Virus-transformed 3T3 Cells Research Articles

Cell Surface Retention Sequence Binding Protein-1 Interacts with the v-sis Gene Product and Platelet-derived Growth Factor β-Type Receptor in Simian Sarcoma Virus-transformed Cells

The cell surface retention sequence (CRS) binding protein-1 (CRSBP-1) is a newly identified membrane glycoprotein which is hypothesized to be responsible for cell surface retention of the oncogene v-sis and c-sis gene products and other secretory proteins containing CRSs. In simian sarcoma virus-transformed NIH 3T3 cells (SSV-NIH 3T3 cells), a fraction of CRSBP-1 was demonstrated at the cell surface and underwent internalization/recycling as revealed by cell surface 125I labeling and its resistance/sensitivity to trypsin digestion. However, the majority of CRSBP-1 was localized in intracellular compartments as evidenced by the resistance of most of the 35S-metabolically labeled CRSBP-1 to trypsin digestion, and by indirect immunofluorescent staining. CRSBP-1 appeared to form complexes with proteolytically processed forms (generated at and/or after the trans-Golgi network) of the v-sis gene product and with a approximately 140-kDa proteolytically cleaved form of the platelet-derived growth factor (PDGF) beta-type receptor, as demonstrated by metabolic labeling and co-immunoprecipitation. CRSBP-1, like the v-sis gene product and PDGF beta-type receptor, underwent rapid turnover which was blocked in the presence of 100 microM suramin. In normal and other transformed NIH 3T3 cells, CRSBP-1 was relatively stable and did not undergo rapid turnover and internalization/recycling at the cell surface. These results suggest that in SSV-NIH 3T3 cells, CRSBP-1 interacts with and forms ternary and binary complexes with the newly synthesized v-sis gene product and PDGF beta-type receptor at the trans-Golgi network and that the stable binary (CRSBP-1.v-sis gene product) complex is transported to the cell surface where it presents the v-sis gene product to unoccupied PDGF beta-type receptors during internalization/recycling.

The in vivo enhancement of IL-1alpha receptor expression on polyoma virus transformed tumor derived cells.

The aim of this study was to evaluate expression of receptor for IL-1 on tumor-derived cells. The in vivo acquisition of an expression of a receptor for Fc ç immunoglobulin on polyoma virus transformed cells has been established by us. We investigated whether a receptor for the IL-1 cytokine, like that for Fc gamma immunoglobulin, could also contribute to the heterogeneity of tumor cell population, as well as to its tumorigenic phenotype. Various clones of polyoma virus transformed 3T3 cells were passaged once in syngeneic mice and resulting tumors explanted and recultured. The expression of receptor for IL-1 was tested on in vitro maintained clones (designated C for culture) and on tumor derived clones (designated CTC - culture-tumor-culture). Expression was determined using a 125I radiolabeled ligand and confirmed by flow cytometry with anti-mouse IL-1 receptor (IL-1R) antibodies. Some CTC clones expressed a higher level of IL-1 receptor than others. A positive correlation between the level of IL-1R and a metastatic phenotype was established with some tumor derived cells. A high IL-1R expressing tumor cell population, sorted by flow cytometry, was considerably more metastatic than the sorted low IL-1 expressing cells. IL-1R expression by tumor derived cells may, contribute to the metastatic phenotype of a tumor cell population.

Nucleotide sequence and tissue distribution of mouse transforming growth factor-α

Transforming growth factor-α (TGFα) is secreted into the medium of the Moloney murine sarcoma virus-transformed 3T3 cell line, 3B11-1C. Using reverse transcription-polymerase chain reaction (RT-PCR), we have amplified, cloned and sequenced a cDNA fragment from this cell line which encodes the full protein-coding region of the mouse TGFα precursor. The deduced amino acid sequence (159 residues) differs from that of human TGFα at 13 sites and from that of rat at 3 sites. In the mouse, TGFα transcripts were detected in a variety of normal adult tissues including two previously unreported sites: the preputial glands and the bladder.

