ATP-resistant variants of transformed mouse fibroblasts.

Addition of ATP to cultures of transformed mouse fibroblasts, 3T6 cells, resulted in cell growth inhibition, whereas the growth of the non-transformed counterparts, 3T3 cells, was only slightly affected. The inhibition was found to be specific for adenine nucleotides, and concentration dependent. At relatively low concentrations (e.g., 1.0 mM) the effect of ATP was cytostatic, whereas at higher concentrations (e.g., 1.0 mM) a cytotoxic effect was exerted. ATP-resistant variants of 3T6 cells were selected by exposure of cultures to gradually elevated concentrations of ATP. The variants were found to resemble the non-transformed counterparts, 3T3 cells, more than the 3T6 parent cells, by the following criteria: ATP-induced alterations in the membrane potential, changes in membrane permeability, cell growth inhibition, and colony formation on soft agar. The data indicate that long exposure of the transformed cells to external ATP results in redifferentiation and reduction in their tumorigenicity.

Modulation of platelet-derived growth factor receptor function in BP3T3, a chemically transformed BALB/c-3T3 cell line.

BP3T3, a clonal benzo(a)pyrene-transformed BALB/c-3T3 cell, has been shown to be conditionally responsive to platelet-derived growth factor (PDGF)-stimulated DNA synthesis. PDGF stimulates DNA synthesis in BP3T3 cell cultures maintained in 0.5% platelet-poor plasma, but pretreatment with 10% serum or 10 micrograms/ml insulin inhibits PDGF-modulated DNA synthesis. BALB/c-3T3 cells remain mitogenically responsive irrespective of pretreatment with serum or insulin. The present study demonstrates that pretreatment with serum or insulin inhibits BP3T3 cell DNA synthesis by affecting receptor function. Insulin and serum, however, act through different mechanisms. Pretreatment with serum for 3 or more days down-modulated the BP3T3 cell PDGF receptor, resulting in both inhibition of PDGF binding and inhibition of PDGF-stimulated receptor autophosphorylation. In contrast, treatment of nontransformed BALB/c-3T3 cells with serum for 3 or more days did not down-modulate the PDGF receptor. Pretreatment of BP3T3 cells with insulin did not inhibit PDGF binding to BP3T3 cells but did inhibit PDGF-stimulatable tyrosine-specific receptor autophosphorylation. This effect was minimal to nonexistent in BALB/c-3T3 cell cultures. It appears likely that pretreatment of BP3T3 cells with insulin either inhibits the tyrosine kinase activity of the PDGF receptor or activates receptor dephosphorylation.

Regulation of transforming growth factor alpha messenger RNA expression in a chemically transformed rat hepatic epithelial cell line by phorbol ester and hormones.

Transforming growth factor alpha (TGF-alpha) is produced by many transformed cells, but little is known about the regulation of its expression. We examined TGF-alpha mRNA levels in a set of cloned neoplastic cell lines derived by chemical transformation of a normal rat liver epithelial cell. The untransformed parental cell line, WB-344, did not express a detectable level of TGF-alpha mRNA, whereas GP6ac, a transformed line capable of autonomous growth in soft agar, expressed TGF-alpha. When GP6ac cells were treated with agents thought to regulate protein kinase C activity, e.g., the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), TGF-alpha mRNA levels increased by 8- to 11-fold. The induction of TGF-alpha mRNA was detectable at 2 h, was maximal at 8-12 h, and declined by 24 h. Angiotensin, bradykinin, epinephrine, and epidermal growth factor also increased TGF-alpha mRNA by 2- to 5-fold. In contrast, parental WB cells neither expressed TGF-alpha mRNA, nor responded to TPA. TPA also increased epidermal growth factor receptor mRNA in GP6ac cells but the effect was less prolonged; maximal levels were seen at 4 h after TPA exposure and returned to control levels by 12 h. TPA increased TGF-alpha mRNA in GP6ac cells, in part, by increasing transcription of the TGF-alpha gene as measured by run-on transcription rates in isolated nuclei. In addition, the induction of TGF-alpha by TPA was blocked by concurrent incubation with agents that inhibit protein synthesis. However, if TPA was present for at least 2 h, subsequent addition of cycloheximide enhanced the effect of TPA. This indicates that the induction of TGF-alpha in GP6ac cells is comprised of at least two phases demarcated by the requirement for protein synthesis. The time course of induction and the sensitivity to inhibition of protein synthesis distinguish the effect of TPA on TGF-alpha mRNA from that of other genes regulated by TPA, e.g., c-myc and c-fos. These data also suggest that chemical transformation of rat liver epithelial cells leads to expression of TGF-alpha mRNA, and that once expressed, TGF-alpha mRNA can be modulated in a protein kinase C-dependent manner.

