Increased collagen synthesis in Kirsten sarcoma virus-transformed BALB 3T3 cells grown in the presence of dibutyryl cyclic AMP

Growth of Kirsten sarcoma virus-transformed BALB 3T3 (Ki-3T3) cells in the presence of dibutyryl cyclic AMP (dbcAMP) resulted in alteration of morphology, inhibition of growth, and increased collagen synthesis as measured by incorporation of ¹⁴C-proline into collagenase-digestible protein. There was an increase in incorporation of ¹⁴C-proline into collagen when expressed not only as dpm per μg DNA or protein, but also as the relative rate of collagen synthesis compared to total cellular protein synthesis, which suggests that an alteration in amino acid transport cannot totally account for the increased incorporation into collagen. The three properties studied were all affected over a concentration range of 0.10 to 1.0 mM dbcAMP, but each had a slightly different dose-response curve. At 0.5 mM dbcGMP or sodium butyrate, there was no affect on growth, morphology, or the relative rate of collagen synthesis indicating specificity for the dibutyryl analog of cAMP. Growth of the parent line, BALB 3T3, was inhibited by 0.5 mM dbcAMP, but the relative rate of collagen synthesis did not increase. These results suggest that although growth, morphology, and collagen synthesis are altered in transformed cells so that they more closely resemble those of the parent line, each property may be regulated independently.     

No activation of new initiation points for deoxyribonucleic acid replication in BALB/c 3T3 cells transformed by Kirsten sarcoma virus.

BALB/c 3T3 cells were transformed by Kirsten sarcoma virus, and five clones were isolated in soft agar. Average replicon sizes of the transformed cell lines were estimated by the method of fiber-autoradiography (J. A. Huberman and A. D. Riggs, J. Mol. Biol.32:327-341, 1968) and found to be the same size as the nontransformed 3T3 cells, analyzed in parallel. The results indicate that, unlike simian virus 40 and Epstein-Barr virus, Kirsten sarcoma virus does not activate new initiation points for cellular deoxyribonucleic acid replication in murine sarcoma virus-transformed BALB/c 3T3 cells.     

Presence of a Kirsten murine sarcoma virus ras oncogene in cells transformed by 3-methylcholanthrene.

Oncogenes have previously been reported in the DNAs of mouse fibroblast lines which had become transformed after in vitro exposure to the carcinogen 3-methylcholanthrene. These oncogenes are now shown to be versions of the cellular Kirsten ras gene and are therefore homologous to oncogenes detected in a variety of human tumor DNAs.     

Dibutyryl cAMP inhibits expression of transformation-related properties in Kirsten murine sarcoma virus transformed Balb/c-3T3 cells despite continued presence of p21v-Ki-ras

We studied the effects of simultaneous treatment with 0.1 mM N6, O2'-dibutyryl cAMP (dbcAMP) and 1 mM theophylline on several transformation-specific properties and on levels of the Kirsten murine sarcoma virus (Ki-MSV) transforming gene product p21v-Ki-ras, in a Ki-MSV-transformed mouse cell line (Balb/c-3T3, clone A31; KA31). The rate of logarithmic growth, cell motility, and final saturation density were reduced in dbcAMP-treated KA31 cultures. Capabilities for anchorage-independent growth were reduced in treated cells, to levels similar to those observed for the untransformed parental A31 cell line. Treatment with dbcAMP had no observable effect on the binding of 125I-labeled epidermal growth factor and did not alter fluorescence staining patterns for actin microfilaments and fibronectin which, although characteristic of normal cells, were also present in KA31 cells. Changes induced by dbcAMP were readily reversible, except for loss of anchorage-independent growth. However, this property was also reversible, provided removal of dbcAMP occurred 48 h prior to inoculation into soft agar medium. Immunoprecipitation with a monoclonal antibody directed against the protein p21v-Ki-ras (Y13-259) revealed the continued presence of this protein in dbcAMP-treated KA31 cells. We, therefore, conclude that cAMP mediates the inhibition of growth-related transformation-specific properties either by acting at steps subsequent to the expression of p21v-Ki-ras or on a pathway independent of p21ras function.     

