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PubMed1989

Tumour induction in fetal brain transplants exposed to the viral oncogenes polyoma middle T and v-src.

Using a neural transplantation model, we have studied the effect of polyoma virus middle T antigen and of the viral src oncogene on the developing rat brain. For this purpose, single-cell suspensions of fetal rat brain were prepared on day 14 of gestation (E14), infected with a replication-defective retroviral vector which carries the middle T oncogene driven by an internal thymidine kinase promoter, and stereotaxically injected into the caudoputamen of adult host rats. With mock-infected donor cells, the transplants developed into an organotypically differentiated population of neurons, astrocytes, oligodendrocytes and other central nervous system cell types and expressed marker proteins of mature neuroectodermal cells, including neuron-specific enolase, synaptophysin, neurofilament protein, glial fibrillary acidic protein and S-100 protein. A high percentage of rats carrying transplants infected with the middle T-encoding vector died within two to six weeks from massive haemorrhage into the transplant. Upon microscopic examination, gross abnormalities of the microvasculature were seen, with formation of large blood-filled spaces lined by a thin layer of proliferating endothelial cells. This effect of middle T was apparently cell type-specific, since the differentiation of neuroectodermal cells within the graft proceeded in a regular fashion. In order to assess further the cell-specificity of the oncogene, analogous experiments were carried out with a retroviral construct in which the middle T cDNA had been replaced by the viral src gene (v-src). Transplants infected with the v-src vector developed astrocytomas, but no vascular abnormalities.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Requirement for receptor-intrinsic tyrosine kinase activities during ligand-induced membrane ruffling of KB cells. Essential sites of src-related growth factor receptor kinases.

We have prepared site-specific antibodies toward human insulin, insulin-like growth factor-I, and epidermal growth factor receptors with chemically synthesized peptides derived from the cDNA-predicted amino acid sequences of these receptors. Two classes of antibodies were produced toward each receptor: one toward the carboxyl termini and the other against the kinase domains containing sequences homologous to the tyrosyl phosphorylation site of the product of src gene (pp60v-src). Both classes of antibodies specifically immunoprecipitated the appropriate 125I-ligand-receptor complexes and [35S]methionine-labeled receptors with almost equal potencies. Antibodies toward the kinase domains inhibited both autophosphorylation and tyrosine kinase activity of the corresponding receptors in a cell-free system, whereas antibodies toward the carboxyl termini did not. Microinjection of the kinase-inhibitory antibodies into the cytoplasm of human epidermoid carcinoma KB cells blocked the ability of the corresponding ligand to induce membrane ruffling. In contrast, these inhibitory antibodies did not block the ability of noncorresponding ligands to induce the same response. Furthermore, control immunoglobulin and antibodies toward the carboxyl termini did not block this biological response. These results support a role for the tyrosine-specific protein kinase activities of these growth factor receptors in mediating their biological effects and suggest that the regions homologous to the tyrosyl phosphorylation site of pp60v-src are important for these kinase activities both in cell-free and intact cell systems.

Amino Acid Sequence↗
PubMed1993

Phosphorylation of Src mutants at Tyr 527 in fibroblasts does not correlate with in vitro phosphorylation by CSK.

In normal fibroblasts, the product of the cellular src gene, p60c-src or Src, is repressed by phosphorylation at its C-terminal tyrosine residue, Tyr 527. Mutations in Src that prevent phosphorylation cause enzymatic activation and malignant transformation. The tyrosine kinases that phosphorylate Src at Tyr 527 in vivo have not been identified, but a tyrosine kinase known as CSK is an excellent candidate. CSK has the unusual ability to phosphorylate Src in vitro only at Tyr 527. To examine whether CSK has the appropriate sequence specificy to explain the phosphorylation of Src at Tyr 527 in fibroblasts, we have made use of a set of C-terminal substitution mutants of Src. These mutants were previously characterized for their levels of Tyr 527 phosphorylation when expressed in Rat2 fibroblasts. The ability of CSK to phosphorylate selected mutants has now been tested, using both in vitro phosphorylation assays and co-expression of CSK with the Src mutants in a heterologous organism, Saccharomyces cerevisiae. We also tested whether the mutant Src molecules could autophosphorylate at Try 527, by examining the phosphorylation state of catalytically active forms expressed in the absence of CSK in yeast cells. The results show that CSK has strict sequence specificity for the normal Src sequence, although it can also phosphorylate the Lck sequence. The other mutant Src molecules tested were not phophorylated by CSK, even though some of these mutants are highly phosphorylated at Tyr 527 in Rat 2 cells. All the mutants that are phosphorylated at Tyr 527 in Rat2 cells are also able to autophosphorylate at Tyr 527. The results suggest that CSK, autophosphorylation, and phosphorylation by kinases other than CSK, may all contribution to repressing Src catalytic activity in fibroblasts.