Loss in transformed cells of cell cycle regulation of expression of a nuclear protein recognized by SLE patient antisera.

The identification of cellular proteins involved in the control of cell proliferation in normal cells is essential for understanding the mechanism underlying growth regulation and cellular transformation. A nuclear protein termed Ki antigen with a relative mobility of 32,000 (Mr 32K) and which is recognized by SLE patient antisera has been identified in cells of human, bovine, and murine origin. Recently, cDNA clones for the bovine and human Ki antigens have been isolated using SLE patient antisera (T. Nikaido, et al., in preparation). The nucleotide sequence predicted a protein of 239 amino acids with a possible nuclear localization signal resembling that identified in SV40 T antigen and other nuclear proteins. Here we show that the expression of Ki antigen is regulated in the normal cell, but not in the transformed cell. Furthermore, in the K-ras temperature-sensitive mutant cell line, ts 371 normal rat kidney (NRK), Ki antigen expression increases several-fold at the permissive temperature relative to the nonpermissive temperature. These results suggest that expression of Ki antigen might be correlated with cellular transformation as well as with cell growth regulation.

Involvement of vimentin in the reverse transformation reaction.

An organized cytoskeleton is required for the cAMP-induced reverse transformation reaction in CHO-K1 cells. In the course of the reaction a considerable fraction of the genome changes its nuclease sensitivity. The current paper presents the following evidence that cAMP-induced phosphorylation of vimentin is an early step in this reaction complex. (i) Vimentin is only slightly phosphorylated in transformed CHO-K1 cells but is heavily phosphorylated in normal fibroblasts. (ii) cAMP addition almost triples the vimentin phosphorylation of CHO-K1 cells but does not change that of normal cells. (iii) Vimentin phosphorylation is one of the earliest phenomena to occur after addition of cAMP to CHO-K1 cells, preceding the cell-stretching reaction and other manifestations of reverse transformation. (iv) Indirect immunofluorescence experiments demonstrate that vimentin appears as a condensed mass in transformed CHO-K1 cells but cAMP addition restores the filamentous structure characteristic of the normal fibroblast. (v) Other transformed cells unresponsive to reverse transformation by cAMP failed to demonstrate increased phosphorylation of vimentin on treatment with cAMP. These results support the proposed scheme that phosphorylation of cytoskeletal elements initiates a large-scale genetic regulatory action in which a substantial change in the spectrum of genome exposure and sequestration occurs. A function for intermediate filaments in reverse transformation is implied.

Novel tyrosine phosphorylations accompany the activation of pp60c-src during chemical carcinogenesis.

Treatment of normal Syrian hamster embryo (SHE) cells in vitro with various chemical carcinogens results in transformed preneoplastic cell lines. Continued passage of these preneoplastic cells gives rise to rare variant cells with enhanced capacity for tumorigenic growth. We have previously shown that tumor-derived SHE cell lines contain an activated proto-oncogene product, pp60c-src. Here we demonstrate that tumor-derived SHE cell lines contain several novel tyrosine phosphoproteins in addition to those found in preneoplastic parent cell lines. A correlation was observed between the activation of endogenous pp60c-src tyrosine kinase specific activity and the presence of new phosphotyrosine-containing proteins. Tyrosine phosphoproteins of approximate Mr 81 kilodaltons (kDa), 55 kDa, and 39 kDa were noted in different tumor-derived cell lines. The 81 kDa and 55 kDa proteins were membrane-associated phosphoproteins, whereas the 39 kDa protein was predominantly cytosolic. Additional signature tyrosine phosphoproteins in individual tumor-derived cell lines apparently were unique to the particular inducing carcinogen or target cell. These studies indicate that during chemical carcinogenesis, activation of the tyrosine kinase proto-oncogene protein pp60c-src coincides with the appearance of novel tyrosine phosphorylations.

