Selection of Revertants of Kirsten Sarcoma Virus Transformed Nonproducer BALB/3T3 Cells

Revertants of Kirsten sarcoma virus transformed nonproducer BALB/3T3 cells (KA31 cells) were isolated after exposing the transformed cells to 5-fluorodeoxyuridine at high cell density, or when suspended in methylcellulose. Revertants were also isolated by treating KA31 cells with the lectin, concanavalin A, which is manyfold more toxic to transformed cells than for normal cells. The revertants resemble BALB/3T3 cells in their morphology and growth characteristics in that they have a low saturation density, fail to grow in 1% calf serum or when suspended in methylcellulose, and cease to synthesize DNA after reaching their saturation density. Infection by murine leukemia virus rescues Kirsten sarcoma virus from only the concanavalin-A-selected variants, though all the revertants are susceptible to infection by leukemia virus. The concanavalin A revertants also become transformed after infection with murine leukemia virus. All the revertants can be transformed by Kirsten sarcoma virus but not by simian virus 40.     

Cell surface glycolipids of transformed NIH 3T3 cells transfected with DNAs of human bladder and lung carcinomas.

Neutral glycolipids and gangliosides of NIH 3T3 cells oncogenically transformed by transfection of DNAs from human lung carcinoma (Lx-1) and human bladder carcinoma (Ej) have been investigated. The chemical quantity and the degree of cell surface exposure of gangliotriaosylceramide (Gg3) were greatly enhanced in NIH 3T3 cells transformed by transfection of DNAs of either Lx-1 or Ej carcinoma cells. An identical but more conspicuous change in cell surface exposure of Gg3 was observed in BALB/c 3T3 cells transformed by murine sarcoma virus Kirsten strain, but the same glycolipid was absent in the original Lx-1 or Ej human carcinomas. The mechanism that defines the chemical quantity and the organization of glycolipids is controlled by multiple factors. These include not only the quantity but also the organization of glycosyl transferases and hydrolases in membranes. This also involves membrane dynamics regulated through a cytoskeletal-membrane conjunction which may determine the degree of glycolipid exposure at the cell surface. The similarity of the chemical and organizational change of a single glycolipid, Gg3, between 3T3 transformants by Kirsten murine sarcoma virus and those by transfection of human cancer DNAs may indicate a common biochemical basis triggered by activation of the oncogene.     

Altered kinase C function in transformed BALB/c3T3 cells

Comparative studies of kinase C function were performed in an untransformed (A31) and the benzo[a]pyrene (BPA31), dimethylbenz[a]anthracene (DA31), and Kirsten sarcoma virus (KA31) transformed BALB/c 3T3 mouse fibroblast cell lines. The 80-kDa kinase C dependent phosphoprotein (pp80), an in vivo marker of kinase C activity, was markedly decreased in the transformed cells although the amount of the 80-kDa substrate protein in the BPA31 cells was similar to that in the untransformed A31 cells. Total cell lysate kinase C levels were lower in the transformed cells but this difference could not account for the reduced pp80 phosphorylation. Increased affinity of kinase C for the membrane fraction in the BPA31 cells may account for decreased phosphorylation of pp80.     

Heparan sulfates of mouse cells. Analysis of parent and transformed 3T3 cell lines.

Heparan sulfate from the surface of a variety of mouse cells at different cell densities was examined by ion-exchange chromatography. The results of this analysis show that: (1) The heparan sulfate from new isolates of Swiss 3T3 cells transformed by SV40 virus (a DNA tumor virus) elutes from DEAE-cellulose at a lower ionic strength than that from the parent cell type. This finding confirms our earlier observation with an established SV40-transformed cell line (Underhill and Keller, '75) and eliminates the possibility that this change is caused by extended passage in culture. (2) For both parent and transformed 3T3 cells, the heparan sulfates from low and high density cultures were the same as judged by chromatography on DEAE-cellulose. This result demonstrates that the transformation-dependent change which we have observed is independent of cell density. (3) The heparan sulfate from Balb/c 3T3 cells transformed with Kirsten murine sarcoma virus (an RNA tumor virus) elutes from DEAE-cellulose prior to that from parent Balb/c 3T3 cells. This result extends the transformation dependent change in heparan sulfate to the Balb/c 3T3 cell line and to cells transformed with an RNA virus.     

