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1.
We have examined culture fluids from a variety of Kirsten murine sarcoma virus (KiMSV) transformed rat and mouse cells for the presence of factors which induce normal Rat-1 cells to assume the transformed phenotype. All KiMSV transformants produced transforming factor (TF). Revertants of KiMSV transformed rat or mouse cells failed to relase TF as did normal rat or mouse cells. Cells transformed by a temperature sensitive mutant of KiMSV produced TF at the permissive temperature but not at the nonpermissive temperature. Further, cells from a spontaneous transformant of Rat-1 cells also produced TF. TF is a small polypeptide which competes for the epidermal growth factor receptor. Its effect upon normal cells is reversible and requires de novo RNA and protein synthesis. Cells treated with TF lose the actin fibers observed in normal fibroblasts, assume a transformed cell morphology, become anchorage independent for growth, grow in low concentrations of serum, grow to a high cell density, and have an increased rate of hexose uptake. 相似文献
2.
Biological and physical modifications of a murine oncornavirus by 2-deoxy-D-glucose. 总被引:2,自引:2,他引:0
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2-Deoxy-D-glucose (2-DG) inhibited the release of transforming Kirsten murine sarcoma-leukemia virus [KiMSV(KiMuLV)] from transformed rat kidney (NRK-K) cells. At a concentration of 30 mM 2-DG, RNA synthesis in NRK-K cells was inhibited by approximately 30 percent and protein synthesis was inhibited by as much as 80 percent of control levels. RNA synthesis was not inhibited in nontransformed normal rat kidney (NRK) cells, although protein synthesis was equally suppressed in NRK and NRK-K cells. After treatment with 2-DG, the release of physical particles of KiMSV(KiMuLV) from NRK-K cels was not reduced as determined by equilibrium density gradient centrifugation and assays for RNA-dependent DNA polymerase of culture fluids. The ability to detect virion-associated radioactivity in equilibrium density gradients was dependent on the conditions of labeling. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of KiMSV(KiMulLV) proteins revealed marked structural alterations after propagation of the virus in 30 mM 2-DG. These alterations may account for the observed loss of transforming ability of KiMSV(KiMuLV). 相似文献
3.
Viral susceptibility of skin fibroblasts from patients with Huntington disease. 总被引:1,自引:1,他引:0
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Cultured skin fibroblasts from patients with Huntington disease (HD) and age-matched controls were tested for susceptibility to vesicular stomatitis virus (VSV) and transformation by Kirsten mouse sarcoma virus (KiMSV). The HD and control cells could not be distinguished on the basis of viral replication, plaque morphology, virus yield, or susceptibility to transformation by KiMSV. These findings suggest that the HD gene product, if expressed within peripheral tissue, does not selectively alter or interfere with viral replication. 相似文献
4.
We demonstrated that the Kirsten murine sarcoma virus (KiMSV) and the Harvey murine sarcoma virus (HaMSV) converted human skin fibroblasts (HSF) into adipocytes. Adipocytic conversion of HSF by KiMSV and HaMSV was dependent on the presence of glucocorticosteroids. The Kirsten murine leukemia virus, the Harvey murine sarcoma [corrected] virus and the amphotropic helper virus (AP292) were ineffective by themselves. Balb murine sarcoma virus and Moloney murine sarcoma virus were, to a lesser degree, able to effect adipocytic conversion of HSF. In contrast, the feline sarcoma virus and the simian sarcoma virus did not cause this conversion. Together, the results suggest a role for certain oncogenes and glucocorticosteroids in the transformation/neodifferentiation of human cells. 相似文献
5.
Characteristic changes in the secreted polypeptides of Kirsten murine sarcoma virus (KiMSV) transformed mouse and rat cell lines could be detected 48 hours after infection of phenotypically normal cells with this virus and correlated with detection of the KiMSV encoded polypeptide p21. 相似文献
6.
