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PubMed1997

Colony-stimulating factor-1 induces cytoskeletal reorganization and c-src-dependent tyrosine phosphorylation of selected cellular proteins in rodent osteoclasts.

Colony-stimulating factor-1 (CSF-1) stimulates motility and cytoplasmic spreading in mature osteoclasts. Therefore, we examined the cellular events and intracellular signaling pathways that accompany CSF-1-induced spreading in normal osteoclasts. To explore the role c-src plays in these processes, we also studied osteoclasts prepared from animals with targeted disruption of the src gene. In normal osteoclasts, CSF-1 treatment induces rapid cytoplasmic spreading, with redistribution of F-actin from a well-delineated central attachment ring to the periphery of the cell. CSF-1 increases membrane phosphotyrosine staining in osteoclasts and induces the phosphorylation of several cellular proteins in cultured, osteoclast-like cells, including c-fms, c-src, and an 85-kD Grb2-binding protein. Src kinase activity is increased threefold after CSF-1 treatment. In src- cells, no attachment ring is present, and CSF-1 fails to induce spreading or a change in the pattern of F-actin distribution. Although c-fms becomes phosphorylated after CSF-1 treatment, the 85-kD protein is significantly less phosphorylated in src- osteoclast-like cells. These results indicate that c-src is critical for the normal cytoskeletal architecture of the osteoclast, and, in its absence, the spreading response induced by CSF-1 is abrogated, and downstream signaling from c-fms is altered.

Amino Acid Sequence↗
PubMed1978

Cellular transformation and the 'morphologic phenotype' of transformed cells.

Expression of the product of the transforming gene (src) of RNA tumour viruses promotes growth and usually alters the adhesion, appearance and surface properties of cultured fibroblasts. The latter group of properties termed the 'morphologic phenotype' of transformed cells is largely due to diminished cell-to-substratum adhesion. The role of cyclic AMP, cell surface protein (CSP), and other factors in producing the 'morphologic phenotype' are discussed. The effects of src expression bear a striking resemblance to the action of peptide hormones such as insulin on appropriate target cells.

Animals↗
PubMed2006

STAT5 phosphorylation in malignant melanoma is important for survival and is mediated through SRC and JAK1 kinases.

Altered signaling pathways are key regulators of cellular functions in tumor cells. Constitutive activation of signal transducer and activator of transcription (STAT)3 and -5 may be involved in tumor formation and progression. We have investigated the role of STAT5 in cutaneous melanoma metastases using various RNA and protein techniques. In melanoma specimens, Stat5b transcripts were upregulated approximately 3.8-fold. In 13 of 21 (62%) human melanoma metastases, STAT5 was phosphorylated in comparison to normal human melanocytes and benign nevi. The STAT5 target gene Bcl-2 was frequently upregulated. The investigation of the underlying mechanism revealed specific STAT5 activation by recombinant human epidermal growth factor (rEGF). rEGF-induced activation of STAT5 occurred in vitro through the non-receptor tyrosine kinases transforming gene (src) of Rous Sarcoma virus and Janus kinase 1. Inhibition of Stat5b expression by small interfering RNA strongly reduced the expression of Bcl-2 and led to decreased cell viability and increased apoptosis in the melanoma cell lines A375 and BLM. Transfection with dominant-negative Stat5b caused enhanced cell death and G1 arrest in A375 cells. Our study identifies phosphorylated STAT5 in melanoma and shows regulation through rEGF; STAT5 may thus act as a survival factor for growth of human melanoma and may represent a potential target for molecular therapy.

Aged↗
PubMed1990

Cellular and molecular aspects of neurocarcinogenesis.