Amino Acid Sequence↗
PubMed1994

Antisense src expression inhibits U937 human leukemia cell proliferation in conjunction with reduction of c-myb expression.

To elucidate the role of pp60c-src in U937 human monoblastoid leukemia cell proliferation, recombinant plasmids containing the src gene or myb gene, which could produce antisense src or antisense myb RNA after dexamethasone treatment, were constructed and transfected into U937 cells (U937-ASRC, U937-AMYB). pp60c-src synthesis in U937-ASRC was diminished by the third day after induction of antisense src RNA and the cell proliferation was reduced, furthermore, the amount of p75c-myb was significantly decreased by the third day. p75c-myb synthesis in U937-AMYB was diminished by the second day after induction of antisense myb RNA and the cell growth was significantly inhibited but the amount of pp60c-src in U937-AMYB was not reduced. These results suggest that a decrease in the amount of pp60c-src leads to an inhibition of p75c-myb expression and subsequent reduction in the U937 cell proliferation.

Cell Division↗
PubMed1987

The structurally distinct form of pp60c-src detected in neuronal cells is encoded by a unique c-src mRNA.

A cellular src (c-src) cDNA clone was isolated from a chicken embryonic brain cDNA library and characterized by DNA sequence analysis. Comparison with the published sequence of a chicken genomic c-src clone indicated that the brain cDNA clone contained an 18-base-pair insertion located between exons 3 and 4 of the c-src gene. The six amino acids encoded by the insertion caused an alteration in the electrophoretic mobility of the c-src gene product similar to that of the structurally distinct form of the src protein detected in neuronal cultures.

Amino Acid Sequence↗
PubMed1983

Inhibition of precartilaginous chick somites by oncogenic virus.

Infection of embryonic chicken notochord-somite explants with Rous sarcoma virus inhibited the in vitro differentiation of somites into cartilage. Visual inspection of the explants revealed that viral infection reduced the size of cartilage nodule formation. Formation of the complex of sulfated proteoglycans with hyaluronic acid was inhibited by RSV infection, and sedimentation analysis of the sulfated proteoglycans showed that very little fast sedimenting proteoglycans were synthesized by RSV-infected explants. The infected explants primarily synthesize a slowly sedimenting sulfated proteoglycan which was chondroitinase resistant. These slow-sedimenting sulfated proteoglycans lack the ability to associate with hyaluronic acid and appear to be noncartilaginous. These effects of RSV are apparently due to the src gene of this virus since the mutant td108, which lacks part of the src gene, has no detectable influence on the chondrogenic differentiation of somite explants. Similarly, infection with RAV-2 as well as with uv-irradiated virus had no detectable effect. The inhibition of synthesis of fast sedimenting proteoglycans was observed at 41 degrees C with explants infected with tsNY68, suggesting that residual activity of transforming gene of this virus at the non-permissive temperature is sufficient for this inhibition in the explants.

Animals↗
PubMed1993

Malignant osteopetrosis: c-src kinase is not reduced in fibroblasts.

Targeted disruption of the c-src gene leads to a severe form of osteopetrosis in mice [2]. As the c-src gene is expressed in all tissues and cells tested, we have analyzed fibroblasts from three individuals with malignant, congenital osteopetrosis for the expression of c-src at the protein level. No differences could be detected in c-src protein and c-src kinase activity levels between fibroblasts from healthy controls and affected individuals. Thus, impairment of c-src function as an etiological factor in human osteopetrosis appears unlikely in the individuals investigated.

CSK Tyrosine-Protein Kinase↗
PubMed1996

The product of the v-src-inducible gene nr-13 is a potent anti-apoptotic factor.