L Kopelovich 《Experimental cell biology》1987,55(5):276-280
Transformation of human skin fibroblasts (SF) by the Kirsten murine sarcoma virus (KiMSV) is associated with their neodifferentiation into preadipose cells. Hydrocortisone or dexamethasone (DX) promote the transformation/neodifferentiation of preadipocytes to fully differentiated adipocytes. 5-Azacytidine (5-Aza-CR) by itself, or together with DX, or immediately after inoculation with KiMSV did not cause adipocytic conversion of human SF. Addition of 5-Aza-CR either before or immediately after exposure of human SF to KiMSV and DX reduced the total number of foci and significantly inhibited expression of adipocyte-containing foci. The results suggest that adipocytic conversion of human SF by KiMSV and DX is specific. This conversion apparently does not involve mechanisms directly related to the effects of 5-Aza-CR on the adipocytic conversion of murine embryo cells. 相似文献
7.
8.
G Carloni A M Venuat L Daya-Grosjean P Nardeux J S Rhim B Azzarone 《FEBS letters》1988,229(2):333-339
The human osteosarcoma cell line Te85 clone F-5 is not tumorigenic in vivo. Its transformation with Kirsten murine sarcoma virus (KiMSV) (KHOS) confers full malignant properties and stable non-tumorigenic revertants of this KHOS cell line have been obtained. Here we show that integration and expression of a single copy of the KiMSV proviral DNA, which is totally lost in the HOS 240S revertant, is responsible for the acquisition of tumorigenicity. Cytogenetic analysis and the absence of a residual LTR copy in the revertant cellular genome suggest that the loss of KiMSV provirus is caused either by chromosomal segregation or by recombination not involving the LTR. In addition analysis of the expression of ras proteins revealed no changes in the pattern of c-ras products and the expression of v-ras only in the KHOS cells. All these data suggest that Te85 and HOS 240S cell lines could represent a human alternative recipient system to rodent cells in studies with oncogenes. 相似文献
9.
Modulation of adenylate cyclase by guanine nucleotides and Kirsten sarcoma virus mediated transformation 总被引:3,自引:0,他引:3
D Saltarelli S Fischer G Gacon 《Biochemical and biophysical research communications》1985,127(1):318-325
Certain tumour cells contain activated ras genes that code for 21 000 dalton proteins (p21). These proteins associate with the inner face of the plasma membrane and bind guanine nucleotides specifically. In order to determine whether p21s have functions similar to other GTP binding proteins, we investigated the regulation, by guanine nucleotides, of adenylate cyclase (AC) activity in membrane preparations isolated from fibroblasts (C127) transformed by a temperature sensitive mutant of Kirsten sarcoma virus (Ts 371). The degree of AC stimulation by GMP P(NH)P increased when these cells were shifted from the permissive temperature (33 degrees C) to the non-permissive temperature (39 degrees C). This effect was more pronounced at low Mg++ and low GMP P(NH)P concentrations. AC stimulation remained unchanged in rat fibroblasts infected with a temperature sensitive mutant of Rous Sarcoma virus. AC activity was depressed in C127 cells infected with wild type KiMSV. Our data illustrate the feasibility of correlating alterations in the AC system with ras gene expression and using such experimental approaches to elucidate the physiological functions of the p21 proteins. 相似文献
10.
Monoclonal antibodies to the p21 products of the transforming gene of Harvey murine sarcoma virus and of the cellular ras gene family. 总被引:47,自引:10,他引:37
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We have isolated eight rat lymphocyte-myeloma hybrid cell lines producing monoclonal antibodies that react with the 21,000-dalton transforming protein (p21) encoded by the v-ras gene of Harvey murine sarcoma virus (Ha-MuSV). These antibodies specifically immunoprecipitate both phosphorylated and non-phosphorylated forms of p21 from lysates of cells transformed by Ha-MuSV. All eight react with the products of closely related ras genes expressed in cells transformed by two additional sarcoma viruses (rat sarcoma virus and BALB sarcoma virus) or by a cellular Harvey-ras gene placed under the control of a viral promoter. Three of the antibodies also react strongly with the p21 encoded by the v-ras gene of Kirsten MuSV. These same three antibodies immunoprecipitate the predominant p21 species synthesized normally in a variety of rodent cell lines, including the p21 produced at high levels in 416B murine hemopoietic cells. This suggests that an endogenous gene closely related to Kirsten-ras is expressed in these cells. The monoclonal antibodies have been used to confirm two properties associated with p21; localization at the inner surface of the membrane of Ha-MuSV-transformed cells, assayed by immunofluorescence microscopy, and binding of guanine nucleotides. 相似文献
11.