Reversible effects of dibutyryl cAMP on laminin and type IV collagen secretion from retinoic acid treated F9 cells

The effects of dibutyryl cAMP on the differentiation of embryonal carcinoma F9 cells were studied mainly using the secretion of laminin and type IV collagen as the marker. For this purpose, F9 cells were labeled with 35S-methionine and radioactive proteins in the medium were analyzed by SDS-polyacrylamide gel electrophoresis. Treatment of F9 cells with retinoic acid alone induced differentiation into cells secreting type IV collagen. The combination of retinoic acid and dibutyryl cAMP stimulated laminin secretion in addition to type IV collagen secretion. This effect of dibutyryl cAMP was observed only 16 h after adding dibutyryl cAMP. Immunofluorescence staining demonstrated that the majority of the cells in culture were converted into cells secreting laminin under these conditions. In contrast to the irreversible effect of retinoic acid, the effect of dibutyryl cAMP on laminin and type IV collagen secretion was reversible at least during the first 5 days of maintaining cells in the medium containing retinoic acid plus dibutyryl cAMP. Removal of dibutyryl cAMP from the culture medium decreased the protein secretion to the basal levels within 2 days. This reversibility was not due to a change in cell number. An in vitro translation assay also suggested the reversible effect of dibutyryl cAMP on the levels of laminin mRNA. Coinciding with variations of the protein secretion, a reversible and homogeneous change in the morphology of retinoic acid generated F9 cells was observed by dibutyryl cAMP.     

Nature of Rous sarcoma virus-specific RNA in transformed and revertant field vole cells.

Cytoplasmic and polyribosomal RNAs from Rous sarcoma virus-transformed and phenotypically reverted field vole cells were fractionated by rate-zonal sedimentation and hybridized with a (3)H-labeled complementary DNA viral probe to determine the size classes of virus-specific RNA present in these cell types. In contrast to Rous sarcoma virus-infected permissive avian cells, only two of three discrete species of virus-specific RNA were detected in the cytoplasm of these vole cells. These included genome-length 35S RNA and a 21S RNA. However, viral 28S RNA, routinely detected in the cytoplasm of productively infected avian cells, could not be found in cytoplasmic RNA from vole cells. In addition, a low-molecular-weight viral RNA sedimenting less than 16S was detected in both infected avian and vole cells. Because of its heterogeneity this latter species is most likely generated from the intracellular degradation of the larger viral RNAs. Both the viral 35S and 21S RNA were also found to be associated with total polyribosomes from these vole cells. Studies were also performed to determine the distribution of both total viral genomic and sarcoma-specific RNA sequences among the size classes of fractionated total polyribosomes. In both vole cell types the majority of cytoplasmic viral RNA sequences were also associated with polyribosomes and were similarly distributed among the size classes of total polyribosomes. Sarcoma-specific sequences were present on both the 35S and 21S RNA species. These data suggest that the expression of the viral transforming gene in revertant field vole cells may be controlled at some stage subsequent to translation of the viral RNA.     

Characterization of hyaluronate binding proteins isolated from 3T3 and murine sarcoma virus transformed 3T3 cells

A hyaluronic acid binding fraction was purified from the supernatant media of both 3T3 and murine sarcoma virus (MSV) transformed 3T3 cultures by hyaluronate and immunoaffinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved the hyaluronate affinity-purified fraction into three major protein bands of estimated molecular weight (Mr,e) 70K, 66K, and 56K which contained hyaluronate binding activity and which were termed hyaluronate binding proteins (HABP). Hyaluronate affinity chromatography combined with immunoaffinity chromatography, using antibody directed against the larger HABP, allowed a 20-fold purification of HABP. Fractions isolated from 3T3 supernatant medium also contained additional binding molecules in the molecular weight range of 20K. This material was present in vanishingly small amounts and was not detected with a silver stain or with [35S]methionine label. The three protein species isolated by hyaluronate affinity chromatography (Mr,e 70K, 66K, and 56K) were related to one another since they shared antigenic determinants and exhibited similar pI values. In isocratic conditions, HABP occurred as aggregates of up to 580 kilodaltons. Their glycoprotein nature was indicated by their incorporation of 3H-sugars. Enzyme-linked immunoadsorbent assay showed they were antigenically distinct from other hyaluronate binding proteins such as fibronectin, cartilage link protein, and the hyaluronate binding region of chondroitin sulfate proteoglycan. The apparent dissociation constant of HABP for hyaluronate was approximately 10(-8) M, and kinetic analyses showed these binding interactions were complex and of a positive cooperative nature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of passage in culture on a clone of BALB/c 3T3 cells transformed by simian virus 40.

Most simian virus 40 (SV40)-transformed BALB/c 3T3 clones employed for biochemical studies have been used without regard to passage level. To determine whether virus-induced properties are stable as a function of passage, we have extensively characterized one transformed clone, FNE, which was isolated after SV40 infection BALB/c 3T3 cells in factor-free medium. From the initial testing at passage 5 and for at least 50 subsequent subcultures, the cells stably maintained many transformed growth properties, including high saturation density, morphology, colony formation on contact-inhibited monolayers, tumorigenicity, and synthesis of viral-specific RNA. However, other properties varied as a function of passage. There was a slight decrease in viral genome equivalents per cell from 1.1 copy/cell at passage 5 to 0.7 copies at passage 40. Initially, the cells were negative for all type C virus; however, cells carried at low density for 13 to 20 passages (65 to 100 generations) began to release an endogenous type C virus that then persisted in the culture. Spontaneous release of type C virus did not occur in control BALB/c 3T3 cells carried under identical culture conditions for 90 passages. When the cultures were releasing type C viruses they stained uniformly and brightly positive for SV40 tumor (T) antigen by immunofluorescence, whereas T antigen staining was variable at early passage. These experiments suggest that subtle but perhaps important differences in viral gene expression can occur as a function of passage; they also demonstrate the importance of evaluating the interactions between SV40 and endogenous type C viruses.     

