Response of cultured rat liver epithelial cell lines to tumour-promoting phorbol esters

The membrane effects of a potent tumour promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA), were studied in a series of cultured rat liver epithelial cell lines. Treatment with TPA resulted in the formation of strand-like aggregates (ridges) of viable cells over the monolayer of IAR 6-1 cells, but not of three other cell lines tested (IAR 20, IAR 6, IAR 6-7). The morphological response of IAR 6-1 to TPA was investigated by determination of phorbol ester receptors, analysis of cellular fucoproteins, surface galactoproteins and iodinatable surface proteins, and specific immunofluorescence for components of the extracellular matrix (fibronectin, laminin-entactin, procollagen type III). A class of specific, saturable, high-affinity receptors for phorbol esters was demonstrated in all four cell lines employing a conventional [20(-3)H]phorbol-12,13-dibutyrate ([3H]PDBu)-binding assay. The dissociation constants were similar in four lines, but the number of receptors per cell in IAR 6-1 cells was about twice that in other lines. Down-regulation of receptors was demonstrated in IAR 20 and IAR 6-1 cells with similar characteristics. Iodinatable surface proteins and galactose-containing surface glycoproteins did not respond to TPA. The distribution of fibronectin, laminin-entactin and procollagen type III was not affected by TPA. A TPA-responsive cell line, IAR 6-1, contained considerably less laminin-entactin than did the other lines. TPA had no influence on metabolic labelling of [3H]fucose-containing cellular glycoprotein in IAR 6-1 cells. One specific protein, with molecular mass of 78 kD, was more heavily labelled with [3H]fucose in IAR 6-1 cells than in the other cell lines. Taken together, the results of this study show that the responsive cells (IAR 6-1) differed from non-responsive ones in having more phorbol ester receptors, increased fucosylation of a specific glycoprotein and decreased deposition of laminin-entactin in the extracellular matrix. These surface properties of IAR 6-1 cells may contribute to their ability to respond to TPA.     

An activator of protein kinase C inhibits gap junction communication between cultured bovine lens cells.

Currently little is known about the regulation of gap junction communication in the lens. We report here on the effects of the protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), on cultured bovine lens cells which appeared to be epithelial in nature. Dramatically reduced intercellular transfer of the fluorescent dye Lucifer yellow was observed when the cultured lens cells were treated with octanol, a known inhibitor of gap junction communication. TPA (4 beta isomer) was also shown to reduce intercellular permeability within these cultures. In contrast, an inactive form of TPA, 4 alpha-TPA, did not decrease dye transfer. Permeability was evaluated in terms of both the number of cells receiving dye and the rate of decrease in fluorescence intensity in the injected cell. The maximum decreases in dye transfer occurred at 2 h of TPA treatment and dye transfer gradually increased to control levels over a time course of many hours. Incubation of cultures with 32Pi and immunoprecipitation using antibodies to the N- and C-terminal regions of connexin43 demonstrated a gap junction phosphoprotein of 43,000 Da. Phosphorylation of connexin43 increased during the first 2 h of TPA treatment. These results suggest that protein kinase C has a direct or indirect effect on gap junction communication in cultured lens cells.     

Disorders of the actin cytoskeleton in transformed epithelial cells

The actin cytoskeleton of 8 transformed epithelial cell lines was studied using electron microscopy of platinum replicas. Seven of these lines belonged to the IAR series of rat liver epithelial cells, being at different stages of neoplastic progression. One cell line (FBT) was derived from the epithelium of bovine fetal trachea. The extent of actin cytoskeleton alteration in cell lines studied has been shown to correlate with other signs of neoplastic transformation. Among various actin-containing cell structures (microfilament bundles, actin meshwork at active edges, cell-cell adherence junctions, and endoplasmic microfilament sheath) the latter was the most sensitive to transformation. The loosening of the sheath and the alteration of its fine structure were observed in all the cell lines. The degree of these changes increased in the following order: FBT; non-tumorigenic IAR lines; IAR lines transformed in vitro; IAR lines obtained from the latter by single or double selection in vivo. The alteration of sheath was the only disturbance of actin cytoskeleton in FBT cells, whereas in other groups of epithelial cell lines some other changes occurred. These involved disruption of actin-containing intercellular junctions, the cell polarization accompanied by progressive shortening of length of the cell active edge containing actin meshwork, and disappearance or reorganization of microfilament bundles.     