Although the morphology of neural tumors induced in rats by N-ethyl-N-nitrosourea (NEU) and related alkylating agents has been extensively investigated, their histogenesis and the molecular basis of malignant transformation are still largely unknown. This review gives an account of the interaction of neurocarcinogenic agents with cellular DNA, the possible role of promutagenic O6-alkyldeoxyguanine and their deficient repair by the cerebral O6-alkylguanine-DNA alkyltransferase. A new experimental model is described in which neural tumors are induced in fetal brain transplants. Pregnant rats received a single iv dose of NEU (50 mg/kg) on the 14th day of gestation. One day later, suspensions were prepared from the fetal forebrain and stereotactically injected into the caudoputamen of adult rats. After additional exposure to NEU of the host animals 8 days and 9 weeks post transplantation, all rats developed brain tumors within the neural graft. Histopathologically, all neoplasms were classified as olidogdendrogliomas. Other neoplasms typically induced by NEU transplacentally (astrocytomas, mixed gliomas, ependymomas) were absent. The selective induction of oligodendrogliomas indicates that neoplastic transformation in the nervous system can occur in a differentiated glial cell or a precursor cell committed to oligodendrocytic differentiation, and that transformation of a pluripotential stem cell is not necessary. Transplacental exposure of the donor fetuses to NEU alone, i.e., without additional postgrafting exposure, did not produce brain tumors in any of the experimental animals indicating that in the microenvironment of fetal brain transplants the multistep development of gliomas requires additional mutational events. Malignant schwanomas perinatally induced by NEU carry a point mutation in the transmembrane domain of the neu gene. The mode of oncogene activation in NEU-induced CNS gliomas has not yet been elucidated. We have used cerebral grafting techniques to study the effects of known oncogenes on the developing nervous system, taking advantage of efficient gene transfer by replication-defective retroviral vectors and of the extraordinary capacity of fetal CNS to differentiate in and fully integrate with the host brain. Rats carrying transplants exposed in vitro to the polyoma medium T-antigen developed endothelial hemangiomas in the graft which often led to fatal cerebral hemorrhage within 13-50 days after transplantation. Introduction of the viral src gene caused astrocytic and mesenchymal tumors after latency periods of 2-6 months. Following infection of fetal donor cells with a vector encoding the v-myc oncogene, only a single embryonal CNS tumor was observed whereas exposure to v-H-ras produced a low incidence of gliomas.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1998

Mutational analysis of the rous sarcoma virus DR posttranscriptional control element.

The direct repeat (DR) sequences flanking the src gene in Rous sarcoma virus are essential posttranscriptional control elements; at least one copy of this sequence is necessary for cytoplasmic accumulation of unspliced viral RNA. These sequences promote Rev-independent human immunodeficiency virus type 1 expression, suggesting they act as constitutive transport elements (CTEs). To determine which regions of this sequence are critical for CTE function, mutations in the downstream DR were generated and tested in a viral deletion construct lacking src and the upstream DR. Two single-point mutations and three different clustered mutations caused substantial reductions in reverse transcriptase activity, Gag protein levels, and unspliced viral RNA in the cytoplasm. Three conserved regions of the CTE, including nucleotides 8844 to 8847, 8862 to 8864, and 8868 to 8870, were most sensitive to inactivation by mutagenesis.

Animals↗
PubMed1980

Specific dephosphorylation of membrane proteins in Rous sarcoma virus-transformed chick embryo fibroblasts.

Chick embryo fibroblasts (CEF) infected with avian sarcoma virus become rapidly transformed as a result of expression of the viral src gene in the form of a single polypeptide of molecular weight 60,000 (pp60src) with protein kinase activity and suggested preferential association with the plasma membrane. Studies with normal avian and mammalian cells have revealed the presence of an antigenically related protein which seems to have similar kinase activity, but which is present at less than 1% of the levels of virally induced src protein found in transformed cells. As dynamic phosphorylation is important in numerous regulatory processes, the phenotypic expression of transformation may arise from an imbalance in one or more regulatory mechanisms that are controlled by protein phosphorylation. The cell membrane is affected during transformation, including its phosphotransferase activity. The latter has been shown using isolated membrane fractions whose properties may be changed during preparation. Therefore, we have compared the phosphorylation state of individual membrane proteins found in intact normal and RSV-transformed cells and report here the identification of two heavily phosphorylated, acidic membrane proteins in normal CEF which are specifically dephosphorylated on transformation by wild-type and temperature-sensitive Rous sarcoma viruses.

