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At least 433 records · Page 24Linked to original sources
PubMed1987

Instability of v-src sequences in nonhuman primate tumors cultured in vitro.

We have analyzed the DNA of marmoset tumors induced and marmoset cells transformed by Rous sarcoma virus (RSV) and derivative viruses of various types. Southern blot hybridization was used to determine the presence of v-src gene sequences. We failed to detect v-src DNA in high-passage cells derived from marmoset tumors induced in vivo or from marmoset cell lines transformed in vitro. The inability to detect src sequences was not related to selection of revertants in culture, since all cell lines retained transformed morphology and cells transformed in vitro retained the ability to induce sarcomas after transplantation into adult allogeneic marmosets. By contrast, we detected integrated proviruses in cells analyzed 32 to 60 days after in vitro transformation. The proviral sequences appeared to be identical to the transforming virus but were apparently unstable and continued to transpose.

Animals↗
PubMed1992

The protein tyrosine kinase p56lck inhibits CD4 endocytosis by preventing entry of CD4 into coated pits.

The lymphocyte glycoprotein CD4 is constitutively internalized and recycled in nonlymphoid cells, but is excluded from the endocytic pathway in lymphocytic cells (Pelchen-Matthews, A., J. E. Armes, G. Griffiths, and M. Marsh. 1991. J. Exp. Med. 173: 575-587). Inhibition of CD4 endocytosis is dependent on CD4 expressing an intact cytoplasmic domain and is only observed in cells where CD4 can interact with the protein tyrosine kinase p56lck, a member of the src gene family. We have expressed p56lck, p60c-src, or chimeras of the two proteins in CD4-transfected NIH-3T3 or HeLa cells. Immunoprecipitation of CD4 and in vitro kinase assays showed that p56lck and the lck/src chimera, which contains the NH2 terminus of p56lck, can associate with CD4. In contrast, p60c-src and the src/lck chimera, which has the NH2 terminus of p60c-src, do not associate with CD4. Endocytosis assays using radioiodinated anti-CD4 monoclonal antibodies demonstrated that coexpression of CD4 with p56lck, but not with p60c-src, inhibited CD4 endocytosis, and that the extent of the inhibition depended directly on the relative levels of CD4 and p56lck expressed. The uptake of mutant CD4 molecules which cannot interact with p56lck was not affected. Measurement of the fluid-phase endocytosis of HRP or the internalization of transferrin indicated that the effect of p56lck was specific for CD4, and did not extend to other receptor-mediated or fluid-phase endocytic processes. Immunogold labeling of CD4 at the cell surface and observation by electron microscopy demonstrated directly that p56lck inhibits CD4 endocytosis by preventing its entry into coated pits.

3T3 Cells↗
PubMed2001

[The role of Src-kinase in the regulation of endocytosis of EGF-receptor complexes. I. Dynamics of EGF internalization, recycling, sorting, and degradation during inhibition of Src-kinase activity].

In the present work, the role of Src-kinase in regulation of different stages of EGF-receptor endocytosis was studied. We used murine fibroblasts with knockout of Src gene and CGP77675, and the inhibitor of Src-family kinases. The absence of Src protein in the cells did not lead to any changes in the rates of 125I-EGF internalization or recycling and caused only slight decrease in the rate of its degradation. At the same time, treatment of the wild type cells with the inhibitor resulted in a small decrease in internalization rate and an increase in recycling. The influence of the inhibitor on 125I-EGF degradation was also more pronounced. But even in this case, the effects were no more than 30% of control values. CGP77675 extended the same effect upon cells of HER14 and HC11 lines. Subcellular fractionation of these cells in Percoll gradient has also demonstrated a slight inhibition of 125I-EGF sorting from early to late endosomes. The more pronounced effect of the Src-family kinase inhibitor on the EGF endocytosis, compared to that of the absence of a single Src protein, suggests a compensating mechanism of the Src-family kinases. A conclusion is made that in spite of a slight influence on practically all stages of intracellular pathway of EGF-receptor complexes, Src-kinases are obviously not the key regulators of their endocytosis.