Tumorigenesis can be induced either by activating cell proliferation or by inhibiting metabolic pathways regulating programmed cell death (apoptosis). There is evidence suggesting that p60(v-src) and other tyrosine kinases protect cells against apoptosis. This effect could contribute to cell transformation by the Rous sarcoma virus. Mechanism of cell death inhibition by p60(v-src) remains largely unknown. We have recently reported that in avian cells p60(v-src) activates the expression of nr-13, a bcl-2-related gene. In this paper, we demonstrate, using the bone marrow derived cell line Baf-3 as an experimental model, that the product of this avian gene (nr-13) is a potent anti-apoptotic factor. In addition, we report that, in quail neuroretinal cells, nr-13 expression is activated upon infection by the Rous sarcoma virus (RSV) but not by other oncogenic retroviruses like FSV or MH2, suggesting that nr-13 is a specific target of v-src. Activation of nr-13 expression may be a key step in cellular transformation by v-src.

Animals↗
PubMed1983

A fps gene without gag gene sequences transforms cells in culture and induces tumors in chickens.

From molecularly cloned DNAs of Fujinami sarcoma virus (FSV) and the Schmidt-Ruppin-A strain of Rous sarcoma virus (SRA), viral DNA was constructed in which fps-specific sequences encoded in FSV replaced the src gene of SRA. A 3' fragment of FSV DNA, from an ATG methionine coding sequence 148 base pairs downstream from the gag-fps junction through the long terminal repeat, was joined to cloned SRA DNA at the translation start site for the src gene. The resultant DNA clone contained the splice acceptor site for src mRNA processing in SRA, but contained no src coding sequences from SRA nor any gag sequences from FSV. All genes for the replication of SRA were retained. Transfection of this cloned viral DNA genome into chicken embryo fibroblasts induced morphological transformation of the cells in culture. However, the morphology of the transformed cells was distinct from that observed in cells infected with wild-type FSV. The transformed cells produced a nondefective transforming virus called F36 which contained a hybrid FSV-SRA long terminal repeat. F36-infected cells produced a protein with the expected molecular weight of 91,000, which had an associated protein kinase activity and was immunoprecipitated by antibodies raised against fps gene determinants but not by antibodies raised against gag or src proteins. Injection of F36 virus into 8-day-old chicks produced tumors at the site of inoculation, detectable within 7 days. These results demonstrated that the gag portion of the gag-fps fusion protein of FSV is not required for transformation or tumorigenesis.

Animals↗
PubMed1979

Restriction endonuclease mapping of unintegrated proviral DNA of Snyder-Theilen feline sarcoma virus: localization of sarcoma-specific sequences.

Extrachromosomal DNA purified from mink cells acutely infected with the Snyder-Theilen strain of feline sarcoma virus (FeSV) was digested with restriction endonucleases, and the DNA fragments were electrophoretically separated, transferred to a solid substrate, and hybridized with radiolabeled DNA transcripts complementary to different portions of the FeSV RNA genome. Major DNA species 8.4 and 5.0 kilobase pairs (kbp) long represent the linear, unintegrated proviruses of Snyder-Theilen feline leukemia virus and FeSV, respectively. Transfection experiments performed with electroeluted DNAs showed that the 8.4-kbp form led to the production of replicating nontransforming virus in mink and cat cells; in contrast, the 5.0-kbp DNA produced helper virus-independent foci of transformation in mouse NIH/3T3 cells and helper virus-dependent foci in mink cells at an efficiency comparable to that obtained with unfractionated extrachromosomal DNA. Sites of restriction endonuclease cleavage for six enzymes were oriented with respect to one another within the FeSV provirus. EcoRI recognized cleavage sites at 0.3 to 0.4 kbp from each terminus of FeSV DNA, reducing the 5.0-kbp DNA to molecules 4.3 kbp long; this enzyme excised a large internal proviral DNA fragment of corresponding size from the DNA of FeSV-transformed mink nonproducer cells. By using DNA transcripts complementary to different portions of the FeSV genome, sarcoma-specific sequences (the FeSV src gene) were positioned within 2.1 and 3.4 kbp from the 5' end of the proviral DNA with respect to the viral RNA genome. The src gene is flanked at both ends by sequences shared in common with feline leukemia virus. The localization of src sequences to this region suggests that a portion of an FeSV polyprotein which contains feline oncornavirus-associated cell membrane antigen (FOCMA-S) is the major product of this gene.

Animals↗
PubMed1979

Heteroduplex analysis of the RNA of clone 3 Moloney murine sarcoma virus.