Animals↗
PubMed1979

Avian sarcoma virus-transformed quail clones defective in the production of focus-forming virus.

Quail embryo fibroblasts were infected at low multiplicity with avian sarcoma virus, and transformed cells were selected by their ability to form colonies in agar. Five clones that failed to produce focus-forming virus were examined for (i) intactness of the integrated proviral DNA, (ii) intracellular viral RNA production, (iii) intracellular viral antigen production, (iv) production of virus particles, and (v) rescue of a functional src gene and of parental host range determinants by superinfection with Rous-associated virus-60, an avian leukosis virus of subgroup E. Deletions in the integrated viral DNA were apparent in three of the five nonproducer clones. In one clone producing focus-forming virus, analysis of the integrated viral DNA revealed an insertion in the region of the genome that codes for src.

Animals↗
PubMed1985

Evolutionary variants of Rous sarcoma virus: large deletion mutants do not result from homologous recombination.

Large deletion (LD) mutants of Prague strain Rous sarcoma virus, subgroup B (PrB), derived by serial undiluted passage through chicken (C/E) cells, were isolated and characterized. Individual LD viruses were initially isolated by cloning in soft agar of infected, chemically transformed quail (QT6) cells. Two regions of the PrB genome were deleted in the formation of the LD virus. This resulted in the junction of gag sequences in p12 to env sequences in gp37, and in the loss of the src gene. DNA restriction analysis of biologically active lambda Charon 27-LD recombinant clones indicated that individual LD viruses contained similar but not identical deletion endpoints. Two LD isolates, LD25 and LD85, were further subcloned into pBR322, and the deletion junctions were examined by DNA sequencing. Although the gag-env deletion endpoints were identical in the two subclones, heterogeneity was observed across the src deletion in that both mutants analyzed had the same 5' endpoint but slightly different 3' endpoints. In all cases, only a single homologous base (always an A residue) was found at the deletion endpoint. S1 nuclease analysis of the RNA from a number of QT6-LD clones gave similar results, indicating that the LD population was composed of viruses with similar but not identical deletion endpoints. Such viruses may have been generated from errors during reverse transcription of the virion RNA with subsequent selection assuring their dominance in the population.

Avian Sarcoma Viruses↗
PubMed1996

Mechanisms of unusually high antioxidant activity of RSV-SR-transformed cells and of its suppression by activated p21ras.

We have previously demonstrated that hamster embryo fibroblasts (HEFs) transformed by Rous Sarcoma virus, Schmidt-Ruppin strain (RSV-SR) are highly resistant to damage by H202 (H2O2R), (in contrast to HEFs transformed spontaneously, or by bovine adenovirus and SV40), while N-ras transfection of RSV-SR transformants leads to suppression of pp6Ov-scr and of H2O2R. In this study we have examined (1) mechanisms of antioxidant activity (AOA) of HEFs transformed by these agents and (2) the possible role of the v-src gene in unusually high AOA of RSV-SR transformants and of activated ras oncogenes in its suppression. All transformants exhibit increased catalase and glutathione peroxidase (GP) activities, while SOD, glutathione and glutathione reductase (GR) were reduced. As compared with other transformants, the significantly higher catalase and the low SOD activities were characteristic of RSV-SR-transformants, while an increase in GP was observed in all types of transformants. Correspondingly, RSV-SR-transformants showed an extremely high H202-catabolizing activity (H2O2CA) and no lipid peroxidation chain reaction (LPCR). N-ras-induced suppression of pp60v-scr of RSV-SR-transformed HEFs coincided with the suppression of catalase, GP, H202 and H202CA. However, suppression of catalase and GP was also observed in N-ras- and Ha-ras-transfected, spontaneously transformed HEFs. Thus, extremely high catalase activity and suppression of LPCR are apparently the main mechanisms of the unusually high H202R of RSV-SR transformants, while its suppression by activated ras oncogenes may also take place in some transformants, free of v-src activity.