Animals↗
PubMed1977

Size and genetic content of viral RNAs in avian oncovirus-infected cells.

Viral complementary DNA (cDNA) sequences corresponding to the gag, pol, env, src, and c regions of the Rous sarcoma virus genome were selected by hybridizing viral cDNA to RNA from viruses that lack the env or src gene or to polyadenylic acid [poly(A)]-containing RNA fragments of different lengths and isolating either hybridized or unhybridized DNA. The specificities, genetic complexities, and map locations of the selected cDNA's were shown to be in good agreement with the size and map locations of the corresponding viral genes. Analyses of virus-specific RNA, using the specific cDNA's as molecular probes, demonstrated that oncovirus-infected cells contained genome-length (30-40S) RNA plus either one or two species of subgenome-length viral RNA. The size and genetic content of these RNAs varied, depending on the genetic makeup of the infecting virus, but in each case the smaller RNAs contained only sequences located near the 3' end of the viral genome. Three RNA species were detected in Schmidt-Ruppin Rous sarcoma virus-infected cells: 39S (genome-length) RNA; 28S RNA, with an apparent sequence of env-src-c-poly(A); and 21S RNA, with an apparent sequence of src-c-poly(A). Cells infected with the Bryan high-titer strain of Rous sarcoma virus, which lacks the env gene, contained genome-length (35S) RNA and 21S src-specific RNA, but not the 28S RNA species. Leukosis virus-infected cells contained two detectable RNA species: 35S (genome-length) RNA and 21S RNA, with apparent sequence env-c-poly(A). Since gag and pol sequences were detected only in genome-length RNAs, it seems likely that the full-length transcripts function as mRNA for these two genes. The 28S and 21S RNAs could be the active messengers for the env and src genes. Analyses of sequence homologies among nucleic acids of different avian oncoviruses demonstrated substantial similarities within most of the genetic regions of these viruses. However, the "common" region of Rous-associated virus-0, an endogenous virus, was found to differ significantly from that of the other viruses tested.

Animals↗
PubMed1983

The effect of quercetin on the phosphorylation activity of the Rous sarcoma virus transforming gene product in vitro and in vivo.

The phosphotransferase activity of the Rous sarcoma virus src gene product, pp60src, was inhibited both in vitro and in vivo by the bioflavonoid quercetin. The Ki for the inhibitory effect was in the range of 6-11 microM under conditions in vitro. The inhibitory effect of quercetin was competitive towards the nucleotides ATP and GTP as substrates for pp60src and was non-competitive towards alpha-casein as the protein substrate of this kinase activity. In contrast, studies in vitro of the phosphotransferase activity of the catalytic subunit of the cAMP-dependent protein kinase showed that this flavonoid did not inhibit the phosphorylation of physiological substrates of this enzyme. In cultured cells the half-maximal inhibition of tyrosine phosphorylation of pp60src as well as the phosphorylation of the Mr = 34000 protein, a physiological substrate of pp60src, was in the range 0.06-0.08 mM.

Animals↗
PubMed1982

Proteins antigenically related to the human erythrocyte glucose transporter in normal and Rous sarcoma virus-transformed chicken embryo fibroblasts.

Antibody raised against the purified human erythrocyte glucose transporter specifically precipitated four proteins from normal and Rous sarcoma virus-transformed chicken embryo cells: a major protein of Mr 41,000 and minor proteins of Mr 68,000, 73,000, and 82,000. The Mr 41,000 and 82,000 proteins were found only in a membrane fraction, not in the soluble fraction, and displayed a heterogeneous mobility on NaDodSO4/polyacrylamide gel electrophoresis, suggesting glycosylation. The Mr 41,000 and 82,000 proteins were increased in amount after malignant transformation in direct proportion to the increase in hexose transport rate, and the increase was dependent on the expression of the src gene product, as revealed with a temperature-conditional src mutant. We suggest that the Mr 41,000 and 82,000 proteins are the glucose transporter of chicken embryo fibroblasts, or a component of the glucose transporter. These experiments provide direct evidence that malignant transformation increases the rate of glucose transport by increasing the number of transporters in the membrane.