Monoclonal antibody to chick embryo hyaluronan-binding protein: Changes in distribution of binding protein during early brain development

A monoclonal antibody, MAb IVd4, that recognizes hyaluronan-binding protein (HABP) from chick embryo brain has been produced and characterized. By immunoblotting, MAb IVd4 was shown to recognize three proteins in chick embryo brain of molecular weight 93, 90, and 69 kDa; this interaction was inhibited by addition of hyaluronan hexasaccharides. Overlay of transblots with [ 3H]hyaluronan showed binding to proteins of similar molecular weight. MAb IVd4 blocked binding of [ 3H]hyaluronan to brain HABP and to simian virus-transformed 3T3 cells, indicating a possible relationship with the 85-kDa hyaluronan receptor of these cells. The distribution of HABP during early brain development was analyzed by immunohistochemistry. Immunoreactivity was uniform in newly formed neuroectoderm but became more concentrated in the roof of the brain during the second day of embryonic development. As the neuroectoderm becomes layered, the HABP was increasingly restricted to the forming plexiform layer, an area enriched in neural cell processes. Immunoreactivity was greatly enhanced by pretreatment of tissue with hyaluronidase, presumably due to removal of hyaluronan bound to the HABP, and was abolished on treatment with hyaluronan hexasaccharide, presumably due to inhibition of HABP-antibody interaction. These results suggest that a hyaluronan receptor is involved in early cellular events in brain development.

Genetic Analysis of the K-rev-1 Transformation-Suppressor Gene

Flat revertants with reduced malignancy in vivo can be isolated from Kirsten sarcoma virus-transformed NIH 3T3 cells (DT line) following transfection with a normal human fibroblast cDNA expression library. We have recovered from one such revertant a 1.8-kb cDNA clone, K-rev-1, that exhibits an activity of inducing flat revertants at certain frequencies (2-5% of total transfectants) when transfected into DT cells. The K-rev-1 cDNA has the capacity to encode a protein with a calculated molecular weight of 21,000, having strong structural similarity to ras proteins (approximately 50% homology), especially in their guanosine triphosphate/guanosine diphosphate-binding, effector-binding, and membrane-attachment domains. Toward understanding the mechanism of action of K-rev-1 protein, we constructed a series of point mutants of K-rev-1 cDNA and tested their biological activities. Substitutions of the amino acid residues in the putative guanine nucleotide-binding regions (Asp17 and Asn116), in the putative effector-binding domain (residue 38), at the putative acylation site (Cys181), and at the unique Thr61 all decreased the transformation-suppressor activity. On the other hand, substitutions including Gly12 to Val12, Ala59 to Thr59, and Gln63 to Glu63 were found to significantly increase the transformation-suppressor activity of K-rev-1. These findings are consistent with the idea that K-rev-1 protein is regulated like many other G-proteins by guanine triphosphate/guanine diphosphate-exchange mechanism probably in response to certain negative growth-regulatory signals.

Genetic analysis of the K-rev-1 transformation-suppressor gene.

Flat revertants with reduced malignancy in vivo can be isolated from Kirsten sarcoma virus-transformed NIH 3T3 cells (DT line) following transfection with a normal human fibroblast cDNA expression library. We have recovered from one such revertant a 1.8-kb cDNA clone, K-rev-1, that exhibits an activity of inducing flat revertants at certain frequencies (2-5% of total transfectants) when transfected into DT cells. The K-rev-1 cDNA has the capacity to encode a protein with a calculated molecular weight of 21,000, having strong structural similarity to ras proteins (approximately 50% homology), especially in their guanosine triphosphate/guanosine diphosphate-binding, effector-binding, and membrane-attachment domains. Toward understanding the mechanism of action of K-rev-1 protein, we constructed a series of point mutants of K-rev-1 cDNA and tested their biological activities. Substitutions of the amino acid residues in the putative guanine nucleotide-binding regions (Asp17 and Asn116), in the putative effector-binding domain (residue 38), at the putative acylation site (Cys181), and at the unique Thr61 all decreased the transformation-suppressor activity. On the other hand, substitutions including Gly12 to Val12, Ala59 to Thr59, and Gln63 to Glu63 were found to significantly increase the transformation-suppressor activity of K-rev-1. These findings are consistent with the idea that K-rev-1 protein is regulated like many other G-proteins by guanine triphosphate/guanine diphosphate-exchange mechanism probably in response to certain negative growth-regulatory signals.

Brief Report: Lessons Learned from Studies on Tumor Suppression by Microcell-Mediated Chromosome Transfer