Control of Intracellular Localization and Function of Cx43 by SEMA3F

Connexin genes are considered to form a family of tumor-suppressor genes. However, the mechanism of connexin-mediated growth control is not well understood. We now provide several lines of evidence which suggest that SEMA3F, a member of the class 3 semaphorin family, which is also reported to be a tumor suppressor, controls the intracellular localization and function of connexin 43 (Cx43). We employed a series of rat liver epithelial cell lines, among which we previously found that the level of expression of malignant phenotypes (IAR20 < IAR27E < IAR6-1 < IAR27F) is inversely related to that of gap junctional intercellular communication (GJIC). When we immunostained SEMA3F and Cx43 in these cell lines, the extent of immunostaining in the plasma membrane of both proteins decreased in the order of IAR20 > IAR27E > IAR6-1 > IAR27F, suggesting a close relationship between Cx43 and SEMA3F. Further studies revealed a partial colocalization of SEMA3F and Cx43 in the plasma membrane of IAR20 cells. We also found that both SEMA3F and Cx43 moved from the cytoplasm to the plasma membrane in a mouse papilloma cell line when E-cadherin became functional after transferring the cells from low- to high-calcium conditions. When SEMA3F gene expression was inhibited by siRNA in IAR20 cells, Cx43 localization in the plasma membrane and GJIC ability were reduced. Moreover, we found that SEMA3F binds with the cytoplasmic loop domain of Cx43, employing the yeast two-hybrid complementation and screening assays. Taken together, these results strongly suggest that SEMA3F directly associates with Cx43 and controls its intracellular localization and function.     

Regulation of cell-cell communication in rat bone cells: the effect of phorbol esters.

The skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent inhibitor of gap junctional intercellular communication. In the present study, the inhibition of cell-cell communication by TPA has been investigated in primary bone cells from newborn rat calvaria, with an emphasis on the involvement of intracellular pH (pH(i)) and cytosolic calcium ([Ca(+2)](i)) in this process. The results show that TPA (5 x 10(-)(8) M) caused a complete inhibition of intercellular communication within 40-60 min. The intercellular communication was fully restored after overnight incubation in the presence of TPA. This effect was found to be associated with an elevation of pH(i). However, neither an increase of pH(i) alone nor exposure to TPA, under conditions preventing pH(i)-shift, were found to affect intercellular communication. It is suggested that the inhibition of intercellular communication, in the presence of TPA, depends on the pH(i)-shift itself rather than on the absolute value of pH(i). In addition, elevation of cytosolic calcium by ionomycin led to the termination of intercellular communication after 30 min. This inhibitory effect was abolished when the cells were incubated for overnight with TPA and then intracellular calcium was elevated by the addition of ionomycin. These results indicate that shift of pH(i) and the increase of intracellular calcium are involved in repression of intercellular communication by TPA.     

Transforming and carcinogenic potential of cadmium chloride in BALB/c-3T3 cells

A large number of workers are potentially exposed to cadmium during mining and processing. Therefore, there is a concern regarding the potential carcinogenic hazards of cadmium to exposed workers. Studies have been performed to determine if cadmium chloride (CdCl(2)) can induce morphological cell transformation, DNA from CdCl(2)-induced transformed cells can transform other mammalian cells, and the transformed cells induced by CdCl(2) can form tumors in nude mice. BALB/c-3T3 cells were treated with different concentrations of CdCl(2) for 72 h. The frequency of transformed foci from each treatment was determined after cells were cultured for 4 to 5 weeks. DNAs from five CdCl(2)-induced transformed cell lines were isolated and gene transfection assay was performed using NIH-3T3 cells. Non-transformed BALB/c-3T3 cells and cells from 10 transformed cell lines induced by CdCl(2) were injected into both axillary regions of nude mice. Mice were screened once a week for the appearance and size of tumors. CdCl(2) caused a statistically significant, concentration-related increase in the transformation frequency. DNA from all five CdCl(2)-induced transformed cell lines tested was found to induce varying degrees of transfection-mediated transformation in NIH-3T3 cells. All 10 CdCl(2)-induced transformed cell lines formed fibrosarcomas in nude mice within 39 days of inoculation. Within this time period, no tumors were found in nude mice injected with non-transformed BALB/c-3T3 cells. These results indicate that CdCl(2) is capable of inducing morphological cell transformation and that the transformed cells induced by CdCl(2) are potentially tumorigenic.     

