Expression of protein kinase C I in NIH 3T3 cells increases its growth response to specific activators

In order to investigate the effects of protein kinase C (PKC) expression on cellular growth and morphology, we established mouse fibroblast cell populations which expressed the rat pkc-γ gene under the control of a retroviral promoter. NIH 3T3 stable transfectants displayed a three-fold increase in total PKC levels. These cells appeared morphologically unaltered but exhibited a stronger mitogenic response to 12-O-tetradecanoylphorbol-13-acetate (TPA) and cardiolipin (CL) as well as enhanced growth in semisolid medium in the presence of TPA. Thus, at these enzyme levels, PKC conferred growth advantages to NIH 3T3 cells only in response to specific activators.     

The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate enhances the proliferative response of Balb/c-3T3 cells to hormonal growth factors.

Stimulation of Balb/c-3T3 cell growth by TPA requires factors found in serum. We examined the interaction between TPA and serum growth factors in the stimulation of cell growth. The number of cells synthesizing DNA (incorporating 3H-thymidine) within 24 to 30 hours after the addition of TPA and the growth factors to density-inhibited Balb/c-3T3 cultures in serum-free medium was determined by autoradiography. With no additions or with TPA (30--300 ng/ml) alone, only 3--7% of cells synthesized DNA. However, TPA synergistically promoted DNA synthesis in combination with each of the defined serum growth fractions, platelet derived growth factor and platelet poor plasma. TPA also synergistically promoted DNA synthesis in combination with purified growth factors including fibroblast growth factor, insulin (10(-6)--10(-5)M), and epidermal growth factor. In all conditions, TPA enhancement of DNA synthesis also resulted in an increase in cell number. Because TPA synergistically enhanced the activity of each growth factor tested, it did not act identically to any of the growth factors.     

Effect of a dominant inhibitory Ha-ras mutation on mitogenic signal transduction in NIH 3T3 cells.

We used a dominant inhibitory mutation of c-Ha-ras which changes Ser-17 to Asn-17 in the gene product p21 [p21(Asn-17)Ha-ras] to investigate ras function in mitogenic signal transduction. An NIH 3T3 cell line [NIH(M17)] was isolated that displayed inducible expression of the mutant Ha-ras gene (Ha-ras Asn-17) via the mouse mammary tumor virus long terminal repeat and was growth inhibited by dexamethasone. The effect of dexamethasone induction on response of quiescent NIH(M17) cells to mitogens was then analyzed. Stimulation of DNA synthesis by epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) was completely blocked by p21(Asn-17) expression, and stimulation by serum, fibroblast growth factor, and platelet-derived growth factor was partially inhibited. However, the induction of fos, jun, and myc by EGF and TPA was not significantly inhibited in this cell line. An effect of p21(Asn-17) on fos induction was, however, demonstrated in transient expression assays in which quiescent NIH 3T3 cells were cotransfected with a fos-cat receptor plasmid plus a Ha-ras Asn-17 expression vector. In this assay, p21(Asn-17) inhibited chloramphenicol acetyltransferase expression induced by EGF and other growth factors. In contrast to its effect on DNA synthesis, however, Ha-ras Asn-17 expression did not inhibit fos-cat expression induced by TPA. Conversely, downregulation of protein kinase C did not inhibit fos-cat induction by activated ras or other oncogenes. These results suggest that ras proteins are involved in at least two parallel mitogenic signal transduction pathways, one of which is independent of protein kinase C. Although either pathway alone appears to be sufficient to induce fos, both appear to be necessary to induce the full mitogenic response.     

Activation of the serum response element and 12-O-tetradecanoylphorbol-13-acetate response element by the activated c-raf-1 protein in a manner independent of protein kinase C