One approach for identifying chromosomes that carry putative tumor-suppressor genes is the introduction of individual, normal human chromosomes to the tumor cells of interest (1-4). In order to identify human chromosomes that can suppress or modulate tumor-associated phentotypes of tumor cells, we performed chromosome transfer experiments via microcell fusion into cell lines derived from a variety of tumors: neuroblastoma (SK-N-MC), fibrosarcoma (HT1080), uterine endometrial carcinoma (HHUA), renal cell carcinoma (YCR), choriocarcinoma (CC1), uterine cervical carcinoma (SiHa), rhabdomyosarcoma (A204), Wilms' tumor (SK-NEP-1), and Kirsten sarcoma virus-transformed NIH 3T3 cells (DT). We first isolated mouse A9 cells containing a single, normal human fibroblast-derived chromosome integrated with pSV2neo plasmid DNA (5). Following fusion of microcells from these A9 cells with tumor cells, we isolated microcell hybrids with the introduction of a specific chromosome and examined their tumorigenicity and in vitro properties (6-11). The following results were obtained (Table 1). The introduction of chromosome 11 suppressed tumorigenicity of HT1080, SiHa, A204, and SK-NEP-1, but not of SK-N-MC, YCR, CC1, and DT, indicating the function of the putative suppressor gene(s) on chromosome 11 is effective only in specific tumors. The tumorigenicity of YCR was modulated by the introduction of chromosome 3p, but not by chromosomes X and 11, supporting a hypothesis that loss and/or mutational inactivation of a gene on 3p may play a crucial role in the development of human renal cell carcinoma.

The cyclin B2 component of MPF is a substrate for the c- mos xe proto-oncogene product

Previous studies from this laboratory have shown that purified MPF from Xenopus eggs contains cyclin B2 complexed with cdc2 kinase. The activation of MPF during oocyte maturation is known to require expression of the c- mos xe proto-oncogene. We show here that immunoprecipitates of either v- mos from Moloney murine sarcoma virus-transformed NIH 3T3 cells or c- mos from Xenopus eggs phosphorylate cyclin B2 in vitro. Phosphopeptide analysis reveals a pattern similar to that observed with cdc2 kinase. Moreover, ablation of c- mos xe from oocytes by antisense oligonucleotide injection reduces the rate of cyclin B2 phosphorylation in oocyte extracts by 40%. These results suggest that the mechanism of activation of MPF by c- mos xe involves phosphorylation of the cyclin component.

2'-5'-oligoadenylate synthetase gene expression in normal and murine sarcoma virus-transformed NIH 3T3 cells

Mouse fibroblasts transformed by murine sarcoma virus (MSV) are highly sensitive to the antiproliferative effect of interferon (IFN) (M. Bakhanashvili, D. H. Wreschner, and S. Salzberg, Cancer Res. 43:1289-1294, 1983). To elucidate the mechanism leading to this IFN sensitivity, the expression of the 2'-5'-oligoadenylate synthetase (2-5A synthetase) gene, the presence of the 2-5A synthetase protein, and the level of its enzymatic activity were determined in IFN-treated and untreated cultures. NIH 3T3 mouse fibroblasts were compared with two different NIH 3T3 clones transformed by MSV. Cultures were treated with 300 IU of beta IFN (IFN-beta) per ml for 16 to 24 h. While no detectable 2-5A synthetase-derived transcripts were seen in untreated NIH 3T3 cells, two size classes of RNA transcripts, i.e., 1.7 and 4.2 kilobases, were detected in IFN-treated cultures. Surprisingly, a similar amount of transcripts were present in untreated transformed cells. However, following IFN treatment, an eightfold increase in the level of RNA was readily detected in these cells, with no change in the size classes. Similar results were obtained with the 2-5A synthetase protein, for which three size classes of 42, 71, and 102 kilodaltons were demonstrated by immunoblotting, and with the enzymatic activity, for which again, the highest level was seen in IFN-treated MSV-transformed cultures. The basal level of 2-5A synthetase gene expression in the transformed cells has biological significance since these cells were more resistant to mengovirus infection than NIH 3T3 mouse fibroblasts. Medium collected from transformed cultures failed to induce 2-5A synthetase activity in NIH 3T3 cells. Furthermore, antibodies directed against mouse IFN-beta failed to inhibit 2-5A synthetase activity detected in transformed cultures. These results suggest that at least IFN-beta secretion is not involved in the elevated level of 2-5A synthetase gene expression in these cells.

Detection of genes with a potential for suppressing the transformed phenotype associated with activated ras genes.

Seven morphologically nontransformed (flat) revertants with reduced tumorigenicity in vivo have been isolated from populations of Kirsten sarcoma virus-transformed NIH 3T3 cells transfected with a cDNA expression library of normal human fibroblasts. Each revertant harbors 1-10 recombinant plasmids per cell and retains a rescuable transforming virus as well as high level expression of v-Ki-ras-specific RNA and the viral oncogene product, p21v-Ki-ras. Transformed phenotypes are suppressed in cell hybrids generated by fusing each revertant to v-Ki-ras-transformed NIH 3T3 cells. From two of the revertant lines, plasmids capable of giving rise to flat secondary transfectants have been recovered. Thus, in some, if not all, of the revertants, transfected cDNAs seem to be responsible for the suppression of specific transformed phenotypes.