Cell contact but not junctional communication (dye coupling) with biliary epithelial cells is required for hepatocytes to maintain differentiated functions

Specific differentiated gene expression and the morphology of adult rat hepatocytes can be maintained for as long as 8 weeks in vitro only when they are cultured in the presence of biliary epithelial cells; when primary hepatocytes are cultured alone, they lose these functions within 2 to 3 days. We obtained evidence suggesting that contact between hepatocytes and biliary epithelial cells is necessary for maintaining hepatocyte functions. We examined whether junctional communication between and among hepatocytes and biliary epithelial cells is required for long-term maintenance of hepatocyte functions, using a dye-transfer method, in three co-cultures: (1) hepatocytes and biliary epithelial cells prepared from Sprague-Dawley rats; (2) hepatocytes from Sprague-Dawley rats and epithelial cells of the IAR 20 line, originally established from BDVI rats; and (3) hepatocytes from BDVI rats and IAR 20 epithelial cells. The established epithelial cell line (IAR 20) and early-passage cultures of biliary epithelial cells maintained hepatocyte-specific functions in culture for 40 and 70 days, respectively, but the latter induced more stable maintenance of albumin secretion. Hepatocytes cultured alone lost their characteristic morphology within 5 to 8 days, and almost no dye transfer was observed. In co-cultures, the capacity of biliary epithelial cells to communicate among themselves remained relatively high throughout the culture period, whereas hepatocytes showed almost no junctional communication at an early phase of culture and first began to communicate after 2 weeks, communication capacity increasing for at least the next 10 days of culture. The most notable finding was that there was no dye transfer between hepatocytes and biliary epithelial cells in any co-culture system. These results suggest that the maintenance of hepatocyte-specific functions requires intercellular contact but probably not gap-junctional communication between hepatocytes and biliary epithelial cells. This system is useful for studying heterotypic cell-cell interactions and the control of gene expression.     

Effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), retinoic acid and diazepam on intercellular communication in a monolayer of rat liver epithelial cells

We have studied the influence of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), the vitamin A derivative retinoic acid and the benzodiazepine diazepam on intercellular communication via established gap junctions in a monolayer of rat liver epithelial cells (RLB) at various times of incubation. Intercellular communication was measured as the transfer of [3H]hypoxanthine-derived nucleotides between RLB hypoxanthine guanine phosphoribosyl transferase+ (HPRT+) and RLB HPRT- cells. TPA only showed transient inhibition of metabolic cooperation: after 4 h of treatment, intercellular communication was reduced to about 40% of the control and longer treatments showed progressively less effect until 24 h of treatment, when no difference was seen between TPA-treated and control preparations. Retinoic acid was a more effective inhibitor: both 3 X 10(-6) M applied for 24 h and 10(-4) M applied for 6.5 h, caused a 50% inhibition of label transfer. The junctional communication could only be blocked at very high concentrations (5 X 10(-4) M) in short-exposure experiments, but this is possibly a consequence of non-specific effects on the cell membrane. When the incubation time was 24 h, a considerable portion of the gap junctions appeared to persist in the 'open' state. Diazepam showed no significant inhibitory effect in the experiments performed.     

Ribosomal protein S6 phosphorylation and morphological changes in response to the tumour promoter 12-O-tetradecanoylphorbol 13-acetate in primary human tumour cells, established and transformed cell lines

The phosphorylation of ribosomal protein S6 in fibroblasts, primary human tumour cells, established and SV40-transformed human cell lines was compared after the addition of 12-O-tetradecanoylphorbol 13-acetate (TPA). In fibroblasts and primary tumour cell cultures, stimulation of S6 phosphorylation was about 4-6-fold. Established and transformed cell lines showed enhanced S6 phosphorylation which was not further stimulated by the addition of TPA. These findings indicated that the influence of TPA on the metabolic pathway, that finally leads to the phosphorylation of protein S6 in cells with a limited lifespan (fibroblasts, primary human tumour cells) can be mimicked by unknown steps also associated with immortalization (establishment function) and the transformed state of the tumour cells. Another interesting observation were morphological changes of the established and SV40-transformed cells which were visible as early as 20 min after the addition of TPA. In fibroblasts and primary tumour cells no changes in morphology were observed, even after 8h incubation.     