Activation of the cellular proto-oncogene product p21Ras by addition of a myristylation signal.

The 21-kD proteins encoded by ras oncogenes (p21Ras) are modified covalently by a palmitate attached to a cysteine residue near the carboxyl terminus. Changing cysteine at position 186 to serine in oncogenic forms produces a nonpalmitylated protein that fails to associate with membranes and does not transform NIH 3T3 cells. Nonpalmitylated p21Ras derivatives were constructed that contained myristic acid at their amino termini to determine if a different form of lipid modification could restore either membrane association or transforming activity. An activated p21Ras, altered in this way, exhibited both efficient membrane association and full transforming activity. Surprisingly, myristylated forms of normal cellular Ras were also transforming. This demonstrates that Ras must bind to membranes in order to transmit a signal for transformation, but that either myristate or palmitate can perform this role. However, the normal function of cellular Ras is diverted to transformation by myristate and therefore must be regulated ordinarily by some unique property of palmitate that myristate does not mimic. Myristylation thus represents a novel mechanism by which Ras can become transforming.

Analysis of mammalian fibroblast transformation by normal and mutated human EGF receptors.

Activation of the EGF receptor (c-erbB) tyrosine kinase has been implicated in tumorigenesis, either by overexpression of the normal receptor in the presence of EGF, or through expression of a truncated receptor lacking the EGF binding domain as in the viral oncogene v-erbB. Here, normal and truncated human EGF receptors expressed in Rat1 fibroblasts were analysed for receptor tyrosine kinase activity and several transformation parameters in comparison with polyoma middle T and EJ-ras. Expression of a truncated EGF receptor lacking the extracellular ligand binding domain induced transformation of immortalized rodent fibroblasts and appears to activate the intrinsic tyrosine kinase. The transformed phenotype becomes enhanced by further truncation of the C-terminal domain containing the tyrosine autophosphorylation sites P1 and P2. Over expression of EGF receptors with an intact extracellular region in transfected Rat1 cells shows EGF dependent transformation, which is reduced by C-terminal truncation. Transformation is dependent on the cellular receptor concentration and can be selected as a stable phenotype. We conclude that expression of receptors with a truncated EGF-binding domain alone is sufficient to transform mammalian fibroblasts, in contrast to chick fibroblasts transformed by v-erbB where additional deletion of C-terminal receptor sequences appears to be an absolute requirement.

Sensitization of human keratinocytes to killing by parvovirus H-1 takes place during their malignant transformation but does not require them to be tumorigenic.

To investigate the antineoplastic activity of parvoviruses, proliferating normal human epidermal cells and a series of established keratinocyte cell lines derived from squamous cell carcinomas or transformed in vitro, were compared for the outcome of H-1 virus infection. All established keratinocyte cell lines were more sensitive to killing by H-1 virus than normal epidermal cells, although to varying extents. Using a step-wise procedure for malignant transformation in vitro, we found that sensitization of transformed epidermal cells to H-1 virus can be dissociated from the acquisition of a tumorigenic phenotype. Thus, spontaneously- or SV40-immortalized human keratinocytes were moderately and highly sensitive to H-1 virus, respectively, and could be made tumorigenic by Harvey-ras oncogene transfection without a major change in their susceptibility to the virus. The capacity of human keratinocytes for replicating and expressing H-1 virus DNA appears to be a revealer of cellular alterations that take place in at least some pathways to malignant transformation but that may be insufficient to confer a tumorigenic potential.

Regulation of ornithine decarboxylase gene expression in normal and transformed hamster embryo fibroblasts following stimulation by 12-O-tetradecanoylphorbol-13-acetate.