Animals↗
PubMed1990

Autocrine factor-independent growth of mammalian fibroblasts established in fully synthetic medium: no v-onc requirement in establishment.

In a previous study Chinese hamster fibroblasts carrying a partially deleted v-src were established in a synthetic medium lacking macromolecular supplements and shown to possess a particular serum-free phenotype hereafter designated sf. In cloning efficiency assays, sf, unlike wild-type, fibroblasts required a threshold cell density to grow from single cells, suggesting autocrine stimulation. In the present study a conditioned medium harvested from sf cells was added to the same sf cells, and the resulting cloning density was found to markedly diminish rather than increase. Sf cells were found to be unable to grow at cloning density because of trypsin damage: sf cells seeded into trypsin inhibitor-containing medium cloned with no requirement for threshold cells and were therefore independent of autocrine secretion from neighboring cells. Their cloning efficiency reached 7.7%; this value could not be improved by subcloning the sf culture, and it diminished when selenium was not added to the assay medium. To determine whether v-src is involved in the sf phenotype, five clones of the parental Chinese hamster fibroblast line not infected with Rous sarcoma virus were explanted into serum-free cultures with no macromolecular additives as in the case of v-src-containing cells. Each clone gave rise to an sf cell line growing indefinitely in synthetic medium like the v-src-containing sf cells, showing that the v-src gene is not required either for the establishment or maintenance of the sf phenotype.

Animals↗
PubMed1982

Role of p60src kinase activity in the induction of neuroretinal cell proliferation by rous sarcoma virus.

Expression of the src gene of Rous sarcoma virus (RSV) in chicken embryo neuroretinal (NR) cells results in morphological transformation and sustained proliferation of a normally resting cell population. We have previously reported the isolation of mutants of RSV which retain full growth-promoting activity while displaying reduced transforming properties. Two such mutants, PA101 and PA104, were used to investigate whether the p60src-associated kinase activity is required for the mitogenic function of src. A comparison of the patterns of phosphorylation of wild-type and mutant p60src revealed that the phosphorylation of tyrosine residues of p60src of PA104 was markedly reduced, whereas the relative amount of phosphotyrosine in p60src of PA101 was comparable to that of the wild-type protein. In vitro kinase activity of p60src immunoprecipitated from NR cells infected with PA101 or PA104 as measured by phosphorylation of the heavy chains of specific immunoglobulin G molecules was 1/10 that of the wild-type molecule. Moreover, when NR cells infected with mutants temperature sensitive for mitogenic capacity were maintained at a temperature either permissive or restrictive for cell growth, quantitation of kinase activity indicated that proliferation of NR cells could not be linked to the absolute level of in vitro kinase activity of p60src. Transformation of NR cells by wild-type RSV resulted in a 10-fold increase in total cellular phosphotyrosine and in the phosphorylation of tyrosine residues of a 34K protein, a possible in vivo substrate for p60src. In contrast, phosphorylation of tyrosine residues of cellular targets was markedly reduced in NR cells infected with PA101 or PA104. These results indicate that the mitogenic capacity of RSV in NR cells does not require elevated levels of p60src kinase activity.

Animals↗
PubMed1986

Effect of transformation by Rous sarcoma virus on the character and distribution of actin in Rat-1 fibroblasts: a biochemical and microscopical study.

Actin has been measured in subcellular fractions from Rat-1 fibroblasts and in Rous sarcoma virus-transformed Rat-1 cells (VIT), using the DNase 1 inhibition assay. The transformed cells showed a significant shift in the actin monomer (G)in equilibrium with polymer (F) equilibrium within the cell cytosol, and a significant increase in actin in the Triton-insoluble cytoskeletal core in comparison with untransformed cells. This incorporation of actin into the cytoskeletal core fraction is associated with a change in filamentous actin assemblies from 'stress fibre' patterns to punctate filament aggregates. These differences have been correlated with changes in morphology, in actin, vinculin and alpha-actinin distribution, in adhesion plaque formation and with the production of pp60v-src-associated protein kinase activity in the transformed cells. Changes in actin distribution and its polymerization in response to src-gene expression may play an important role in the determination of the transformed cell characteristics.