Transforming growth factors released from Kirsten sarcoma virus transformed cells do not compete for epidermal growth factor membrane receptors 总被引:2,自引:0,他引:2
Kirsten murine sarcoma virus (KiMSV)-transformed rat kidney cells (KNRK) release small polypeptides (Mr 12,500-15,300) into the culture medium that are capable of stimulating normal rat kidney cells (NRK) to form colonies in soft agar. The transforming growth factors (TGFs) did not compete with epidermal growth factor (EGF) for its receptor and did not induce specific phosphorylation of EGF receptor on NRK cell membranes. These properties differ from the TGFs isolated by other investigators. Our data further establish the heterogeneity of the materials produced by transformed cells that induce transformation-specific changes in normal cells. 相似文献
12.
V-K-ras transformation induces reversion to an earlier developmental form in adult rat adrenal cells
Nelly Auersperg Ian A. MacLaren Mina J. Bissell 《Differentiation; research in biological diversity》1990,43(1):29-36
The basis for specific changes in differentiation that accompany ras-oncogene-mediated transformation are not understood. When short-term cultures of fibroblast-like cells from adult rat adrenal glands were transformed with Kirsten murine sarcoma virus (KiMSV), the original stromal characteristics of the target cells (metachromatic extracellular matrix, high collagen production, collagen incorporation into pericellular matrix and a fibroblastic morphology and growth pattern) diminished. In contrast, parenchymal differentiation markers (neutral lipid, delta 5, 3 beta-hydroxysteroid dehydrogenase, 21-hydroxylase and epithelial morphology) were enhanced. These changes in differentiation were initiated concurrently with the over-expression of the transforming protein v-p21, but were unrelated to the levels of v-p21 expression. They were independent of the immortalizing component of transformation mediated by v-K-ras, because they did not take place in spontaneously immortalized lines derived from the same target cells, unless the lines were also transformed with KiMSV. In normal embryonic development, stromal and parenchymal adrenocortical cells arise by divergent differentiation pathways from a common, multipotential mesenchymal precursor. The transformation-induced modulation from a predominantly stromal to a more-parenchymal phenotype is thus reminiscent of reversion to a more primitive, bipotential developmental stage. 相似文献
13.
Epidermal growth factor receptor metabolism and protein kinase activity in human A431 cells infected with Snyder-Theilen feline sarcoma virus or harvey or Kirsten murine sarcoma virus. 总被引:8,自引:0,他引:8
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When human A431 cells, which carry high numbers of epidermal growth factor (EGF) receptors, are exposed to EGF, the total content of phosphotyrosine in cell protein is increased, the EGF receptor becomes phosphorylated at tyrosine, and new phosphotyrosine-containing 36,000- and 81,000-dalton proteins are detected. We examined the properties of A431 cells infected with Snyder-Theilen feline sarcoma virus, whose transforming protein has associated tyrosine protein kinase activity, and Harvey and Kirsten sarcoma viruses, whose transforming proteins do not. In all cases, the infected cells were more rounded and more capable of anchorage-independent growth than the uninfected cells. EGF receptors were assayed functionally by measuring EGF binding and structurally by metabolic labeling and immunoprecipitation. In no case did infection appear to alter the rate of EGF receptor synthesis, but infection reduced EGF receptor stability by about 50% for cloned Harvey sarcoma virus-infected cells and by 80% for cloned feline sarcoma virus-infected cells. The corresponding reductions in EGF binding were 70 and 90%, respectively. The proteins of feline sarcoma virus-infected A431 cells contained an increased amount of phosphotyrosine, and the 36,000- and 81,000-dalton phosphoproteins were detected. The EGF receptor was not detectably phosphorylated at tyrosine, however, unless the cells were exposed to EGF. The Harvey and Kirsten sarcoma virus-infected cells did not exhibit elevated levels of phosphotyrosine either in the total cell proteins or in the EGF receptor, nor were the 36,000- and 81,000-dalton proteins detectable. However, these phosphoproteins were found in the infected cells after EGF treatment. Thus, all of the infected A431 cells exhibited reduced EGF binding and increased degradation of EGF receptors, yet their patterns of protein phosphorylation were distinct from those of EGF-treated A431 cells. 相似文献
14.