Animals↗
PubMed1983

Studies on the structure and organization of avian sarcoma proviruses in the rat XC cell line.

The structure and arrangement of the multiple provirus copies of avian sarcoma virus in a rat XC cell line were studied by restriction endonucleases. The following observations were made: (i) the majority of the proviruses integrated randomly with respect to cell DNA; (ii) no gross deletions or rearrangements in the proviruses were observed; (iii) two types of proviruses (type I and type II) could be distinguished on the basis of restriction endonuclease cleavage sites; (iv) the virus rescued from these cells was derived from type II provirus, which has a novel EcoRI site between the env and pol genes; (v) most of the provirus units contained the src gene.

Animals↗
PubMed1987

pp60c-src has less affinity for the detergent-insoluble cellular matrix than do pp60v-src and other viral protein-tyrosine kinases.

A difference in affinity for a Nonidet P-40-insoluble cellular matrix was observed between the products of the viral and cellular src genes. It has previously been demonstrated that pp60v-src is associated with a detergent-insoluble matrix containing the cellular cytoskeleton (J. G. Burr, G. Dreyfuss, S. Penman, and J. M. Buchanan, Proc. Natl. Acad. Sci. USA 77:3484-3488, 1980). We observed a similar association of the transforming proteins of Fujinami sarcoma virus (P130gag-fps) and Yamaguchi 73 avian sarcoma virus (P90gag-yes), both of which are tyrosine-specific protein kinases. However, we found that the endogenous c-src product, pp60c-src, was not tightly bound to the detergent-insoluble matrix. This does not appear to have been due to differences in the cytoskeleton between transformed and nontransformed cells since pp60c-src was also solubilized by nonionic detergent in cells transformed by Rous sarcoma virus. This difference in the affinities of the v-src and c-src products for cytoskeletal proteins may contribute to the inability of pp60c-src to transform cells.

Avian Sarcoma Viruses↗
PubMed1986

Structure, expression, and chromosomal location of the human c-fgr gene.

The nucleotide sequence of seven exons of the human c-fgr gene, a cellular homolog of the oncogene of Gardner-Rasheed feline sarcoma virus, was determined. Twenty-six independent genomic clones were obtained from a human gene library with a DNA clone of Y73 avian sarcoma virus oncogene, v-yes, as a probe under relaxed hybridization conditions. Restriction mapping and partial sequence analyses revealed that two of these clones were derived from the c-fgr gene, distinct from the c-yes gene. Interestingly, the splicing points of the c-fgr gene were identical with those of the c-src gene throughout the seven exons, suggesting that the two proto-oncogenes were generated by gene duplication of an ancestral gene containing intervening sequences. On RNA blot hybridization the major transcript was found to be 2.6 kilobase long. Two additional transcripts of 3.5 and 4.7 kilobases were also detected. Furthermore, karyotype analysis of several human-mouse hybrid cells and Southern blot analyses of DNAs of the hybrids with a human c-fgr locus-specific probe showed that this gene is located on chromosome 1.

Animals↗
PubMed1993

Herbimycin A, a pp60c-src tyrosine kinase inhibitor, inhibits osteoclastic bone resorption in vitro and hypercalcemia in vivo.