Heteroduplex analysis of the RNA isolated from purified virions of clone 3 Moloney murine sarcoma virus (M-MSV) hybridized to cDNA's from Moloney murine leukemia virus (M-MLV) and clone 124 M-MSV shows that the main physical component of clone 3 RNA is missing all or most of the 1.5-kilobase (kb) clone 124 M-MSV specific sequence denoted beta s (S. Hu et al. Cell 10:469--477, 1977). This sequence is either deleted in clone 3 RNA or substituted by a very short (0.3-kilobase) sequence. In other respects, clone 3 and clone 124 RNAs show the same heteroduplex structure relative to M-MLV. Since beta s is believed to contain the src gene(s) of clone 124 RNA, this result leaves as an unresolved question the nature of the src gene(s) of the clone 3 M-MSV RNA complex.

Base Sequence↗
PubMed1997

A complex intronic splicing enhancer from the c-src pre-mRNA activates inclusion of a heterologous exon.

The mouse c-src gene contains a short neuron-specific exon, N1. To characterize the sequences that regulate N1 splicing, we used a heterologous gene, derived from the human beta-globin gene, containing a short internal exon that is usually skipped by the splicing machinery. Various fragments from the src gene were inserted into the globin substrate to measure their effects on the splicing of the test exon. These clones were transiently expressed in neuronal and nonneuronal cell lines, and the level of exon inclusion was measured by primer extension. Several sequences from the N1 exon region induced the splicing of the heterologous exon. The most powerful effect was seen with a sequence from the intron downstream of the N1 exon. This sequence acted as a strong splicing enhancer, activating splicing of the test exon when placed in the intron downstream. The enhancer was strongest in neuronal LA-N-5 cells but also activated splicing in nonneuronal HEK293 cells. Deletion and linker scanning mutagenesis indicate that the enhancer is made up of multiple smaller elements that must act in combination. One of these elements was identified as the sequence UGCAUG. Three copies of this element can strongly activate splicing of the test exon in LA-N-5 neuroblastoma cells. These component elements of the src splicing enhancer are also apparently involved in the splicing of other short cassette exons.

Animals↗
PubMed1989

Localization of cellular src mRNA during development and in the differentiated bipolar neurons of the adult neural retina in Xiphophorus.

The expression of the c-src gene in embryonic and adult tissue of the teleost fish Xiphophorus helleri was analyzed by in-situ hybridization. The highly conserved fish c-src gene was found to be expressed at high levels in midterm embryos, where c-src mRNA was localized in developing neurons of the sensory layer of the differentiating retina and in the developing brain. In adult tissues the expression of c-src was found to persist in certain cell types of the brain and the neural retina, especially in the bipolar cells of the inner nuclear layer, which are postmitotic, fully differentiated mature neurons. Thus c-src in Xiphophorus appears to be a developmentally regulated proto-oncogene which is important for neuronal differentiation during organogenesis, but whose persistence of expression in certain terminally differentiated neurons strongly suggests a particular maintenance function for c-src in these cells as well.

Animals↗
PubMed1997

Src tyrosine kinase mediates stimulation of Raf-1 and mitogen-activated protein kinase by the tumor promoter thapsigargin.

Thapsigargin is a non-phorbol ester-type tumor promoter that elevates the intracellular Ca2+ (Ca(i)2+) levels by blocking the microsomal Ca2+ ATPase. At present, the consequence of this Ca(i)2+ increase and the nature of the tumorigenicity of thapsigargin still remain to be elucidated. Previously, we demonstrated that thapsigargin activates the mitogen-activated protein (MAP) kinase via Ca(i)2+ but independently of protein kinase C or Ca2+ influx. Here, we show that thapsigargin also rapidly stimulates the Src tyrosine kinase. Transfection of a v-Src gene into a hippocampal cell line (H19-7) renders a constitutive activation of MAP kinase, whereas transfection of a kinase-deficient Src mutant blocks the activation by thapsigargin, suggesting that Src is required for the thapsigargin-induced MAP kinase activation. Cotransfection of a dominant-inhibitory Raf-1 and the v-Src genes into H19-7 cells results in an inhibition of the otherwise constitutively elevated MAP kinase activity, suggesting that Raf-1 is required for the Src-dependent activation of MAP kinase. Similarly, in the LA-90 cells, expression of a temperature-sensitive allele of v-Src constitutively activates Raf-1 and MAP kinase, whereas expression of a dominant-inhibitory Raf-1 mutant abolishes the MAP kinase activation induced by either v-Src or thapsigargin treatment. Together, these results suggest that thapsigargin stimulates MAP kinase signaling via Src and Raf-1. The activation of this Src-MAP kinase pathway suggests a biochemical mechanism for the tumorigenic nature of thapsigargin.