Actins↗
PubMed1984

Transformation by Rous sarcoma virus prevents acetylcholine receptor clustering on cultured chicken muscle fibers.

Acetylcholine receptors aggregate in the membrane of cultured chicken myotubes; the process of receptor clustering can be stimulated by exogenous factors that we, among others, have begun to characterize. Chicken myoblasts transformed by temperature-sensitive mutants of Rous sarcoma virus, such as tsNY68, fuse to form multinucleated myotubes at 42 degrees C, the nonpermissive temperature for transformation. However, tsNY68-infected myotubes do not cluster acetylcholine receptors at 42 degrees C, even in the presence of active clustering agents. This phenomenon is not merely a result of viral infection, since myotubes infected with a transformation-deficient viral mutant, td107A, behave like noninfected myotubes with respect to receptor clustering; thus, the effects of tsNY68 on the clustering process must be mediated by the src gene product. These experiments may provide a method of identifying essential elements of acetylcholine receptor clusters.

Animals↗
PubMed1995

ERF: an ETS domain protein with strong transcriptional repressor activity, can suppress ets-associated tumorigenesis and is regulated by phosphorylation during cell cycle and mitogenic stimulation.

ERF (ETS2 Repressor Factor) is a novel member of the ets family of genes, which was isolated by virtue of its interaction with the ets binding site (EBS) within the ETS2 promoter. The 2.7 kb ubiquitously expressed ERF mRNA encodes a 548 amino acid phosphoprotein that exhibits strong transcriptional repressor activity on promoters that contain an EBS. The localization of the DNA-binding domain of the protein at the N-terminus and th repression domain at the C-terminus is reminiscent of the organization of ELK1-like members of the ets family; however, there is no significant homology between ERF and ELK1 or any other ets member outside the DNA-binding domain. The repressor activity of ERF can antagonize the activity of other ets genes that are known transcriptional activators. Furthermore, ERF can suppress the ets-dependent transforming activity of the gag-myb-ets fusion oncogene of ME26 virus. Although ERF protein levels remain constant throughout the cell cycle, the phosphorylation level of the protein is altered as a function of the cell cycle and after mitogenic stimulation. The ERF protein is also hyperphosphorylated in cells transformed by the activated Ha-ras and v-src genes and the transcription repressor activity of ERF is decreased after co-transfection with activated Ha-ras or the kinase domain of the c-Raf-1 gene, indicating that ERF activity is probably regulated by the ras/MAPK pathway. Consistent with the in vivo phosphorylation and inactivation by ras, ERF is efficiently phosphorylated in vitro by Erk2 and cdc2/cyclin B kinases, at sites similar to those detected in vivo. Furthermore, a single mutation at position 526 results in the loss of a specific phosphopeptide both in in vivo and in vitro (by Erk2) labeling. Substitution of Thr526 for glutamic acid also decreases the repression ability of ERF. Our data suggest a model in which modulation of ERF activity is involved in the transcriptional regulation of genes activated during entry into G1 phase. Obstruction of the ERF repressor function by the transactivating members of the ets family of genes (i.e.gag-myb-ets) may be essential for the control of genes involved in cell proliferation and may also underlie their tumorigenic effects.

3T3 Cells↗
PubMed1985

Immunoprecipitation of insulin receptors from cultured human lymphocytes (IM-9 cells) by antibodies to pp60src.

The family of tyrosine-specific protein kinases includes proteins encoded by retroviral oncogenes as well as receptors for insulin and several growth factors. Antibodies to pp60src, the protein encoded by the src oncogene of Rous sarcoma virus (RSV), can specifically immunoprecipitate affinity-labeled insulin receptors from cultured human lymphocytes (IM-9 cells). This precipitation is specifically inhibited by the src gene product purified from RSV-transformed rat cells. These observations provide evidence that there is structural homology between the insulin receptors and pp60src.