Growth stimulation of 3T3 fibroblasts by Cystatin

Treatment of cultures of mouse 3T3 fibroblasts with Cystatin C, a thiol-proteinase inhibitor isolated from chicken egg white, resulted in an enhanced rate of cell proliferation. This stimulation was demonstrated using two independent assay systems: (a) assessment of total cell number and (b) measurement of [ 3H]thymidine incorporated into acid-precipitable DNA. In both assays, the dose-response curves of Cystatin stimulation showed a rising function that plateaued at a concentration of ~120 μg/ml. The addition of Cystatin to cultures of Kirsten murine sarcoma virus-transformed 3T3 cells also enhanced DNA synthesis in these target cells. Control experiments showed that the presence of Cystatin did not alter the level of binding of radioactively labeled epidermal growth factor and platelet derived growth factor to 3T3 cells. These results argue against the possibility that the observed growth stimulation by Cystatin was due to growth factor contamination of the Cystatin preparation.

Use of a cloned multidrug resistance gene for coamplification and overproduction of major excreted protein, a transformation-regulated secreted acid protease.

Malignantly transformed mouse fibroblasts synthesize and secrete large amounts of major excreted protein (MEP), a 39,000-dalton precursor to an acid protease (cathepsin L). To evaluate the possible role of this protease in the transformed phenotype, we transfected cloned genes for mouse or human MEP into mouse NIH 3T3 cells with an expression vector for the dominant, selectable human multidrug resistance (MDR1) gene. The cotransfected MEP sequences were efficiently coamplified and transcribed during stepwise selection for multidrug resistance in colchicine. The transfected NIH 3T3 cell lines containing amplified MEP sequences synthesized as much MEP as did Kirsten sarcoma virus-transformed NIH 3T3 cells. The MEP synthesized by cells transfected with the cloned mouse and human MEP genes was also secreted. Elevated synthesis and secretion of MEP by NIH 3T3 cells did not change the nontransformed phenotype of these cells.

Use of a Cloned Multidrug Resistance Gene for Coamplification and Overproduction of Major Excreted Protein, a Transformation-Regulated Secreted Acid Protease

Malignantly transformed mouse fibroblasts synthesize and secrete large amounts of major excreted protein (MEP), a 39,000-dalton precursor to an acid protease (cathepsin L). To evaluate the possible role of this protease in the transformed phenotype, we transfected cloned genes for mouse or human MEP into mouse NIH 3T3 cells with an expression vector for the dominant, selectable human multidrug resistance (MDR1) gene. The cotransfected MEP sequences were efficiently coamplified and transcribed during stepwise selection for multidrug resistance in colchicine. The transfected NIH 3T3 cell lines containing amplified MEP sequences synthesized as much MEP as did Kirsten sarcoma virus-transformed NIH 3T3 cells. The MEP synthesized by cells transfected with the cloned mouse and human MEP genes was also secreted. Elevated synthesis and secretion of MEP by NIH 3T3 cells did not change the nontransformed phenotype of these cells.

Correlation of anionic residue mobility at the filopodial plasma membranes with multilayer formation in transformed 3T3 cells.

Simian virus 40-transformed 3T3 (SV40-3T3) cells formed multilayers on a Falcon dish and had numerous filopodial projections, some of which intertwined with those of adjacent cells, in contrast to the few projections of their nontransformed counterparts. When these cells were incubated with polycationic ferritin (0.5 mg/ml), ferritin particles, representative of anionic sites, were spread widely on their surfaces at 4 degrees C, while they formed clusters at 37 degrees C, especially on filopodial surface areas opposing adjacent projections in SV40-3T3 cells. These findings demonstrate an increase in the mobility of molecules with anionic residues on filopodial plasma membranes in SV40-3T3 cells, thus suggesting a role for these projections in the formation of multilayered cell aggregates.

Hyaluronate inhibition of cell proliferation.

The proliferation of rabbit synovial cells, 3T3 cells, or simian virus-transformed 3T3 cells in cell culture was inhibited by the addition of hyaluronate to the culture medium. This effect was markedly dependent on the molecular weight and concentration of the hyaluronate. At the molecular weight and concentration of hyaluronate present in normal synovial fluid, proliferation was inhibited. At lower molecular weights or concentrations, as found in rheumatoid synovial fluid, hyaluronate was significantly less inhibitory. Thus, the changes in synovial fluid hyaluronate that are associated with arthropathies may contribute to a favorable environment for rheumatoid pannus expansion.

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)

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.