We have compared the regulation of ornithine decarboxylase (ODC) gene expression in primary cultures of hamster embryo fibroblasts and in two independently transformed hamster embryo cell lines. Previous studies have demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA) can greatly potentiate the serum growth factor induction of ODC enzyme activity in transformed cells, but not in normal hamster embryo fibroblasts. Treatment of either normal or transformed cells with both TPA and serum yielded greater accumulations of ODC mRNA than with either treatment alone, which is consistent with changes at the protein level. However, treatment of the transformed cells with TPA and serum resulted in a greater increase in steady state levels of ODC mRNA than that observed using normal fibroblasts. The time course for the induction of ODC mRNA was similar for both normal and transformed cells with maximal accumulations 4-8 h after treatment. Studies with actinomycin D further suggests that ODC mRNA is comparatively long-lived in both normal and transformed cells. The accumulation of ODC mRNA after stimulation with TPA and serum is blocked by cycloheximide in normal hamster fibroblasts suggesting that this induction is dependent upon protein synthesis. In contrast, cycloheximide did not affect the accumulation of ODC mRNA under similar treatment conditions in transformed cells. This altered regulation of ODC gene expression in transformed hamster embryo fibroblasts cannot be explained by either gene rearrangement or the amplification of an ODC gene. These data suggest that transformation of hamster embryo cells results in a loss of cellular control over ODC gene regulation which includes an alteration in the requirement for protein synthesis for ODC mRNA accumulation.

Effect of growth state, tumor promoters, and transformation upon intercellular communication between C3H/10T1/2 murine fibroblasts.

Diminished intercellular communication has been associated with heightened sensitivity of cultured cells to morphological transformation and enhancement of transformation by tumor promoters. Microinjection of Lucifer yellow dye was employed to evaluate intercellular communication between transformable C3H/10T1/2 murine fibroblasts under a variety of culture conditions. Intercellular communication assayed by dye transfer from injected cells to surrounding cells in contact occurred in logarithmically growing cultures, declined to very low levels as confluence was attained, and then resumed upon the formation of mature confluent monolayers. Dye-transfer networks of 50 or more cells resulted from injection of single monolayer cells. Freeze-fracture electron microscopy confirmed the presence of gap junction structures in confluent cultures. Treatment with the initiating agent N-methyl-N'-nitro-N-nitrosoguanidine and/or the tumor promoter 2,3,7,8-tetrachlorodibenzo-p-dioxin did not inhibit intercellular communication between C3H/10T1/2 cells during 6-week transformation experiments. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate produced a transient inhibition of dye-coupling upon first introduction to cultures and prolonged the period of diminished dye-coupling at the attainment of confluence, but did not inhibit subsequent interactions between monolayer cells. A simple relationship thus could not be established between levels of dye-coupling within monolayers and focus formation events. Curiously, although the cells of foci in early phases of development did not exhibit dye-transfer capacity, dye-coupling was observed in mass cultures of most transformed cell lines cloned from foci. Co-cultivation of communication-competent transformed cells with nontransformed cells to produce reconstructed foci generally resulted in a cessation of dye-transfer by transformed cells. An often reversible loss of communication competence thus accompanies the growth of transformed C3H/10T1/2 cells as foci and may constitute an adaptive response which facilitates focus growth in the presence of intercellular communication between monolayer cells.

Desensitization of the Ca2+-mobilizing system to serum growth factors by Ha-ras and v-mos.

