Sarcoma Virus-transformed 3T3 Cells Research Articles

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.

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)

Protamine inhibits platelet derived growth factor receptor activity but not epidermal growth factor activity.

Protamine sulfate blocked 125I-PDGF binding to its specific physiological receptor on Swiss mouse 3T3 cells. Reduced 125I-PDGF binding in the presence of protamine sulfate correlated directly with a protamine sulfate dose-dependent decrease in the PDGF-dependent incorporation of [3H]-thymidine into 3T3 cells and a decreased PDGF-stimulated tyrosine-specific protein kinase activity in isolated membrane preparations of 3T3 cells. Protamine sulfate blocked 125I-PDGF binding to simian sarcoma virus transformed cells (SSV-NIH 3T3 and SSV-NP1 cells) and to nontransformed cells in a manner qualitatively identical to unlabelled PDGF. In contrast, protamine sulfate enhanced the specific binding of 125I-EGF by increasing the apparent number of EGF receptors on the cell surface. The increase in 125I-EGF receptor binding was not prevented by cycloheximide nor by actinomycin D. Protamine sulfate did not affect 125I-EGF binding to membranes from 3T3 cells or the EGF-stimulated 3T3 cell membrane tyrosine specific protein kinase activity, suggesting that protamine sulfate may have exposed a population of cryptic EGF receptors otherwise not accessible. Protamine sulfate was fractionated into four active fractions by Sephadex G-50 gel filtration columns; the half maximum inhibition concentration of 125I-PDGF binding to 3T3 cells of protamines I and II (MW approximately 11,000 daltons and 7,000 daltons, respectively) is approximately 0.4 microM. Protamine II (MW approximately 4,800 daltons) was equally active (half maximum inhibition concentration approximately 0.4 microM); protamine IV (MW approximately 3,300 daltons) was substantially less active (half maximum inhibition concentration approximately 2.8 microM). These investigations have extended previous observations that protamine sulfate is a potent inhibitor of PDGF binding and establish that protamine sulfate blocks PDGF binding at the physiological receptor, preventing PDGF initiated biological activities. Protamine sulfate can be used as a reagent to separate the influence of PDGF and EGF on cells with high specificity and has been used to demonstrate that the receptors on simian sarcoma virus transformed 3T3 cells qualitatively respond identically to protamine sulfate as to unlabelled PDGF and are likely identical to those on nontransformed 3T3 cells.

Purification by reverse-phase high-performance liquid chromatography of an epidermal growth factor-dependent transforming growth factor

Transforming growth factors (TGF) are low molecular weight, acid-stable polypeptides that confer a malignant phenotype on nonneoplastic cells. TGF from murine sarcoma virus transformed 3T3 cells were isolated by acid/ethanol extraction, Bio-Gel P-30 chromatography, and reverse-phase high-performance liquid chromatography. Using a μBondapak C 18 column with an acetonitrile gradient, TGF activity can be resolved into two peaks, one of which requires epidermal growth factor (EGF) to induce colony formation of indicator cells in soft agar. Subsequent rechromatography of the EGF-dependent TGF on a μBondapak CN column using an n-propanol gradient resulted in a 430-fold purification over the acid/ethanol extract and showed soft agar activity at a concentration of 4 ng/ml in the presence of 2 ng/ml EGF. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed one main band with an apparent molecular weight of 13,000.

Dihydrocytochalasin B disorganizes actin cytoarchitecture and inhibits initiation of DNA synthesis in 3T3 cells

Dihydrocytochalasin B (H 2CB) disrupts the actin structure of Swiss/3T3 mouse fibroblasts and inhibits the ability of serum growth factors to stimulate DNA synthesis in quiescent cultures. Low doses of H 2CB (2–10 × 10 −7 M) added to serum-arrested cells reversibly block initiation of DNA synthesis by serum; by epidermal growth factor and insulin; or by epidermal growth factor, fibroblast growth factor and insulin. H 2CB is effective only when added to cells within 8–10 hr after stimulation. Low doses of H 2CB cause cell rounding and a loss of actin microfilament bundles, but they do not interfere with glucose or thymidine transport. These results suggest that stimulation of 3T3 cells involves at least one obligatory actin-mediated step. Transformed cells appear to obviate this step, for H 2CB does not inhibit the entry into S phase of SV40-transformed or Moloney murine sarcoma virus-transformed 3T3 cells synchronized by mitotic shake-off.

Procollagen mRNA metabolism during the fibroblast cell cycle and its synthesis in transformed cells

Procollagen mRNA was isolated from mouse embryos and used for the synthesis of a highly labelled cDNA probe complementary to collagen mRNA. This probe was used for the investigation of procollagen mRNA metabolism during the cell cycle of 3T6 mouse embryo fibroblasts in culture. Titration hybridization experiments revealed that procollagen mRNA was present throughout the cell cycle following stumulation of confluent monolayers. Procollagen mRNA levels of sparse cultures appeared similar to those of unstimulated monolayers. The fluctuating levels of collagen synthesis during the cell cycle can be ascribed to changes in the amount of collagen mRNA present. In mouse sarcoma virus transformed 3T3 cells only 20--30% of the amount of procollagen mRNA in 3T3 cells is present indicating that the decline in collagen synthesis is due to mRNA availability.