Cross Reactions↗
PubMed1986

A nonviral, virus strain-specific antigen expressed on rat cells transformed by avian sarcoma virus.

Five of six rat sarcomas, induced by the Schmidt-Ruppin (SR) strain of avian tumor virus, expressed a Mr 60,000 tumor cell surface antigen (TSA), immunoprecipitable from non-ionic detergent extracts. Expression of the antigen was exclusive to rat cells transformed by the SR virus strain. Moreover, expression of TSA appeared restricted by cell type. The five TSA-positive SR-transformed rat cell lines tested were apparently of fibroblastic origin, but cultured rat cerebral endothelial cells (RCE-T1), transformed by SR virus, showed no expression of TSA. However, the antigen emerged on cultured tumors obtained after histoincompatible transplantation of these cells into newborn rats of another strain (tumor digest cells). Investigation of TSA for immunological relationship to viral structural antigens and the src gene product indicated that the TSA is distinct from any of these and more probably derives from a virus-directed alteration in a host molecule.

Animals↗
PubMed1978

Nucleotide sequences related to the transforming gene of avian sarcoma virus are present in DNA of uninfected vertebrates.

We have detected nucleotide sequences related to the transforming gene of avian sarcoma vius (ASV) in the DNA of uninfected vertebrates. Purified radioactive DNA (cDNAsarc) complementary to most of all of the gene (src) required for transformation of fibroblasts by ASV was annealed with DNA from a variety of normal species. Under conditions that facilitate pairing of partially matched nucleotide sequences (1.5 M NaCl, 59 degrees), cDNAsarc formed duplexes with chicken, human, calf, mouse, and salmon DNA but not with DNA from sea urchin, Drosophila, or Escherichia coli. The kinetics of duplex formation indicated that cDNAsarc was reacting with nucleotide sequences present in a single copy or at most a few copies per cell. In contrast to the preceding findings, nucleotide sequences complementary to the remainder of the ASV genome were observed only in chicken DNA. Thermal denaturation studies of the duplexes formed with cDNAsarc indicated a high degree of conservation of the nucleotide sequences related to src in vertebrate DNAs; the reductions in melting temperature suggested about 3--4% mismatching of cDNAsarc with chicken DNA and 8--10% mismatching of cDNAsarc with the other vertebrate DNAs.

Animals↗
PubMed1990

Identification of a novel neuronal C-SRC exon expressed in human brain.

Neuronal cells are known to express at least two different forms of the C-SRC proto-oncogene as a consequence of alternative splicing events which add an 18-nucleotide exon (the NI exon) between C-SRC exons 3 and 4. Here we report that a second neuronal exon of C-SRC is also present between C-SRC exons 3 and 4. This neuronal exon (the NII exon) of C-SRC was isolated from human adult and fetal brain-derived cDNAs and contains 33 nucleotides capable of encoding 11 amino acids (Gln-Thr-Trp-Phe-Thr-Phe-Arg-Trp-Leu-Gln-Arg). The human NI exon was located approximately 390 nucleotides from the end of C-SRC exon 3, whereas the NII exon was approximately 1,000 nucleotides from the beginning of C-SRC exon 4. Analysis of human brain RNA revealed that the NII exon is utilized primarily in conjunction with the NI exon to yield transcripts capable of encoding C-SRC products possessing 17 additional amino acids. These splicing events, which occur between the NI and NII exons, are predicted to alter the sixth amino acid encoded by the NI exon from an arginine to a serine residue, producing a potentially novel phosphorylation site. Analysis of the different C-SRC RNA transcripts revealed that the level of C-SRC RNA containing both NI and NII exons is similar in adult and fetal brain tissue, whereas the level of C-SRC RNA containing only the NI exon or the nonneuronal form of C-SRC RNAs is significantly higher in fetal brain tissues. These results indicate that the expression and splicing pattern of the C-SRC gene are developmentally regulated in the human brain.

Adult↗