Transfection of the cDNA encoding the activated c-raf-1 protein or addition of 12-O-tetradecanoylphorbol-13-acetate (TPA) or dibutyryl cAMP to NIH/3T3 cells activated the c-fos gene enhancer linked to the chloramphenicol acetyltransferase or luciferase reporter gene. Prolonged treatment of NIH/3T3 cells with phorbol 12,13-dibutyrate caused down-regulation of protein kinase C. In these cells, addition of TPA did not stimulate the c-fos gene enhancer any more, but transfection of the c-raf-1 cDNA or addition of dibutyryl cAMP still stimulated the c-fos gene enhancer to the same extent as those induced in the control cells. Transfection of the c-raf-1 cDNA or addition of TPA to NIH/3T3 cells stimulated the serum response element and TPA response element but not the cAMP response element. In contrast, addition of dibutyryl cAMP to NIH/3T3 cells stimulated the cAMP response element but not the serum response element or TPA response element. These results indicate that the activated c-raf-1 protein stimulates the serum response element and TPA response element in a manner independent of protein kinase C and cAMP-dependent protein kinase. Since the c-fos gene enhancer has been shown to contain the serum response element and cAMP response element, it is most likely that the c-raf-1 protein is involved in the regulation of c-fos gene expression through the serum response element.     

Induction of tumor promotor-inducible genes in murine 3T3 cell lines and tetradecanoyl phorbol acetate-nonproliferative 3T3 variants can occur through protein kinase C-dependent and -independent pathways.

We isolated a group of genes that are rapidly and transiently induced in 3T3 cells by tetradecanoyl phorbol acetate (TPA). These genes are called TIS genes (for TPA-inducible sequences). Epidermal growth factor (EGF), fibroblast growth factor (FGF), and TPA activated TIS gene expression with similar induction kinetics. TPA pretreatment to deplete protein kinase C activity did not abolish the subsequent induction of TIS gene expression by epidermal growth factor or fibroblast growth factor; both peptide mitogens can activate TIS genes through a protein kinase C-independent pathway(s). We also analyzed TIS gene expression in three TPA-nonproliferative variants (3T3-TNR2, 3T3-TNR9, and A31T6E12A). The results indicate that (i) modulation of a TPA-responsive sodium-potassium-chloride transport system is not necessary for TIS gene induction either by TPA or by other mitogens and (ii) TIS gene induction is not sufficient to guarantee a proliferative response to mitogenic stimulation.     

Expression of rat BTG(3) gene, Rbtg3, is regulated by redox changes

The Rbtg3 gene was isolated by PCR (polymerase chain reaction) cloning from the cDNA library of Rat1 fibroblasts that were stimulated with TPA (12-O-tetradecanoylphorbol-13-acetate) or various growth factors for 3h and was found to be a rat homologue of mouse BTG3 and human ANA genes. The Rbtg3 gene had unique DNA sequences in the 5'-UTR and 3'-UTR that contained four ATTTA and one TTATTTA(T/A)(T/A) nonamer motif, and also a polyA addition site. Nucleotide homology of Rbtg3 with BTG3 and ANA was 88.5 and 76.6%, respectively. Expression of Rbtg3 was investigated in SD rats as well as cell lines derived from mouse--SW3T3, NIH3T3 fibroblasts--and rat--Rat1, 3Y1 fibroblasts and PC12--cells. Rbtg3 was highly expressed in brain but barely in lung, kidney, thymus and spleen. The constitutive expression level was high in SW3T3, Rat1 and 3Y1 fibroblasts, but very low in NIH3T3 fibroblast and PC12 cells. However, in all cells tested, Rbtg3 was proved to be one of the primary response genes superinduced by TPA (50ng/ml)+cycloheximide (CHX, 10 microgram/ml). Expression of Rbtg3 was induced by H(2)O(2) (500mM) up to fourfold in PC12 cells and was blocked by pretreatment of NAC (N-acetyl-L-cysteine, 10mM). The induction was ninefold in 3Y1 fibroblasts by menadione (25mM) treatment for 1h, whereas it was reduced to a third of the control level in SW3T3 fibroblast by the same treatment. Rbtg3 was not expressed in NIH3T3 cells but minimally regulated by redox changes as compared with rapid and strong induction of TIS21/BTG2 mRNAs after TPA or H(2)O(2) stimulation. The above results indicate that Rbtg3 is one of many redox-regulated genes as well as a primary response gene.     

Growth factor requirements of oncogene-transformed NIH 3T3 and BALB/c 3T3 cells cultured in defined media.