Immunomorphological research on the changes in the focal contacts of cultured cells transformed by Kirsten sarcoma virus

Alterations of focal contacts (FC) of NRK cultured cells transformed by Kirsten's sarcoma virus and by ts-mutant of this virus were examined immunomorphologically using a new antibody-exclusion method. In nontransformed NRK cells the FC are arranged in the form of a network near the cell edges; numerous single contacts are seen in the center. The FC are always present in both KNRK and tsKNRK cells at permissive temperature. However, their quantity is drastically reduced, especially in the center of the cells, as is often their size. It is suggested that expression of the product of viral oncogene interferes with maturation of the FC continuously formed at the cell edge.     

Control of K+ influx in 3T3 cells transformed by a conditional mutant of Rous sarcoma virus

Mouse 3T3 cells transformed by a conditional mutant of Rous sarcoma virus (LA90) can assume either a normal or a transformed phenotype, depending on the temperature of cultivation. These cells (LA90) were arrested at the G0/G1 phase of the cell cycle by starvation for serum growth factors at the nonpermissive temperature (39 degrees C). Release from the G0/G1 phase by serum growth factors resulted in a rapid stimulation of Rb+ influx. To investigate whether the stimulation of Rb+ influx is obligatory for cell proliferation, the cultures were released from the G0/G1 phase by a temperature decrease in the absence of serum. A temperature decrease from 39 to 32 degrees C activated the viral pp60src gene mitogenic activity. Under these conditions, no rapid stimulation of Rb+ influx was observed. These results suggest that the rapid stimulation of Rb+ influx induced by serum growth factors is not an essential signal for cell release from the G0/G1 phase. However, a delayed increase in Rb+ influx concomitant with an increase in the cell content of K+ was observed in the cultures released from the G0/G1 phase by temperature decrease in the absence of serum growth factors. We found that the LA90 cells incubated at the permissive temperature (32 degrees C) secreted a mitogenic activity into the medium. Moreover, the conditioned medium from cultures incubated at 32 degrees C, but not at 39 degrees C, stimulate Rb+ influx in G0/G1 cells. These results indicate that Rous sarcoma virus pp60src induces a slow autocrine secretion of a mitogenic activity. This mitogenic activity slowly modulates the K+ content. Therefore, the slow elevation in cellular content of K+ is proposed to be an obligatory event for proliferation in normal and transformed cells.     

High-affinity cytochalasin B binding to normal and transformed BALB/3T3 cells.

To study the molecular basis of changes in sugar uptake rate in cultured mouse fibroblasts with different physiological states, we have measured the high affinity binding of [3H] cytochalsin B, a potent sugar transport inhibitor, to actively growing and contact inhibited Balb/3T3 cells as well as to 3T12 and SV3T3 cells. Binding was the same whether the cells were detached from dishes with EDTA or trypsin. The amount of drug bound to intact cells measured with a centrifugation assay was essentially the same as that bound to cell sonicates measured with equilibrium dialysis. Cytochalasin B binding to intact cells was extremely rapid and reversible over a wide range of drug concentrations, and was not affected by 0.1 M D--glucose in the assay medium. Actively growing and contact inhibited 3T3 cells had a similar number of high affinity cytochalasin B binding sites per cell, while 3T12 and SV3T3 cells had one third to one fourth the number of sites per cell. However, the number of sites per mug cellular protein appeared to be similar for cells in all of the physiological states examined.     

The uncoupled regulation of fibronectin and collagen synthesis in Rous sarcoma virus transformed avian tendon cells.

We have investigated the regulation of fibronectin and procollagen synthesis in normal and Rous sarcoma virus transformed primary avian tendon cells. These two proteins interact at the cell periphery and both are reportedly lost upon transformation. We thus examined whether their synthesis was coordinately regulated in Rous sarcoma virus-infected cells. It was found that while the synthesis of both pro alpha 1 and pro alpha 2 peptides was reduced upon transformation, the synthesis of fibronectin was not altered. Nevertheless, long term radiolabeling demonstrated that fibronectin levels were reduced in transformed cells. It is concluded that the reduction in levels of these components at the surface is brought about by different mechanisms; collagen levels being regulated by procollagen synthesis and fibronectin levels by degradation and/or release into the culture medium. The possibility is discussed that fibronectin is lost from the cell periphery of primary avian tendon cells as a consequence of decreased levels of anchoring collagen molecules.     