Animals↗
PubMed1987

Comparative studies on the src proto-oncogene and its gene product pp60c-src in normal and neoplastic tissues of lower vertebrates.

1. Oncogenes appear to play an important role in the physiology of transformed as well as of normal cells. 2. The src oncogene is by far the most investigated oncogene in birds and mammals with respect to the biochemical characteristics of its tyrosine kinase activity, although the specific function of this enzymatic activity still remains to be uncovered. 3. Systematic studies on the src related kinase activity in lower animals are lacking. 4. To contribute to a better understanding of the function of the c-src gene, we performed a comparative study on lower chordates. 5. We were able to demonstrate the presence of c-src related sequences in Acrania, Cyclostomata, cartilagenous and bony fish. 6. By performing the pp60src-specific immune complex assay we detected a tyrosine specific kinase activity, that shows the same biochemical properties as the pp60c-src from higher vertebrates. 7. The level of kinase activity is regulated in an organ specific manner. 8. In lymphocystis tumors of flat-fish and in stomatopapilloma of freshwater eels a considerable amount of pp60c-src kinase activity was found, which, however, never exceeded the levels found in the normal brain.

Animals↗
PubMed1991

Molecular and biological properties of hamster tumor cell lines transformed with B77 virus: expression of v-src does not correlate with metastatic potential.

Three hamster tumor cell lines (B77Hep, B77H1De and ML cl 3.1) were investigated with the aim to determine some biological and molecular properties of cells which are connected with invasive and metastatic ability. Except B77H1De all cell lines exhibited high metastatic capacity in syngeneic adult animals. Analysis of cell lines revealed a relationship between metastatic ability and growth properties (growth rate, saturation density and colony formation in soft agar). Southern blot analysis of genomic DNA samples from high metastatic cell line (B77Hep) and low metastatic (B77H1De) showed that the metastatic potential of these cell lines did not depend on the number of integrated proviral copies. Northern blot analysis was used to determine the level of mRNA encoded by v-src gene in B77Hep and B77H1De cell lines. We found a good correlation between the number of integrated proviral copies and the level of v-src gene expression in investigated cell lines, but not with their metastatic potential. No proviral sequences were found in genomic DNA isolated from ML cl 3.1 cell line. In cell lines used in this study we found differences in expression of endogenous proto-oncogenes c-myc and c-fos.

Animals↗
PubMed1982

Cells transformed by Rous sarcoma virus release transforming growth factors.

Chicken embryo fibroblasts and hamster BHK cells transformed by Rous sarcoma virus (RSV) release in their culture media growth factors which enhance markedly anchorage-independent colony formation in gelified medium, at the restrictive temperature (41 degrees 5 C), of chicken embryo fibroblasts (CEF) infected by RSV mutants with a ts mutation of the src gene. This action is not observed with uninfected CEF, and, therefore, appears to require some expression of the viral src gene in the target cells. The enhancing factors are proteins related to the family of the transforming growth factors (TGFs) by their molecular weight (about 20 kd), their heat and acid resistance, and their sensitivity to dithiothreitol. They do not compete with 125I EGF for binding on the EGF receptors of the membrane of A431 cells. As chicken embryo fibroblasts are devoid of EGF receptors, their activity is not potentiated by EGF.

Animals↗
PubMed1982

Expression of neuronal markers in chick and quail embryo neuroretina cultures infected with Rous sarcoma virus.

Cultures of neuroretina (NR) cells from 7-day chick and quail embryos were infected with ts NY-68, a thermosensitive mutant of Rous sarcoma virus (RSV) which transformed NR cells at 36 degrees C. The following differentiation markers for neurones were studied: tetanus toxin-binding sites at the cell surfaces, presence of synapses, and the specific activity of the enzymes choline acetyltransferase (CAT) and glutamic acid decarboxylase (GAD). Appearance of synapses and expression of CAT were similar in control and transformed cultures. Tetanus toxin-binding cells were observed in transformed primary cultures and also in quail NR subcultures. GAD-specific activity was markedly stimulated in chick and quail primary cultures transformed by ts NY-68 and further increased in subcultures of ts NY-68-transformed quail NR cells. Stimulation of GAD activity is controlled by the transforming (src) gene of RSV since it was not observed in cultures infected with RAV-1, a leukosis virus which lacks the src gene. These data show that infection of chick and quail NR cultures with RSV results in the transformation of cells with neuronal markers.

Animals↗