Retention of transformant specific type III collagen in dibutyryl cAMP treated Kirsten sarcoma virus transformed BALB 3T3 cells and in a flat revertant

AbstractWe previously reported that Kirsten sarcoma virus transformed BALB 3T3 (Ki‐3T3) cell cultures contained mainly type I collagen and about 30% of another type designated by us as Y and which appears to be type III collagen, [α1 (III)]3. Clones of BALB 3T3 which exhibited contact‐inhibition were found to contain mainly type I collagen [α1(I)]2α2, and about 25% of another type (X) which was composed of three α1 chains differing from those of type III (Hata, R. and B. Peterkofsky, 1977 Proc. Nat. Acad. Sci. (U.S.A.), 74: 2933—2937). Since dibutyryl 3′:5′ cyclic adenosine monophosphate (dbcAMP) increases collagen synthesis and alters other transformation specific properties of Ki‐3T3 cells, we determined whether treatment of Ki‐3T3 cells with this compound restored the normal collagen phenotype. We also analyzed the collagen of a revertant of Ki‐3T3 which exhibits properties similar to those of the dbcAMP treated transformant. Procollagen labeled with radioactive proline was isolated from the medium or cells of cultures and was converted to collagen with pepsin; the collagen was analyzed by carboxymethyl cellulose (CMC) chromatography or gel electrophoresis under denaturing conditions. Ki‐3T3 cells treated with 0.5 mM dbcAMP continued to accumulate type III collagen but there was an increase in the number of α1 chains eluting from CMC columns in the same position as α1 (I) suggesting increased accumulation of type X collagen. Although the revertant was similar to dbcAMP treated cells in that it exhibited a flattened morphology and a high relative rate of collagen synthesis, the collagen profile was similar to that of the transformant, consisting mainly of types I and III. These results indicate that accumulation of type III collagen is unaffected by dbcAMP but suggest that cAMP may be involved in the regulation of type X collagen. The failure of dbcAMP or reversion to affect the occurrence of type III collagen supports the mechanism of cell selection as a means of explaining the specific occurrence of type III collagen in sarcoma virus transformed 3T3 cells.

Studies on virus induction by 5-bromodeoxyuridine in nonproducer murine sarcoma virus-transformed 3T3 cells

Treatment with 5-bromodeoxyuridine (BrdU) of nonproducer BALB 3T3 cells (KA31 cells) transformed by the Kirsten strain of murine sarcoma virus (Ki-MSV) induced the synthesis of DNA polymerase-containing particles which band in sucrose gradients at a density of 1.16 and utilize poly(I) (dC) 12–18, a template-primer specific for RNA-directed DNA polymerase. In order to establish optimal conditions for virus induction, the induction process was studied by analysis of the culture fluid for polymerase activity and the induced cells for viral proteins by immunofluorescence and virus particles by electron microscopy. By varying the concentration and time of exposure to BrdU, it was found that the maximal level of polymerase activity was induced when cells were treated with 20 μg of BrdU for a 24-hr period, 24 hr after seeding. Under these conditions about 15% of the cells were induced to synthesize viral antigens detected with anti-Moloney MSV rat serum. The level of polymerase activity in the culture fluid and viral antigen in the cells became maximal at 3 days after BrdU treatment and decreased rapidly. Electron microscopy showed a large increase in the proportion of cells containing incomplete virus particles and in the number of particles per cell at 2 and 3 days after BrdU treatment. At 3 days after treatment, 82% of the cells contained incomplete virus particles within dilated vesicles of the endoplasmic reticulum as compared to 18% for untreated cultures. A high level of virus production could be maintained for at least 15 days by continuous maintenance of cells on 4 μg/ml of BrdU. It is not clear whether the mechanism involves continuous reinduction of virus or overcoming the normal inhibition of virus multiplication in these cells. Under these conditions, BrdU reversed the rounded, transformed appearance of KA31 cells to a flat fibroblastic morphology.

Chromatin fractionation of normal and virus-transformed cells in culture

A simple technique for the obtaining of purified chromatin fractions from mammalian cells in culture is described. The procedure involves the isolation of clean nuclei in 0.30 M sucrose, 1.5 mM MgCl 2, 0.2 mM CaCl 2, 0.01 M Tris HCl pH 7.2, followed by sonication and differential centrifugation to collect specific chromatin fractions. Heterochromatin of SV-40 and Rous sarcoma virus transformed 3T3 cells showed a 6- to 7-fold enrichment in satellite DNA while a 2- to 3-fold enrichment in repetitive DNA was obtained in established and RSV transformed cells of Microtus agrestis. This method will facilitate the search for the site of integration of oncogenic viruses in the chromatin of mammalian cells.