An elevation of the intracellular pH and a rise in the cytoplasmic Ca2+ concentration are considered important mitogenic signals which are observed after stimulation by various growth factors. In a preceding report it was demonstrated that the expression of Ha-ras or v-mos in cells transfected with Ha-ras or v-mos, respectively, leads to an activation of the Na+/H+ antiporter and a concomitant rise in intracellular pH (W. Doppler, R. Jaggi, and B. Groner, Gene 54:145-151, 1987). This report describes the effect of the Ha-ras and v-mos oncogenes on intracellular Ca2+ release. The expression of Ha-ras in NIH 3T3 cells carrying a glucocorticoid-inducible transforming Ha-ras gene caused a desensitization of the Ca2+-mobilizing system to serum growth factors. The induction of p21ras in cells carrying the corresponding glucocorticoid-inducible proto-oncogene did not affect the Ca2+ response to growth factors. Conditions leading to the expression of the transforming Ha-ras gene but not those causing the induction of the normal Ha-ras gene yielded an increase in phosphatidylinositol turnover and a concomitant rise in inositol phosphates. Results similar to those obtained with the transforming Ha-ras gene were seen after the expression of v-mos. The data are consistent with a mechanism in which expression of the transforming Ha-ras gene leads to a release of Ca2+ from intracellular stores via elevated levels of inositol trisphosphate.

Subnuclear localization and antitransforming activity of N-myc:beta-galactosidase fusion proteins.

N-myc expression is under stage- and tissue-specific regulation in mammalian development, but its function is totally unknown. We sought agents to block N-myc activity in order to infer from the effect the possible function of N-myc in the apparently complex processes. As candidates for such agents, we tested fusion genes encoding N-myc:beta-galactosidase fusion proteins for their effects on the formation of transformed foci of rat embryo primary fibroblasts as the result of transfection with N-myc and activated H-ras. One of the gene constructs very efficiently antagonized N-myc activity, as assessed by its effect on focus formation, but did not appreciably affect cell viability. The product of this gene was not only targeted to the nucleus but also accumulated in subnuclear loci which may represent the sites where normal N-myc proteins reside. The occurrence of antagonistic effect at a low stoichiometric ratio suggested that the fusion protein gene competed with the N-myc gene in a fashion analogous to a dominant negative mutation.

Transforming activity of ras proteins translocated to the plasma membrane by a myristoylation sequence from the src gene product.

Ras p21 proteins exert their biological functions when associated to the inner surface of the plasma membrane. This association is mediated by a lipid molecule which is covalently attached to the protein by a thioester bond through a cysteine at residue 186, at the carboxy end of the molecule. Deletion or substitution of the critical Cys186 residue of the Harvey-ras protein leads to ras-p21 mutants lacking the ability to translocate to the membrane and devoid of transforming activity (Willumsen et al., 1984a, 1984b). We have been able to regenerate both localization to the plasma membrane as well as transforming activity of such mutant ras p21 proteins by fusion of the amino-terminal 15 residues of the v-p60src protein, responsible for the covalent binding of myristic acid and its membrane association. Thus, while translocation to the plasma membrane is necessary for function of the transforming Harvey-ras p21 protein, it appears to be independent of a specific membrane insertion mechanism.

An acid protease secreted by transformed cells interferes with antigen processing.

The major excreted protein of malignantly transformed mouse fibroblasts (MEP), which is the precursor to lysosomal cathepsin L, was used to study the effect of exogenous acid proteases on antigen processing. When MEP and native pigeon cytochrome c were added to Chinese hamster ovary (CHO) cells expressing transfected major histocompatability complex class II gene products, the antigen-specific T-cell hybridoma 2B4 did not respond to the antigen. MEP appears to destroy the antigen in an acid compartment of the presenting cell because: (a) MEP is only active as a protease under acid conditions; (b) mannose 6-phosphate inhibited the internalization of MEP and blocked its effect on antigen processing; (c) the destruction required the simultaneous entry of the antigen and MEP into the cells; and (d) cytochrome c fragment 66-104 which does not need to be processed stimulated 2B4 in the presence of MEP. These results support the hypothesis that antigen processing requires internalization of the antigen into an acidic compartment, and they provide a new model for the investigation of the contribution of acid proteases to the reduced immunocompetence of tumor-bearing animals.

Separation of immortalization and T24-ras oncogene cooperative functions of adenovirus E1a.