Density-dependent expression of keratins in transformed rat liver cell lines

Immunomorphological examination of the distribution of three keratins in cultured rat liver-derived epithelial cell lines of the IAR series was performed in order to find out the effects of neoplastic evolution on the expression of these epithelium-specific markers. Specific monoclonal antibodies were used to reveal various intermediate filament proteins: keratins with molecular masses of 55, 49 or 40 kD (K55, K49 or K40), and vimentin. The expression of keratins was negligible in sparse and dense cultures of non-transformed lines, which had typical epithelial morphology. The examined keratins were also absent in the sparse cultures of transformed lines, which have lost partially or completely the morphological features of epithelia. However, cells in dense cultures of most transformed lines contained numerous keratin filaments. It is suggested that the paradoxical increase of keratin expression after transformation is due to increased saturation density of transformed cultures; this high density favours the expression. As shown by the experiments with culture wounding, the effects of density are local and reversible. While K55 was present in all the cells of dense cultures, the expression of the other two keratins was dependent on the cell position within these cultures. It is suggested that the expression of the latter two keratins, besides high cell density, also requires the presence (K40) or the absence (K49) of cell-substratum contacts. Possible mechanisms of the effects of cell density on the expression of keratins are discussed.     

Genomic instability in silica- and cadmium chloride-transformed BALB/c-3T3 and tumor cell lines by random amplified polymorphic DNA analysis

Our earlier studies using random amplified polymorphic DNA (RAPD) analysis have shown genetic instability in human lung cancer tissues. Here we have investigated the potential for genetic instability in silica- and cadmium chloride (CdCl2)-transformed BALB/c-3T3 cell lines. Non-transformed, transformed BALB/c-3T3 cells, and tumor cell lines (obtained by injecting nude mice with transformed cell lines) were analyzed for genomic changes. DNAs from 10 different transformed clones and their corresponding tumor cell lines were amplified individually by RAPD analysis using 10 arbitrary primers. DNA from non-transformed BALB/c-3T3 cells was used as a control to compare genetic alterations, if any, between non-transformed, transformed and tumor cell populations. PCR products from RAPD were electrophoretically separated on agarose gels and the banding profiles were visualized by ethidium bromide staining. Five of the 10 primers tested revealed genomic changes in silica-transformed cell lines when compared to non-transformed BALB/c-3T3 cells. Comparison of all 10 transformed and tumor cell lines showed varied degrees of genomic changes using all 10 primers. CdCl2-transformed cell lines displayed fewer genomic changes, only three of 10 primers showed a positive result. CdCl2-transformed cells and their corresponding tumor cell lines showed specific banding pattern differences in six of the 10 samples tested with six of the 10 primers. Changes in band intensity were the most commonly observed changes both in silica- and CdCl2-transformed and tumor cell lines. The results seem to indicate a progressive change in genomic rearrangements which may directly or indirectly be associated with progression of tumorigenesis.     

Modifications of microfilaments and microtubules induced by two hepatic tumor promoters,phenobarbital and biliverdin in non-transformed and transformed hepatic cell lines

Microfilaments and microtubules are components of the cytoskeleton which could be implicated in neoplastic transformation. We studied the effect of two hepatic tumor promoters, phenobarbital (PB) and biliverdin (BV), on microfilaments and microtubules of non-transformed (Cl₃) and transformed (FV) hepatic epithelial cells. Cl₃ non-transformed cells cultured in the presence of 1 × 10^–6M BV for 48 h showed a loss of F-actin, fragmentation of actin and the appearance of star-like structures in the cytoplasm, as well as loosening of the peripheral bundle of actin, and some ruffling of cell membranes. In Cl₃ cells exposed to 0.2 × 10^–3M PB a similar disappearance of F-actin staining and a very prominent ruffling of cell membrane were observed. BV and PB also produced in these cells modifications of microtubules characterized by a disappearance of centrosome staining in numerous cells, a condensed ring of tubulin around the nucleus and a depolymerized aspect of the microtubular network. All these modifications of microfilaments and microtubules closely resembled those observed in FV transformed cells in the absence of any treatment (Solvent DMSO only). We did not observe an effect of BV and PB on FV cells.The present data demonstrate that the cytoskeleton of non-transformed epithelial liver cells is sensitive to the action of liver tumor promoters suggesting that it might play a role as to yet be defined in the promotion mechanism.Abbreviations PB phenobarbital - BV biliverdin - TPA 12-0-tetradecanoyl-phorbol 13 acetate - GGT gamma-glutamyl-transpeptidase - DMSO dimethylsulfoxyde     

Effect of thioridazine on gap junction intercellular communication in connexin 43-expressing cells