An apparently unique isozyme of lactate dehydrogenase has been reported associated with transformation by Kirsten sarcoma virus, which was also expressed in human cancer. This isozyme was designated LDHk (Anderson, G.R., and Kovacik, W.P., Jr., (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 3209-3213; Anderson, G. R., Kovacik, W. P., Jr., and Marotti, K. R. (1981) J. Biol. Chem. 256, 10583-10591). However, preparations of LDH5 from human placenta and from HeLa cells were later shown to exhibit some of the properties ascribed to LDHk9 and the identify of LDHk as a unique isozyme was questioned (Morin, M. E., and Hance, A. J., (1983) J. Biol. Chem. 258, 2864-2869). Saavadra and Anderson (Saavedra, R. A., and Anderson, G. R. (1983) Science (Wash. D.C.) 221, 291-292) refuted the arguments of Morin and Hance (Morin, M. E., and Hance, A. J. (1983) J. Biol. Chem. 258, 2864-2869) by claiming that commercial preparations of human placental LDH5 were contaminated with LDHk. Re-evaluation of the unique properties which distinguish LDHk from conventional LDH5 indicates that the two isozymes may not be different. Highly purified preparations of LDHk exhibit a single Mr = 34,000 polypeptide subunit on sodium dodecyl sulfate-acrylamide gels, yet retain activity detectable as both LDHk and LDH5. Attempts to separate LDHk and LDH5 by column chromatography or by continuous electrophoresis on a variety of solid support matrices were unsuccessful. Enzyme activity identified as LDHk in imidazole-borate-buffered gels migrating toward the cathode was detected as LDH5 activity on re-electrophoresis. LDH5 activity identified by electrophoretic migration toward the anode in Tris-glycine-buffered gels also recorded as LDHk when re-electrophoresed toward the cathode in imidazole-borate-buffered gels. Quantitative assays of enzyme activity recovered from the two-gel assay systems, as well as re-electrophoresis of isozyme-enriched preparations, indicated that cross-contamination of isozymes was not responsible for the results obtained. 相似文献
15.
T Y Shih P E Stokes G W Smythers R Dhar S Oroszlan 《The Journal of biological chemistry》1982,257(19):11767-11773
The transforming protein coded for by the onc gene (v-rasHa) of Harvey murine sarcoma virus (Ha-MuSV) is the 21,000-dalton protein (p21) which is the immediate agent responsible for the virus-induced malignant transformation of normal cells. The p21 proteins of Ha-MuSV and the closely related Kirsten murine sarcoma virus are heavily phosphorylated in vivo. In the partially purified Ha-MuSV p21, the protein shows a guanine nucleotide-binding activity and, in addition, a very unique autophosphorylating activity at a threonine residue using as phosphoryl donor GTP but not ATP. In the present study, we compared the tryptic peptide maps of the Ha-MuSV p21 phosphorylated in vivo and in vitro. The results show that the major phosphorylation site is identical. Since the GTP-specific phosphorylation is very unique and distinct from all other known protein kinases, the present observation suggests that the in vitro enzymatic activity is responsible for the p21 phosphorylation in vivo. We have analyzed the amino acid sequence surrounding the major phosphorylation site of the Ha-MuSV p21 by automated Edman degradations of the tryptic phosphopeptides. Threonine residue 59 from the initiator methionine residue 1 of the p21 protein is the phosphorylated amino acid residue, and the surrounding amino acid sequence is NH2...-Thr-Cys-Leu-Leu-Asp-Ile-Leu-Asp-Thr-Thr(P)-Gly-Gln-Glu-Glu-Tyr-...COOH. The p21 proteins of both the Ha-MuSV and the closely related Kirsten murine sarcoma virus share the same phosphopeptide. The amino acid sequence of the phosphorylation site is distinct from all other known protein kinases. 相似文献
16.