Properties of a sarcoma-growth-factor-like peptide from cells transformed by a temperature-sensitive sarcoma virus

Serum-free conditioned media was collected from three sarcoma virus-transformed cell lines and an untransformed cell line. All three virally transformed lines produced and released growth factors into their serum-free media. The major activity in all cases, whether the cells were transformed by Moloney sarcoma virus (MSV) or Kirsten sarcoma virus (KiSV), or whether they were mouse or rat, was a sarcoma-growth-factor (SGF)-like activity with an apparent molecular weight of 10,000. The SGF-like pools from a Moloney sarcoma virus-transformed mouse 3T3 cell and a Kirsten sarcoma virus-transformed NRK cell were further purified by carboxymethyl cellulose chromatography. The elution profiles of these peptides were very similar. The serum-free conditioned media from the untransformed cells showed no detectable growth stimulating activity. The temperature sensitivity of an SGF-like growth factor from the supernate of a NRK cell transformed by a wild-type Kirsten sarcoma virus (KiSV) was compared with that of the SGF-like activity from the supernates of a NRK cell transformed by a ts-mutant of KiSV that is temperature sensitive with respect to transformation (ts-371 Cl 5). Neither the cells transformed by the wild-type sarcoma virus nor those transformed by the temperature sensitive virus released a SGF-like activity that was temperature sensitive under the conditions of the assays.     

Isolation of infectious xenotropic mouse type C virus by transfection of a heterologous cell with DNA from a transformed mouse cell

An endogenous xenotropic type C virus has been isolated from a Kirsten sarcoma virus-transformed BALB/c mouse cell line by transfection of a mink fibroblast cell with the DNA from the transformed cells. The results indicate that transfection may be used as a technique to isolate this endogenous type C virus without the need to chemically induce the cell line containing the provirus prior to attempting to isolate the virus.     

Partial Transcription of Murine Type C Viral Genomes in BALB/c Cell Lines

The mouse cell line, BALB/c 3T3, and its derivatives transformed either spontaneously or by treatment with a variety of external agents, were analyzed for cytoplasmic RNA complementary to DNA products prepared from the Kirsten strain of murine sarcoma-leukemia virus, and from an endogenous type C virus of BALB/c 3T3. Although none of these cell lines spontaneously releases complete type C virions, they all contain RNA which is partially homologous to a portion of the 35S RNA isolated from these viruses. The parental cell line, BALB/c 3T3, contains a low level of viral-related RNA, and there is an increased amount of this RNA in some of the transformed cells. The RNA detected represents only a fraction of the viral RNA found in virus-producing cells. The formation of RNA:DNA hybrids was detected by equilibrium centrifugation in Cs(2)SO(4) density gradients and by analysis with a single-strand-specific nuclease from Aspergillus oryzae. Viral DNA products prepared either from an endogenous reaction with whole virus in the presence of actinomycin D or from purified 70S viral RNA as template using avian myeloblastosis virus DNA polymerase yield comparable data. In addition, all of the BALB/c lines examined produce detectable levels of murine type C virus group-specific antigen.     

Selective inhibition of cell growth and associated changes in glycolipid metabolism induced by monovalent antibodies to glycolipids

Monovalent antibodies directed against N-acetylhematoside are growth inhibitory for BALB/3T3 and NIL hamster fibroblasts but not their transformed counterparts. Within a similar dose range antibodies directed against globoside have no effect on cell growth. Inhibition of 3T3 cell growth by anti-hematoside correlates with a specific change in the metabolism of hematoside within the cell membrane. Following antibody treatment the radiolabeling of hematoside is elevated for cell in logarithmic growth but reduced relative to control at final saturation density. This effect is not observed for 3T3 cells transformed by Kirsten murine sarcoma virus. It is suggested that cell surface glycolipids may play a role in the control of normal cell growth in vitro.     

Guanylate cyclase and cyclic guanosine 3':5'-monophosphate phosphodiesterase activities and cyclic guanosine 3':5'-monophosphate levels in normal and transformed fibroblasts in culture.