Propagation of electrical activity between myocytes in the heart requires gap junction channels, which contribute to coordinated conduction of the heartbeat. Some antipsychotic drugs, such as thioridazine and its active metabolite, mesoridazine, have known cardiac conduction side-effects, which have resulted in fatal or nearly fatal clinical consequences in patients. The physiological mechanisms responsible for these cardiac side-effects are unknown. We tested the effect of thioridazine and mesoridazine on gap junction-mediated intercellular communication between cells that express the major cardiac gap junction subtype connexin 43. Micromolar concentrations of thioridazine and mesoridazine inhibited gap junction-mediated intercellular communication between WB-F344 epithelial cells in a dose-dependent manner, as measured by fluorescent dye transfer. Kinetic analyses demonstrated that inhibition by 10 μmol/L thioridazine occurred within 5 min, achieved its maximal effect within 1 h, and was maintained for at least 24 h. Inhibition was reversible within 1 h upon removal of the drug. Western blot analysis of connexin 43 in a membrane-enriched fraction of WB-F344 cells treated with thioridazine revealed decreased amounts of unphosphorylated connexin 43, and appearance of a phosphorylated connexin 43 band that co-migrated with a “hyperphosphorylated” connexin 43 band present in TPA-inhibited cells. When tested for its effects on cardiomyocytes isolated from neonatal rats, thioridazine decreased fluorescent dye transfer between colonies of beating myocytes. Microinjection of individual cells with fluorescent dye also showed inhibition of dye transfer in thioridazine-treated cells compared to vehicle-treated cells. In addition, thioridazine, like TPA, inhibited rhythmic beating of myocytes within 15 min of application. In light of the fact that the thioridazine and mesoridazine concentrations used in these experiments are in the range of those used clinically in patients, our results suggest that inhibition of gap junction intercellular communication may be one factor contributing to the cardiac side-effects observed in some patients taking these medications.     

Cell and toxicant specific phosphorylation of conexin43: effects of lindane and TPA on rat myometrial and WB-F344 liver cell gap junctions

Previous studies showed that the pesticide lindane (gamma-hexachlorocyclohexane) inhibits gap junction intercellular communication in rat myometrial cells. The present study tested the hypothesis that lindane and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibit gap junction communication in rat myometrial and liver WBr-F344 cells by the common mechanism of increasing phosphorylation of the gap junction protein connexin43. We evaluated changes of connexin43 phosphorylation using Western blot of standard SDS-PAGE gels and cell immunostaining, and we monitored gap junction communication using microinjection and transfer of Lucifer yellow dye. Exposure of rat myometrial cells to lindane or TPA nearly abolished dye transfer but did not alter the electrophoretic mobility of connexin43, and neither lindane nor TPA increased phosphorylation of connexin43 as assessed by immunoblot with anti-phospho-connexin43 (S368) antibody. However, TPA increased punctate immunofluorescence staining of phospho-connexin43 (S368) in myometrial cells whereas lindane had no such effect. In WBr-F344 cells, lindane and TPA inhibited dye transfer. Lindane increased immunostaining for phospho-connexin43 (S368) in WBr-F344 cells without altering the abundance, electrophoretic mobility or phosphorylation of connexin43 as detected in immunoblots. TPA intensified a slower migrating connexin43 band and increased phospho-connexin43 (S368) in immunoblots, and intensified phospho-connexin43 immunostaining at WBr-F344 cell interfaces and nuclear regions. These results show that phosphorylation of connexin43 at serine 368 occurred in cell and toxicant specific manners and was independent of changes in electrophoretic mobility in standard SDS-PAGE gels. Moreover, lindane inhibited gap junction communication in myometrial cells by a mechanism that was not be explained by changes in phosphorylation of connexin43.     

Morphology,cell-cell interactions,and migratory activity of IAR-2 epithelial cells transformed with the <Emphasis Type="Italic">RAS</Emphasis> oncogene: Contribution of cell adhesion protein E-Cadherin