An adenovirus 2 E1a gene coding for a protein of 243 (243R) amino acids can efficiently immortalize primary rat kidney (BRK) cells and cooperate with the activated cellular ras oncogene (T24 ras). A mutant (47-0) of the 243R gene that maps between amino acid residues 47-50 within a region that is highly conserved among the various adenovirus serotypes was found to be severely defective in immortalization. Despite the defect in immortalization, mutant 47-0 had the ability to cooperate with T24 ras in oncogenic transformation. These results suggest that the immortalization and the oncogene cooperation functions of the 243R are separable. Our results further suggest that the requirement for a separate immortalization function can be circumvented by oncogenic transformation and that the immortalization of cells transformed by E1a and T24 ras may be a secondary consequence of transformation by these two oncogenes.

Structural and functional domains of the myb oncogene: requirements for nuclear transport, myeloid transformation, and colony formation.

The v-myb oncogene of avian myeloblastosis virus causes acute myelomonocytic leukemia in vivo and transforms only myeloid cells in vitro. Its product, p48v-myb, is a nuclear protein of unknown function. To determine structure-function relationships for this protein, we constructed a series of deletion mutants of v-myb, expressed them in retroviral vectors, and studied their biochemical and biological properties. We used these mutants to identify two separate domains of p48v-myb which had distinct roles in its accumulation in the cell nucleus. We showed that the viral sequences which normally encode both termini of p48v-myb were dispensible for transformation. In contrast, both copies of the highly conserved v-myb amino-terminal repeat were required for transformation. We also identified a carboxyl-terminal domain of p48v-myb which was required for the growth of v-myb-transformed myeloblasts in soft agar but not for morphological transformation.

Ligand activation of overexpressed epidermal growth factor receptors transforms NIH 3T3 mouse fibroblasts.

The cell surface receptor for the mitogenic peptide epidermal growth factor (EGF) is involved in control of normal cell growth and may play a role in the genesis of human neoplasia such as squamous carcinoma and glioblastoma. Soft-agar growth and focus-formation experiments with NIH 3T3 mouse fibroblasts transfected with an expression plasmid demonstrated the ligand-dependent transforming potential of the human EGF receptor without structural alterations. Activation of overexpressed normal receptor alone appears to be sufficient for transformation of NIH 3T3 cells in vitro.

Transformation/neodifferentiation of human skin fibroblasts by acute oncornaviruses and dexamethasone.

We demonstrated that the Kirsten murine sarcoma virus (KiMSV) and the Harvey murine sarcoma virus (HaMSV) converted human skin fibroblasts (HSF) into adipocytes. Adipocytic conversion of HSF by KiMSV and HaMSV was dependent on the presence of glucocorticosteroids. The Kirsten murine leukemia virus, the Harvey murine sarcoma [corrected] virus and the amphotropic helper virus (AP292) were ineffective by themselves. Balb murine sarcoma virus and Moloney murine sarcoma virus were, to a lesser degree, able to effect adipocytic conversion of HSF. In contrast, the feline sarcoma virus and the simian sarcoma virus did not cause this conversion. Together, the results suggest a role for certain oncogenes and glucocorticosteroids in the transformation/neodifferentiation of human cells.

Tyrosine phosphorylations in vivo associated with v-fms transformation.

The role of tyrosine-specific phosphorylation in v-fms-mediated transformation was examined by immunoblotting techniques together with a high-affinity antibody that is specific for phosphotyrosine. This antiphosphotyrosine antibody detected phosphorylated tyrosine residues on the gp140v-fms molecule, but not gP180v-fms or gp120v-fms, in v-fms-transformed cells. This antibody also identified a number of cellular proteins that were either newly phosphorylated on tyrosine residues or showed enhanced phosphorylation on tyrosine residues as a result of v-fms transformation. However, the substrates of the v-fms-induced tyrosine kinase activity were not the characterized pp60v-src substrates. The phosphorylation of some of these cellular proteins and of the gp140fms molecule was found to correlate with the ability of v-fms/c-fms hybrids to transform cells. In addition, immunoblotting with the phosphotyrosine antibody allowed a comparison to be made of the substrates phosphorylated on tyrosine residues in various transformed cell lines. This study indicates that the pattern of tyrosine phosphorylation in v-fms-transformed cells is strikingly similar to that in v-sis-transformed cells.