To investigate the role of guanosine 3':5'-monophosphate (cyclic GMP) in cultured cells we have measured guanylate cyclase and cyclic GMP phosphodiesterase activities and cyclic GMP levels in normal and transformed fibroblastic cells. Guanylate cyclase activity is found almost exclusively in the particulate fraction of normal rat kidney (NRK) and BALB 3T3 cells. Enzyme activity is stimulated 3- to 10-fold by treatment with the detergent Lubrol PX. However, enhancement of guanylate cyclase by fibroblast growth factor could not be demonstrated under a variety of assay conditions. In both NRK and BALB 3T3 cells guanylate cyclase activity is low during logarithmic growth and increases as the cells crowd together and growth slows. Guanylate cyclase activity is undetectable in homogenates of NRK cells transformed by the Kirsten sarcoma virus (KNRK cells) either in the presence or absence of Lubrol PX. Guanylate cyclase activity is also greatly decreased in NRK cells transformed by Moloney, Schmidt-Ruppin, or Harvey viruses. BALB 3T3 cells transformed by RNA viruses (Kirsten, Harvey, or Moloney), by a DNA virus (SV40), by methylcholanthrene, or spontaneously, all have diminished but readily detectable guanylate cyclase activity. Cyclic GMP phosphodiesterase activity is found predominately in the soluble fraction of NRK cells. This activity increases slightly as NRK cells enter the stationary growth phase. Cyclic GMP phosphodiesterase activity is undetectable in two clones of KNRK cells under a variety of assay conditions, and is decreased relative to the level present in NRK cells in a third KNRK clone. However, both Moloney- and Schmidt-Ruppin-transformed NRK cells have a phosphodiesterase activity similar to that found in NRK cells. Boiled supernatant from both NRK and KNRK cells is observed to appreciably enhance the activity of activator-deficient phosphodiesterase from bovine heart. This result indicates that the absence of cyclic GMP phosphodiesterase activity in KNRK cells is not due to a loss of the phosphodiesterase activator. The intracellular concentration of cyclic GMP is found to be very low in transformed NRK cells when compared to levels measured in confluent NRK cells. The low levels of cyclic GMP in transformed NRK cells reflect the greatly decreased guanylate cyclase activity observed in these cells. These results do not appear to support the suggestion that cyclic GMP promotes the growth of fibroblastic cells.     

Stimulation of cell proliferation by vitamin A derivatives on murine sarcoma virus-transformed mouse cells in serum-free culture

The effect of retinoids (Rds) on cell proliferation was studied in serum-free culture condition, using non-transformed and transformed derivatives of BALB 3T3. Cell proliferation of an SV40-transformed line was inhibited significantly by Rd treatment. However, proliferation of two cell lines that were transformed by a Kirsten and Moloney strain of murine sarcoma virus (MSV) and produced growth factor into culture medium, was remarkably stimulated by Rds. Addition of serum masked both the inhibitory and stimulatory effects of Rds.     

Transformation of BALB/3T3 cells by simian virus 40 causes a decreased synthesis of a collagen-binding heat-shock protein (hsp47)

The synthesis of a major collagen-binding heat-shock protein of molecular weight 47,000 (hsp47) was shown previously to be decreased after transformation of chick embryo fibroblasts by Rous sarcoma virus (Nagata, K., and Yamada, K. M. (1986) J. Biol. Chem. 261, 7531-7536; and Nagata, K., Saga, S., and Yamada, K. M. (1986) J. Cell Biol. 103, 223-229). In this paper, further study demonstrated that the total amount and the synthesis of hsp47 are also decreased by a factor of three in BALB/3T3 cells transformed by simian virus 40 (SV40). Higher synthesis was observed for BALB/3T3 cells in the resting state compared to those in the proliferating state. The synthesis of hsp47 in SV40-transformed cells, however, was consistently lower than that in normal cells irrespective of the cell density. Pulse label and chase experiments revealed that hsp47 was stable in the cells for at least 6 h and that there was no difference between normal and transformed BALB/3T3 cells in terms of the half-life. Decreases in the amount and the synthesis of hsp47 by transformation apparently correlate with the decreased synthesis of collagen in transformed cells. Immunoprecipitation using rat monoclonal antibody against hsp47 demonstrated the association of hsp47 with intracellular procollagen. Immunofluorescence studies using anti-hsp47 monoclonal antibody and anti-collagen antibody confirmed the co-localization of hsp47 and procollagen in both nonshocked and heat-shocked cells. Furthermore, we determined the biochemical characteristics of hsp47 of heat-shocked cells.     

Resistance to oncogenic transformation in revertant R1 of human ras-transformed NIH 3T3 cells.