Since absence of expression of the c-src gene product in mice indicates that the pp60c-src tyrosine kinase is required and essential for osteoclastic bone resorption, we tested the effects of the antibiotic herbimycin A, which is an inhibitor of pp60c-src on osteoclastic bone resorption in vitro and on hypercalcemia in vivo. We examined the effects of herbimycin A on the formation of bone resorbing osteoclasts in mouse long-term marrow cultures, on isolated rodent osteoclasts and on bone resorption in organ cultures of fetal rat long bones stimulated by parathyroid hormone. We found that herbimycin A in concentrations of 1-100 ng/ml inhibited bone resorption in each of these systems. We determined the effects of herbimycin A (100 ng/ml) on src tyrosine kinase activity in mouse marrow cultures and found that it was decreased. Herbimycin A also decreased elevated blood calcium levels that were induced either by repeated subcutaneous injections of recombinant human interleukin-1 alpha or by a human tumor. There was no evidence for toxicity in any of these culture systems or in mice treated with herbimycin A. A different tyrosine kinase inhibitor that does not inhibit pp60c-src was used as a control and caused none of these effects. These data suggest that pp60c-src tyrosine kinase inhibitors may be useful pharmacologic inhibitors of osteoclastic bone resorption and hypercalcemia.

Animals↗
PubMed1985

Neoplastic transformation of normal and carcinogen-induced preneoplastic Syrian hamster embryo cells by the v-src oncogene.

The ability of cloned Rous sarcoma virus (RSV) DNA encoding the v-src oncogene to neoplastically transform normal, diploid Syrian hamster embryo (SHE) cells was examined. Transfection of RSV DNA into early passage SHE cells resulted in a low but significant number of tumors when treated cells were injected into nude mice. Tumors formed with a low frequency (two tumors out of ten sites injected) and only after a long latency period (14 weeks). In contrast to the normal SHE cells, several different carcinogen-induced preneoplastic immortal SHE cell lines were highly susceptible to transformation by the v-src oncogene to the neoplastic phenotype. Tumors formed with high efficiency and a short latency period (less than 3 weeks). Further studies were performed to determine the basis for the inefficient transformation of the normal SHE cells. NeoR clones isolated after cotransfection of SHE cells with pSV2-neo and RSV DNAs were neither morphologically altered nor immortal and did not contain detectable levels of the v-src gene product. These results suggest that neoplastic transformation by v-src DNA in the normal cells is initially suppressed. However, cells from a v-src-induced tumor expressed v-src RNA, and antibody to v-src protein precipitated from the tumor cells a 60,000-molecular-weight protein which displayed protein kinase activity. Karyotypic analyses confirmed that the tumor was derived from Syrian hamster cells and suggested that it was clonal in nature. These results indicate that the v-src oncogene was primarily responsible for neoplastic transformation of SHE cells. In contrast to the results with the v-src oncogene, our previous studies showed that v-Ha-ras oncogene alone is unable to induce neoplastic transformation of SHE cells. Furthermore, the v-myc oncogene was able to compliment v-Ha-ras to neoplastically transform SHE cells, while cotransfection with v-src plus v-myc did not increase the incidence of tumors.

Animals↗
PubMed1992

Rat cellular mutants for expression of mRNA from the long terminal repeat of murine retrovirus.

Previously we isolated revertants from a rat cell line transformed by recombinant murine retrovirus containing the v-src gene. These mutant cell lines, R78 and R107, showed low src-kinase activity, but retained wild-type transforming retrovirus, suggesting that a cellular gene involved in viral gene expression was mutated. Southern and Northern hybridization analyses showed that the expression of viral mRNAs from the integrated proviral DNA was reduced in these mutant cells. DNA transfection experiments with various transforming genes and promoters revealed that the mutant cell lines were resistant to transformation by transforming genes expressed under the long terminal repeat (LTR) of Moloney murine leukemia virus (Mo-MuLV). In contrast, these cell lines could be efficiently transformed by the same transforming genes with human metallothionein promoter, polyomavirus promoter-enhancer, and c-H-ras promoter. Transient expression assays using plasmids containing the CAT gene under the LTR of Mo-MuLV also showed that CAT activity expressed under the LTR in these mutant cells was lower than that in the parental cell line, No. 7. These results suggest that cellular mutations of R78 and R107 cells affect specific transcription from the LTR of Mo-MuLV. Studies using various constructs of the LTR CAT indicated that the region responsible for the repression was located in a fragment (-328 to -150) of the LTR containing the 72-bp repeat enhancer. Somatic cell hybridization experiments showed that the mutant phenotype of these mutant cell lines is dominant to that of the parental cell line.