Dissociation between transformed and differentiated phenotype in rat thyroid epithelial cells after transformation with a temperature-sensitive mutant of the Kirsten murine sarcoma virus.

Differentiated rat thyroid epithelial cells, infected in vitro with a temperature-sensitive mutant of the Kirsten murine sarcoma virus, expressed at the permissive temperature (33 degrees C) some phenotypic properties typical of transformed cells, including morphological features, colony formation in agar, and induction of tumors in newborn animals. Specific functional markers of these differentiated cells, i.e., synthesis/secretion of thyroglobulin, synthesis of thyroglobulin mRNA and iodide uptake, were blocked during growth at 33 degrees C. Normal morphology, failure to grow in agar, and the requirement of hormones for optimal growth were all restored after shifting to the temperature nonpermissive for transformation (39 degrees C), though the typical differentiated functions remained blocked. Infection with a leukemia helper virus clone (Moloney or Kirsten murine leukemia virus) did not lead to the loss of the differentiated phenotype of rat epithelial thyroid cells, thus demonstrating that the loss of the differentiated phenotype is caused by the sarcoma virus component. These results indicate that the expression of some of the phenotypic properties of transformed differentiated rat thyroid epithelial cells is under the direct control of the p21 thermosensitive activity, whereas the block in the expression of two typical differentiation markers of thyroid epithelial cells is irreversible and probably controlled by different mechanisms.     

Effect of mycoplasma on polyamine synthesis and levels in NIH 3T3 cells and in cells transformed by Rous sarcoma virus

Abstract Infecting NIH 3T3 cells with different species of mycoplasmas resulted only in a slight decrease in ornithine decarboxylase (ODC) activity and in the appearance of cadaverine in the infected cells. Similarly, the presence of mycoplasma in NIH 3T3 cells infected with a temperature-sensitive mutant of Rous Sarcoma virus did not bring about any significant changes either in the pattern of ODC activity or in putrescine levels, when transferred to the permissive temperature. This indicates that mycoplasmal contamination of cultures may not significantly change the putrescine metabolism in host cells. On the other hand, the presence of cadaverine in cultured cells may be attributed to contamination by mycoplasma.     

Karyotype analysis and quantitation of viral transforming genes in Rous sarcoma virus transformed, revertant, and retransformed field vole cells

Comparative studies of the number of cellular chromosomes and viral genes, including the gene required for malignant transformation, were performed on several clones of Rous sarcoma virus-transformed, revertant, and spontaneously retransformed field vole cells. The results of these studies indicate that no appreciable differences in either total viral gene equivalents or transforming gene sequences can be detected between transformed and revertant cell types, even though considerable differences in the number of certain chromosomes exist among the clones tested. Furthermore, no increase in the amount of total genes or transforming gene sequences accompanies retransformation of revertant clones, including clones that exhibited significant increases in chromosome number following retransformation.     

Bifunctional activity of transforming growth factor type beta on the growth of NRK-49F cells, normal and transformed by Kirsten murine sarcoma virus

Transformation of rat NRK-49F cells (49F) by Kirsten murine sarcoma virus (Ki-MSV) renders these cells (Ki-49F cells) capable of autonomous anchorage independent (AI) growth. As compared to nontransformed 49F cells, the transformation by Ki-MSV does not modify the cell response to transforming growth factor-beta (TGF-beta) in monolayer conditions, but alters it in A I growth conditions. The growth of nontransformed or Ki-MSV-transformed adherent 49F cells is slowed down by porcine TGF-beta, and this effect is reversed by epidermal growth factor (EGF). This decrease in the cell growth rate, induced by TGF-beta, does not affect the cloning efficiency of untransformed and transformed adherent 49F cells. Contrarily, porcine TGF-beta decreases the A I cloning efficiency of Ki-49F cells in agar-gelled medium; this effect is only partly reversed by EGF, which does not synergise with TGF-beta to enhance the A I growth as in the case of untransformed 49F cells. Media conditioned by 49F cells, Ki-49F cells, and chicken embryo fibroblasts contain a latent TGF-beta whose capacity to promote the A I growth of 49F cells and to inhibit that of Ki-49F cells is unmasked by acidification. The same situation exists concerning TGF-beta from human platelets. Neutral extracts are inefficient in both tests of promotion and inhibition of A I growth and contain an acid-activable component with an apparent molecular weight of 600 kd. In acid extracts, a 5-9 kd apparent molecular weight component is responsible for the A I growth enhancement of 49F cells and the A I growth inhibition of Ki-49F cells. Further purification by reverse phase chromatography shows that both activities strictly coelute at the same point (32%) of an acetonitrile gradient. These results indicate that TGF-beta is present in physiological conditions as a latent form which requires activation for inhibiting the A I growth of transformed cells as well as for enhancing that of 49F cells.