Evidence that the Rous sarcoma virus transforming gene product is associated with glycerol kinase activity 总被引:7,自引:0,他引:7
This communication provides biochemical, immunological, and genetic evidence that pp60src, the Rous sarcoma virus transforming gene product, is associated with glycerol kinase activity. Our investigations demonstrated that the compound phosphorylated by pp60src or by glycerol kinase (EC 2.7.1.30) from Candida mycoderma share the same electrophoretic and chromatographic mobilities. The glycerol kinase and protein kinase activities of pp60src were inhibited similarly by preincubation with immune IgG. Both activities were reduced 6-9-fold in pp60src preparations derived by immunoaffinity chromatography from cells which were infected with NY68, a temperature-sensitive transformation mutant of Rous sarcoma virus. The thermolability at 41 degrees C of the glycerol kinase activity of pp60src from the mutant virus-infected cells was greater (t/2 = 1.3 min) than the same activity in pp60src preparations from wild type virus-infected cells (t/2 = 4.8 min). 相似文献
17.
Isozyme patterns of leucine aminotransferase were studied in connection with glucose transport and DNA synthesis during the activation and deactivation of the transforming gene product in rat kidney cells transformed by one Rous sarcoma virus mutant (which has a temperature-sensitive lesion in its transforming gene. On temperature shift-down of confluent transformed cells grown at 40 degrees C in the presence of fresh serum, isozyme III of leucine aminotransferase appeared in 12--20 h, with increasing amounts from 24 to 48 h. Upon temperature shift-up, isozyme I became the predominant form in these cells within 4 days, the major change occurring within the first 24 h. The rate of protein turnover was similar to the rate of loss of isozymes I and III during temperature shift-down and shift-up, respectively. A stimulation of incorporation of [3H]thymidine into DNA was observed within 8--12 h after temperature shift-down of the transformed cells. For the maintenance of stimulated DNA synthesis for at least 16 h, continued exposure to the permissive temperature is not necessary. Stimulation of glucose transport occurred prior to the stimulation of [3H]thymidine incorporation. The isozymes of leucine aminotransferase also changed during the in vitro differentiation of Yaffee L6A cells in such a way that isozyme I represented the major part of this enzyme in the fused myotube, and isozyme III was more predominant in the less differentiated state (mononucleated cells). 相似文献
18.
19.
D C Fish D B Djurickovic R J Huebner 《Journal of immunology (Baltimore, Md. : 1950)》1979,123(6):2658-2663
Fischer 344 rats were specifically hyperimmunized with allogeneic, nonvirus-producing [Kirsten murine sarcoma virus (KiMSV)] or syngeneic, virus-producing [KiMSV (Rasheed)] rat tumors. Spleen cells taken from these rats adoptively transferred protection against a 100 to 1,000 X rat tumor dose50 cell challenge with several different transplantable rat tumors. Protection was obtained with spleen cells after removal of adherent cells and macrophages but not peritoneal cells. The spleen cells were not directly cytotoxic but required more than 3 days residence in the recipient before protecting the recipient against challenge. No protection against tumor cell challenge was observed when spleen cells were lethally x-ray irradiated before injection into nontreated rats. Spleen cells taken from rats immunized with normal histocompatibility antigens did not protect in this test system. 相似文献
20.
Action of temperature-sensitive mutants of myeloproliferative sarcoma virus suggests that fibroblast-transforming and hematopoietic transforming viral properties are related 总被引:9,自引:4,他引:5
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The myeloproliferative sarcoma virus is molecularly related to the Moloney sarcoma virus (Pragnell et al., J. Virol. 38:952-957, 1981), but causes both fibroblast transformation in vitro and leukemic changes--including spleen focus formation--in adult mice. The fibroblast transforming properties of myeloproliferative sarcoma virus were used to select viral temperature-sensitive mutants at 39.5 degrees C, the nonpermissive temperature. These mutants are temperature sensitive in the maintenance of the transformed state. This was also shown by cytoskeletal changes of the infected cells at permissive and nonpermissive temperatures. Viruses released from cells maintained at both the permissive and nonpermissive temperature are temperature sensitive in fibroblast transformation functions. All temperature-sensitive mutants show only a low reversion rate to wild-type transforming function. The myeloproliferative sarcoma virus temperature-sensitive mutants are inefficient in causing leukemic transformation (spleen enlargement, focus formation) in mice at the normal temperature. A method to maintain a low body temperature (33 to 34 degrees C) in mice is described. One temperature-sensitive mutant was checked at low body temperature and did not induce leukemia. These data thus indicate that the same or related viral functions are responsible for hematopoietic and fibroblast transformation. 相似文献