Analysis of hexose transport in untransformed and sarcoma virus-transformed mouse 3T3 cells by photoaffinity binding of cytochalasin B

The effect of simian virus 40 transformation on the hexose transport system in mouse embryo fibroblast Swiss 3T3 cells was examined. The concentration of hexose transporters was estimated by measuring d-glucose-inhibitable cytochalasin B binding. The binding of cytochalasin B to the plasma membranes of simian virus 40-transformed mouse 3T3 cells (SV3T3 cells) was significantly greater than that of 3T3 cells. On the other hand, cytochalasin B binding to the microsomal membranes of SV3T3 cells was decreased, and the total amount of binding to plasma and microsomal membranes was not significantly changed in both cell lines. The electrophoretic analysis demonstrated that both hexose-transporter components of M r 46 000 and M r 58 000 affinity labeled were responsible for an increase in the hexose transport by viral transformation. These results suggested that the higher hexose-transport activity of transformed cells is caused by a redistribution of transporter from intracellular membranes to plasma membranes.

Processing and lysosomal localization of a glycoprotein whose secretion is transformation stimulated.

The major excreted protein (MEP) of transformed mouse fibroblasts is a mannose 6-phosphate-containing glycoprotein whose synthesis and secretion are increased in malignantly transformed 3T3 cells and whose synthesis is increased by treatment of 3T3 cells with tumor promoters or growth factors. When pulse-labeled extracts from Kirsten virus-transformed NIH 3T3 (KNIH) cells were immunoprecipitated using an antibody against secreted MEP, one cellular protein was immunoprecipitated that had the same molecular weight and tryptic peptide map as the secreted protein. Pulse-chase labeling experiments showed that 50-60% of this 39,000-mol-wt form was secreted in transformed cells. Of the 40-50% remaining, approximately 5% was processed into two lower molecular weight forms (29,000 and 20,000) which are sequestered within the cell. Similar processing of these proteins was observed in the nontransformed parent NIH 3T3 (NIH) cells. However, in NIH cells, much less of the synthesized MEP was secreted. Measurements of steady-state levels of these three forms of cellular MEP by Western blot immunolocalization revealed approximately fourfold more MEP in KNIH cells than in NIH cells as well as differences in the relative distribution of MEP forms in transformed and nontransformed cells. Subcellular fractionation of KNIH cells on a Percoll gradient demonstrated a distribution of total MEP similar to that of several lysosomal enzymes. The light lysosomal/Golgi peak from these gradients contained both the precursor 39,000-mol-wt form of MEP and the 20,000-mol-wt form, whereas the heavy lysosomal peak was enriched in the 20,000-mol-wt form. The distribution of MEP forms was found to be similar in NIH cells except that the 29,000-mol-wt form was also seen to be enriched in the heavy lysosomal peak. This biochemical localization of MEP was confirmed by immunolocalization with light and electron microscopy. These data support the hypothesis that MEP is a lysosomal protein that is secreted by transformed cells.

Coordinate regulation of the levels of type III and type I collagen mRNA in most but not all mouse fibroblasts.

A mouse genomic clone was isolated by cross-hybridization with a DNA fragment which codes for the NH2-propeptide of chick alpha1(III) collagen. The region of cross-hybridization within the mouse clone was localized, its sequence determined, and an exon coding for the NH2-propeptide of mouse alpha1(III) collagen was identified. This DNA fragment hybridizes to an RNA species of approximately 5300 nucleotides, slightly larger than the major alpha2(I) collagen RNA species. The mouse type III collagen probe was used to examine the effect of transformation on alpha1(III) collagen RNA levels in mouse fibroblasts. The levels of type III and type I collagen mRNA levels were compared in control and sarcoma virus-transformed murine cell lines, as well as in NIH 3T3 cells transformed by members of the human ras oncogenes. The levels of type III RNA decreased about 10-15-fold in Moloney sarcoma virus-transformed cells and in a cell line transformed with a v-mos-containing plasmid, but showed only a 50% decrease in a Kirsten murine sarcoma virus-transformed BALB 3T3 cell line, and increased 4-fold in a Rous sarcoma virus (RSV)-transformed BALB 3T3 cell line. In contrast, the levels of alpha2(I) collagen mRNA are 8- to 10-fold lower in all these cell lines when compared to untransformed cells. NIH 3T3 cells transformed with two human ras oncogenes showed decreased levels of alpha2(I) and alpha1(III) mRNAs. In contrast to the RSV-transformed mouse cell line, RSV-transformed chick embryo fibroblasts contained much smaller amounts of type III RNA than control chick embryo fibroblasts. We conclude that the levels of alpha1(III) and alpha2(I) collagen mRNA are often but not necessarily coordinately regulated by transformation in mouse cells.