Animals↗
PubMed1990

Functional expression of dihydropyridine-insensitive calcium channels during PC12 cell differentiation by nerve growth factor (NGF), oncogenic ras, or src tyrosine kinase.

1. Recombinant retroviruses were used to introduce a temperature-sensitive v-src gene and oncogenic c-Ha-ras into PC12 cells, and stable cell lines expressing these genes were established. 2. As previously reported, expression of v-src (Alema et al., 1985) or c-Ha-ras (Noda et al., 1985) in PC12 cells results in neurite outgrowth resembling that induced by NGF. We report here that v-src but not oncogenic c-Ha-ras induces a stable morphologic neuronal differentiation similar to treatment with NGF. Oncogenic c-Ha-ras-induced neurite outgrowth is not stable with long-term culture, rather the cells revert to an undifferentiated morphology with altered cell cycle kinetics. 3. The stable neuronal phenotype induced by v-src and NGF is characterized by the functional expression of dihydropyridine-insensitive calcium currents.

Animals↗
PubMed1989

Expression of proto-oncogene products during drug-induced differentiation of a neuroblastoma cell line SK-N-DZ.

Four human neuroblastoma cell lines exhibited differences in their ability to differentiate into neuron-like cells in response to three different treatments, serum deprivation, or additions of dibutyryl cyclic-AMP or retinoic acid. Expression of N-myc gene product was reduced in neuroblastoma cell line SK-N-DZ differentiated by retinoic acid as compared with untreated cells. On the contrary, expression of c-src gene product, pp60c-src, was considerably enhanced in differentiated SK-N-DZ cells. Tyrosine phosphorylation of several cellular proteins was found to be enhanced in differentiated cells. Alteration in expression of these proto-oncogene products might be important in the differentiation of neuroblastoma cells into neuron-like cells.

Bucladesine↗
PubMed1980

Characterization of viral polyproteins in cells transformed and producing Moloney murine sarcoma virus-124.

The viral proteins of Moloney murine sarcoma virus-transformed mouse cells (G8-124), that overproduce the sarcoma virus relative to helper leukemia virus, were examined by immunoprecipitation, sizing by gel electrophoresis and peptide mapping. Using antisera to the viral core proteins, a p10-deficient core polyprotein of 63 000 daltons (P63gag) was detected which was found to be a product of the MuSV-124 genome. Helper virus proteins Pr67gag, gPr85gag, Pr200gag-pol, and gPr83env were also detected and characterized. An unusual helper virus precursor polyprotein of 93 000 daltons was also detected and characterized. It was labeled with [3H]fucose, suggesting that it was an intermediate precursor containing gp70 on which further modification of the core oligosaccharide had occurred relative to gPr83env. The usual viral proteins were also found in sarcoma virus particles and they were undistinguishable in size from those of Moloney murine leukemia virus (cl-1 strain). These experiments together with pulse-labeling experiments performed in the presence of the arginine analog canavanine, which prevents proteolytic cleavage, suggest that the product derived from the Mo-MuSV-specific sequences must be expressed as a separate gene product since we were unable to detect in transformed cells a polyprotein containing both viral (e.g., 'gag', 'pol' or 'env') and non-viral components. Cell-free protein synthesis studies performed with Mo-MuSV-124 genomic RNA have confirmed this interpretation and have identified three size classes of polypeptides as translation products of the sarcoma virus genome. They are P63gag, P42-P38 and P23. Intracellular p63gag and cell-free synthesized P63gag, appear to have identical sizes, antigenic determinants and tryptic peptides. The P42-P38 proteins contain core protein determinants. P23 is not related to the products of the replication genes of MuLV. Thus, P23 is a candidate for the 'src' gene product of Mo-MuSV-124.