We report conditions for the efficient growth of NIH 3T3 and BALB/c 3T3 cells cultured in a defined medium supplemented with either platelet-derived growth factor (PDGF) or pituitary-derived fibroblast growth factor (FGF). The oncogenes v-mos, v-src, v-sis, and c-H-ras Val 12 can induce morphological transformation of these cells and can release them from the mitogen requirement for growth, while the oncogene v-fos cannot abrogate the PDGF-FGF requirement. The radically different behavior of normal and transformed NIH 3T3 cells in PDGF-FGF-free defined medium can form the basis of a sensitive new fibroblast transformation assay.     

Reduced induction of c-fos but not of c-myc expressions in a nontumorigenic revertant R1 of Ej-ras-transformed NIH/3T3 cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA)

It has been reported that both c-fos and c-myc mRNAs are induced in NIH/3T3 cells after 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. We have studied the effect of TPA on the expression of c-fos and c-myc in EJ-ras-transformed NIH/3T3 and its nontumorigenic flat revertant R1 cells. Although TPA treatment induces c-myc mRNA, as in the case of NIH/3T3 cells, the induced level of c-fos mRNA is greatly reduced not only in slow-growing EJ-ras-transformed NIH/3T3 but also in quiescent R1 cells. In addition, serum-induced c-fos expression is also reduced in EJ-ras-transformed NIH/3T3 and R1 cells. These observations suggest that the pathway from TPA to c-fos gene is different from that to c-myc gene and that the former pathway is down-regulated in association not with the transformed phenotype, but with EJ-ras expression, and it is possible that this reduced induction of c-fos is not specific to TPA.     

Characteristics of 30-, 63-, and 89-kilodalton proteins whose secretion from mouse fibroblasts is altered by beta-interferon

Quiescent mouse BALB/c-3T3 cells were treated with beta-interferon to induce the secretion of proteins of 30 and 89 kDa and with a platelet-derived growth factor preparation to induce the secretion of a 63-kDa protein. To label the secreted proteins the cultures were supplemented with [35S]methionine after addition of the inducer. The proteins in the culture fluid were fractionated resulting in a radioactively pure 63-kDa protein and 30- and 89-kDa protein preparations with residual minor radioactive impurities. The secreted 89-kDa protein shared at least one characteristic with some interferon-induced cell-associated enzymes: it bound double-stranded RNA tightly. The 63-kDa protein was undetectable in the culture fluid from resting BALB/c-3T3 cells and was barely or not at all detectable in the culture fluids from growing BALB/c-3T3 and NIH 3T3 cells, respectively. The protein was, however, among the three major constitutively secreted proteins in the case of growing Kirsten murine sarcoma virus-transformed NIH 3T3 cells. Treatment with 1000 units/ml beta-interferon decreased the accumulation of the 63-kDa protein in the culture fluid of quiescent BALB/c-3T3 cells which had been treated with a platelet-derived growth factor preparation by over 80% and that in the culture fluid of Kirsten murine sarcoma virus-transformed NIH 3T3 cells by about 50%. This decrease was not a consequence of an inhibition of cell growth.     

Phorbol ester TPA inhibits the stimulation of bumetanide-sensitive Na+/K+/Cl- transporter by different mitogens in quiescent BALB/c 3T3 mouse fibroblasts

In this study we examined the effect of the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the bumetanide-sensitive Na+/K+/Cl- transporter in quiescent BALB/c 3T3 cells. We have shown that exposure of quiescent BALB/c 3T3 cultures to phorbol ester did not inhibit the basal bumetanide-sensitive Rb+ influx or efflux. In fact, at high concentration (100 ng/ml), TPA slightly stimulated the bumetanide-sensitive Rb+ influx and efflux. However, when the quiescent cultures were stimulated by serum or by defined growth factors, the stimulated fraction of the bumetanide-sensitive Rb+ influx was drastically inhibited by exposure of the cells to the phorbol ester TPA. Based on the above findings, we propose that activation of protein kinase C by the phorbol ester TPA does not inhibit the Na+/K+/Cl- cotransport activity; however it does suppress only the growth-factors-stimulated fraction of the cotransport in quiescent BALB/c 3T3 cells. These data propose that activation of kinase C has a regulatory feedback effect on the stimulation of the Na+/K+/Cl- cotransport activity by growth factors.