A flat revertant, R1, was isolated from human activated c-Ha-ras-1 (hu-ac-Ha-ras) gene-transformed NIH 3T3 cells (EJ-NIH 3T3) treated with mutagens. R1 contained unchanged transfected hu-ac-Ha-ras DNA and expressed high levels of hu-ac-Ha-ras-specific mRNA and p21 protein. Transfection experiments revealed that NIH 3T3 cells could be transformed by DNA from R1 cells but R1 cells could not be retransformed by Kirsten sarcoma virus, DNA from EJ-NIH 3T3 cells, hu-ac-Ha-ras, v-src, v-mos, simian virus 40 large T antigen, or polyomavirus middle T antigen. Somatic cell hybridization studies showed that R1 was not retransformed by fusion with NIH 3T3 cells and suppressed anchorage independence of EJ-NIH 3T3 and hu-ac-Ha-ras gene-transformed rat W31 cells in soft agar. These results suggest that the reversion and resistance to several oncogenes in R1 is due not to cellular defects in the production of the transformed phenotype but rather to enhancement of cellular mechanisms that suppress oncogenic transformation.     

Increment of DNA topoisomerases in chemically and virally transformed cells

The activities of topoisomerases I and II were assayed in subcellular extracts obtained from nontumorigenic BALB/c 3T3 A31 and normal rat kidney (NRK) cell lines and from the same cells transformed by benzo[a]pyrene (BP-A31), Moloney (M-MSV-A31) and Kirsten (K-A31) sarcoma viruses, and simian virus 40 (SV-NRK). The enzymatic activity of topoisomerase I was monitored by the relaxation of negatively supercoiled pBR322 DNA and by the formation of covalent complexes between 32P-labeled DNA and topoisomerase I. Topoisomerase II activity was determined by decatenation of kinetoplast DNA (k-DNA). It was found that nuclear and cytoplasmic type I topoisomerase specific activities were higher in every transformed cell line than in the normal counterparts. These differences cannot be attributed to an inhibitory factor present in A31 cells. When chromatin was treated at increasing ionic strengths, the 0.4 M NaCl extract showed the highest topoisomerase I specific activity. Moreover, in this fraction the transformed cells exhibited the most significant increment in the enzymatic activity as compared with nontransformed cultures. Spontaneously transformed A31 cells showed topoisomerase I activity similar to that of extracts of cells transformed by benzo[a]pyrene. Topoisomerase II specific activity was also increased in SV-NRK cells, as judged by the assay for decatenation of k-DNA to yield minicircle DNA.     

A comparison of membrane glycoconjugates from mouse cells transformed by murine and primate RNA sarcoma viruses.

Kirsten murine sarcoma virus (Ki-MSV) transformed Balb/eT3 mouse cells (K-Balb) were found to have altered membrane glycoconjugates compared to normal Balb/3T3 cells. There were reduced amounts of mono- and disialogangliosides, GM1 and GD1a, and activity of the specific galactosyltransferase required for synthesis of these gangliosides was reduced to between 0 and 18.5% of normal in the several K-Balb clones examined. When fucose-labeled glycopeptides derived from the surfaces of Balb/3T3 and K-Balb cells were compared by gel filtration chromatography, the glycopeptides from the transformed cells were enriched in earlier eluting components. These differences were also observed when the glycopeptides were derived from the entire cell and were diminished when the surface or cellular glycopeptides from Balb/3T3 and K-Balb were digested with neuraminidase prior to chromatographic analysis. Changes in these membrane sialoglycolipids and sialoglycopeptides were not influenced by Rauscher leukemia virus infection. In marked contrast, these changes in membrane glycoconjugates were not observed in Wooley monkey sarcoma virus (WSV) transformed Balb/3T3 cells (W-Balb). Although W-Balb cells like K-Balb were transformed by tissue culture criteria, their ganglioside composition, galactosyltransferase activity, and glycopeptide patterns were similar to normal Balb/3T3. These findings have potential implications concerning the role of these complex carbohydrates in the phenotypic alterations of transformed cells.     

Enucleation of normal and transformed cells

A quantitative analysis based on centrifugal force requirements for enucleation was developed to examine the response of a number of untransformed and transformed cell lines to cytochalasin mediated enucleation. Examination of the extent of cell enucleation as a function of centrifugal force resulted in a series of response curves demonstrating that enucleation g force requirements varied between Balb/c 3T3, Swiss 3T3, and Kirsten sarcoma virus transformed Balb/c 3T3 (3T3-K). A four times greater centrifugal force was required to reach 50% enucleation for transformed Balb/c 3T3-K when compared to Swiss 3T3. A qualitative correlation could be observed between ease of enucleation and the existence of a well-formed stress fiber network. A comparison of cytochalasin B and D suggested that cytochalasin D was far more effective in the enucleation of transformed cells. Experiments with 2-deoxyglucose and monensin provided evidence that decreasing cellular ATP levels, either directly or potentially by uncoupling ion transport from ATP generation, can decrease the efficiency of enucleation. It is suggested that the organization of the cytoskeleton is affected by the altered cellular ATP levels which can affect the centrifugal requirements of enucleation.     