The destruction of stable cell-cell adhesion and the acquisition of the ability to migrate are consistent stages of neoplastic evolution of tumor cells of epithelial origin. We studied the morphological and migration characteristics of epithelial cells of IAR1162 and IAR1170 clones derived from a mixed culture of N-RasV12 oncogene-transformed IAR-2 cell line. It was found that the oncogenic RAS can cause two types of morphological changes in IAR-2 epithelial cells. Cells of one type (IAR1162 clones) underwent epithelial-mesenchymal transition: they stopped to express E-cadherin, acquired fibroblast-like morphology, and did not form tight junctions. Cells of the other type (IAR1170 clones) retained a morphology close to the morphology of nontransformed progenitor cells, assembled E-cadherin-based adherens junctions and tight junctions, and formed a monolayer in confluent culture. However, in both IAR1162 and IAR1170 cells, the oncogenic RAS caused the destruction of marginal actin bundle and the reorganization of cell-cell adherens junctions. RAS-transformed IAR1162 and IAR1170 epithelial cells acquired the ability to migrate on a flat substrate as well as through narrow pores in membranes of migration chambers. A videomicroscopic study of transformed epithelial cell cultures demonstrated the instability of cell-cell contacts and the independent nature of cell migration. IAR1170 epithelial cells, which had E-cadherin-based adherens junctions, were also able to move as a group (collective migration). 1162D3 cells, which lost the ability to express endogenous E-cadherin as a result of Ras-transformation, were transfected with a plasmid carrying the CDH1. As a result of transfection, clones of cells with different levels of expression of exogenous E-cadherin were obtained. The high level of expression of exogenous E-cadherin in transformed epithelial cells led to a decrease in the rate of migration on a two-dimensional substrate of the cells that were in contact with neighboring cells but almost had no effect on the migration of single cells, at the same time increasing the number of cells that migrated through the pores in migration chambers. Thus, the destruction of marginal actin bundle and the change in the spatial organization of cell-cell adherens junctions, irrespective of the presence or absence of E-cadherin, was accompanied by destruction of stable cell-cell adhesion and the appearance of cell motility in Ras-transformed epithelial cells. The retaining of E-cadherin in cell-cell adhesion junctions affects the motility of transformed epithelial cells and plays an important role in their collective migration.     

Antimetabolic activity of L-ascorbic acid in human and animal tumors

1. The effect of high concentrations of L-ascorbic acid on the growth of some human and animal transformed and non-transformed cell lines has been investigated. Directly implemented into culture of transformed cell lines it decreased [3H]thymidine, [3H]uridine and [3H]leucine incorporation into cells. Vitamin C inhibited DNA synthesis by transformed cells 3-4 times more efficiently than by normal cells. 2. In vivo treatment of athymic nude mice bearing human mammary carcinoma with 500 mg/kg L-ascorbic acid for the first 15 days markedly inhibited the growth of tumor cells. 3. As determined by alkaline elution, both DNA strand breaks and DNA cross links were observed in mammary carcinoma cells treated with vitamin C. DNA-DNA and DNA-protein cross links in cells treated with L-ascorbic acid were revealed by the proteinase K assay. Removal of vitamin C caused an immediate onset of spontaneous repair of single or double stranded DNA breaks. If, however, vitamin was reintroduced into cell culture, this spontaneous repair was reversed. 4. Our results indicate an antimetabolic activity of L-ascorbic acid in human and animal transformed cells, probably due to lethal damages in DNA.     

Establishment of two rabbit mammary epithelial cell lines with distinct oncogenic potential and differentiated phenotype after microinjection of transforming genes.

The goal of this work was to establish an assay for transformation of epithelial cells. Two epithelial cell lines were obtained after microinjecting transforming genes into primary rabbit mammary secretory cells. The cell lines were analyzed for their oncogenic potential and for the maintenance of a differentiated phenotype. A fully transformed cell line, which retained epithelial cell organization, was obtained by coinjecting simian virus 40 DNA and the activated human c-Ha-ras gene. The proliferation rate of these cells was high, with a doubling time of 16 h. Their growth was anchorage independent, and they had lost contact inhibition. The cells were tumorigenic in nude mice, but had no metastatic potential. Both microinjected DNAs were efficiently transcribed and translated, in contrast to the casein genes, which were expressed in primary cells but not in the transformed cell line. An immortalized cell line established after injection with simian virus 40 DNA alone was characterized by a moderate rate of proliferation with a doubling time of approximately 30 h. The growth of these cells was contact inhibited and anchorage dependent. The cells were not tumorigenic in nude mice. The viral DNA was expressed during early passages, as shown by the presence of the large T antigen in cell nuclei, but not at later passages. A high number of lactogenic hormone receptors were found associated with the cell surface. Despite the presence of these receptors, no induction of genes coding for milk proteins was observed after addition of prolactin. These data demonstrate that this assay system can be used to assess the immortalizing and transforming potential of candidate oncogenes in epithelial cells.