Animals↗
PubMed1979

Chromosome 1 contains the endogenous RAV-0 retrovirus sequences in chicken cells.

We have developed a structurally unique probe which can be used to determine the chromosomal location of nonreiterated genes in vertebrate organisms by the method of in situ hybridization. The probe consists of several specific RNA molecules attached by means of poly(A) . poly(BrdUrd) hybrids to 125I-labeled DNA of high molecular weight. The probe can be synthesized with a variety of RNA molecules, giving it versatility for detecting a variety of genes irrespective of gene size, copy frequency, and host genome complexity. Using this probe prepared with retrovirus genomic RNAs, we have physically mapped all three detectable endogenous genomes of Rous-associated virus type 0 (RAV-0) in Spafas gs- chf- (group-specific antigen negative, chicken helper factor negative) chicken fibroblasts to specific sites on chromosome 1. This finding suggests that these multiple nontranscribed RAV-0 genomes evolved through gene duplication of an original RAV-0 genome. The endogenous src gene coding for a 60,000-dalton protein also has been localized to one of the small macrochromosomes, 10, 11, or 12, in both chicken and Japanese quail cells. The results presented here are consistent with and greatly extend previously reported data obtained by using both chromosome fractionation and restriction endonuclease techniques and thus support the soundness of this hybridization approach.

Animals↗
PubMed1993

Tyrosine phosphorylation of alpha-tubulin is an early response to NGF and pp60v-src in PC12 cells.

Neuronal differentiation is accompanied by extensive reorganization of the cytoskeleton to initiate the extension of neuritic processes. We have used the rat PC12 pheochromocytoma cell line to examine the role of protein tyrosine kinase activity in the induction of these events. Immunoblotting with phosphotyrosine antibodies revealed that tyrosine phosphorylation of alpha-tubulin in PC12 cells occurred within 10 min of nerve growth factor (NGF) treatment. Tyrosine phosphorylation of alpha-tubulin also occurred on induction of pp60v-src expression in a PC12 cell line (PC12-B9) harboring an inducible v-src gene under transcriptional control of the mouse metallothionine I gene promoter. Two tyrosine phosphorylated proteins in NGF- and pp60v-src induced PC12 cells were identified as alpha-tubulin isoforms by comigration with alpha-tubulin on two-dimensional gel electrophoresis, and by immunoprecipitation with phosphotyrosine antibodies followed by immunoblotting with a monoclonal antibody specific for alpha-tubulin. These results demonstrate that alpha-tubulin is an in vivo tyrosine kinase substrate, which is phosphorylated as an early event in the neuronal differentiation pathway of PC12 cells in response to NGF or pp60v-src. Tyrosine phosphorylation of alpha-tubulin could conceivably alter microtubule dynamics during induction of neurite extension.

Animals↗
PubMed1989

Src homology 2 domain deletion mutants of p60v-src do not phosphorylate cellular proteins of 120-150 kDa.

We have constructed seven deletions in the src homology 2 (SH2) domain of the Rous sarcoma virus src gene and have expressed them and wild-type v-src (wt v-src) in Rat 1 fibroblasts. Transfected cells containing mutant DNAs have reduced focus forming activity when compared to cells containing the wt v-src DNA. In most cases, established cell lines that express these mutants have altered growth properties in soft agar. The src proteins isolated from mutant cell lines have reduced tyrosine kinase activity. We also see differences in the phosphorylation of cellular proteins in vivo. Unlike the wt protein kinase, the SH2 domain mutant kinases do not phosphorylate a set of cellular proteins ranging in size from 120-150 kDa.

Animals↗