Polypeptide maps of cells infected with murine type C leukemia or sarcoma oncovirus

The polypeptide composition of murine fibroblast cells and the effect of infection by RNA sarcoma and leukemia viruses were analyzed by two-dimensional gel electrophoresis and tryptic peptide mapping. The polypeptide maps of NIH Swiss mouse embryo fibroblasts (NIH/3T3) and BALB/c mouse embryo fibroblasts (BALB/3T3) were very similar except for two major polypeptides of about 65,000 and 75,000 daltons which were not detected in BALB/3T3 cells. NIH/3T3 cells infected with either Rauscher or Gross oncoviruses and outbred Swiss mouse embryo fibroblasts (3T3 FL) showed two major polypeptrides of about 73,000 and 80,000 daltons not found in uninfected NIH/3T3 cells. The 3T3 FL cells, although uninfected, were also found to contain a high concentration of envelope glycoprotein of an endogenous oncovirus. 3T3 FL cells transformed by Moloney sarcoma virus showed changes in many polypeptides, including several major components: the disappearance or modification of a component of 60,000 daltons, an increased concentration and shift in pl of a glycoprotein of 48,000 daltons, and the apparent loss of several smaller polypeptides. None of the major changes of the transformed cells were associated with cell surface proteins labeled by lactoperoxidase-catalyzed iodination.     

Nuclear transport in 3T3 fibroblasts: effects of growth factors, transformation, and cell shape

Nucleocytoplasmic transport of fluorescent-labeled macromolecules was investigated in transformed and nontransformed 3T3 fibroblasts. Insulin and epidermal growth factor enhanced transport three-fold after 1-2-h incubation with nontransformed adhering fibroblasts; no enhancement of transport was observed for spherical unattached fibroblasts. The concentration of growth factor for maximal enhancement was 3-10 nM. Nuclear transport for Kirsten murine sarcoma virus-transformed BALB/c 3T3 fibroblasts, however, was maximally enhanced before addition of growth factors; addition of insulin or epidermal growth factor causes no additional transport enhancement. Transformation also minimizes cell shape effects on macromolecular nuclear transport. These results provide evidence that protein growth factors and oncogenic transformation may use a similar mechanism for activation of nuclear transport.     

Retrovirus transformation associated secretory phosphoproteins of mouse and mink cells.

Using antisera to major excreted protein (MEP) of Kirsten sarcoma virus transformed NIH 3T3 (KNIH) cells, we have identified phosphoproteins from the media of feline sarcoma virus (FeSV) transformed mink cells. These secretory phosphoproteins from FeSV-transformed mink cells are of 35 kDa M.W. and they do not have autophosphorylation activity. A comparative analysis of MEP from the media of transformed mouse and mink cells was performed on the basis of proteolytic cleavage products and acid hydrolyzed products of the phosphoproteins. While a marked difference was observed in the peptide map, a common 32P-linked molecule was observed following acid hydrolysis of both species of phosphoproteins.     

The chick chorioallantoic membrane as a model system for the study of tissue invasion by viral transformed cells.

The chick chorioallantoic membrane (CAM) was used as an assay system to investigate the the invasive properties of viral transformed NIH/3Y3 cells. Scanning electron microscopy demonstrated that single Kirsten sarcoma virus (KiSV)-transformed cells passed between the epithelial cells of the CAM ectoderm within 6 hr of application, while viable NIH/3T3 cells did not penetrate the ectoderm within 24 hr. The transformed cells entered the mesoderm of the CAM and formed tumors of proliferating cells. The application of 5 X 10(5) KiSV-transformed cells resulted in the formation of donor cells resulted in the formation of the donor cell tumors within 5 days in 43% of the membranes. No tumors were formed when as many as 5 X 10(6) NIH/3T3 cells were applied to the membrane. NIH/3T3 cells transformed by the Abelson leukemia virus or the Moloney sarcoma virus also ivaded the CAM and formed tumors of proliferating cells within the mesoderm, while cells infected with the Moloney leukemia virus did not. NIH/3T3 cells inoculated onto the CAM 8 days after infection and transformation with KiSV formed tumors with a frequency similar to that of KiSV transformed cells that have been passaged in culture for many generations. Cells that formed invasive tumors within the mesoderm also attracted loops of host blood vessels.