturation density of pure cultures of 10T1/2 cells or of their transformed counterparts. Conditioned medium removed from IBX-treated 10T1/2 cells was not growth inhibitory for transformed cells, indicating a requirement for cell-cell contact. IBX caused a dose-dependent increase in intracellular cAMP in confluent 10T1/2 cells and a more pronounced increase in cAMP concentration in the culture medium of these cells. The dose-response effects of IBX on growth inhibition of malignant cells in mixed cultures appear to correlate well with its ability to elevate cAMP levels. Thus, IBX increased the capacity of 10T1/2 cells to cause reversible growth arrest of transformed cells and appears to act in a manner analogous to the previously reported effects of serum.