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PubMed1980

Activation of cellular genes by avian RNA tumor viruses.

We demonstrated previously that chicken embryo fibroblasts accumulate approximately 100 copies of embryonic globin RNA after transformation by Rous sarcoma virus. Here we demonstrate that the globin gene in chicken embryo fibroblasts is activated by infection with two other oncogenic retroviruses, avian erythroblastosis virus and strain MC-29 of avian myeloblastosis virus, which contain transforming genes unrelated in nucleotide sequence content to each other or to the Rous sarcoma virus src gene. In addition, we have measured the genetic complexity of transformation by using established techniques for determining the number of different RNA sequences in specific populations of cells. Our results indicate that transformation of chicken embryo fibroblasts by Rous sarcoma virus results in the accumulation of RNA from approximately 1000 average-sized new transcription units.

Alpharetrovirus↗
PubMed2003

Regulation of cytochrome c oxidase activity by c-Src in osteoclasts.

The function of the nonreceptor tyrosine kinase c-Src as a plasma membrane-associated molecular effector of a variety of extracellular stimuli is well known. Here, we show that c-Src is also present within mitochondria, where it phosphorylates cytochrome c oxidase (Cox). Deleting the c-src gene reduces Cox activity, and this inhibitory effect is restored by expressing exogenous c-Src. Furthermore, reducing endogenous Src kinase activity down-regulates Cox activity, whereas activating Src has the opposite effect. Src-induced Cox activity is required for normal function of cells that require high levels of ATP, such as mitochondria-rich osteoclasts. The peptide hormone calcitonin, which inhibits osteoclast function, also down-regulates Cox activity. Increasing Src kinase activity prevented the inhibitory effect of calcitonin on Cox activity and osteoclast function. These results suggest that c-Src plays a previously unrecognized role in maintaining cellular energy stores by activating Cox in mitochondria.

Animals↗
PubMed1994

Expression of Bruton's agammaglobulinemia tyrosine kinase gene, BTK, is selectively down-regulated in T lymphocytes and plasma cells.

The gene mutated in the human disease, X-linked agammaglobulinemia (XLA), is related to the Src gene family of cytoplasmic protein-tyrosine kinases and is designated Btk (Bruton's agammaglobulinemia tyrosine kinase; formerly Atk/Bpk; the human gene is denoted BTK, using capital letters according to the kinase nomenclature). We have recently reported that this gene is expressed in B lymphocytes and that the specific mRNA was undetectable in T cells using Northern blotting. Further analyses of different sources of B and T lymphocytes confirmed this pattern. However, BTK transcripts were undetectable in four plasmacytoma lines. Moreover, as virtually normal amounts of BTK transcripts were found in PBMC from two patients carrying a point mutation in BTK, despite low B cell numbers, we anticipated that the gene would also be expressed in cells of other lineages. The erythroleukemia cell line K-562, the promyelocytic line HL-60 and the histiocytic lymphoma line U-937 were found to have BTK mRNA levels comparable to B cells. BTK mRNA was also detected in monocytes from healthy donors as well as in the human immature basophilic cell line KU812, in the human mast cell leukemia cell line HMC-1 and in the CD34 expressing myeloblast KG-1. A similar expression pattern was obtained when BTK protein was analyzed by immunoprecipitation and Western blotting. Using a polymerase chain reaction-based analysis, a small amount (less than 1% of the level in B cells) of BTK mRNA was identified in T lymphocytes. Our findings are compatible with a general expression of the BTK gene in hematopoietic cells, except in T lymphocytes and plasma cells, in which the transcript level is selectively down-regulated.

Agammaglobulinemia↗
PubMed1983

Nucleotide sequences of the Drosophila src and abl homologs: conservation and variability in the src family oncogenes.

Two cloned Drosophila sequences, Dash and Dsrc, were previously isolated by hybridization to the viral oncogenes v-abl and v-src. We report that the Drosophila DNA sequences are more than 50% homologous to 700 base pairs of the vertebrate oncogenes that are essential for kinase activity of the v-abl and the v-src gene products. Alignment of the predicted amino acid sequences in the homologous regions shows amino acid sequence identities of 74% between Dash and the 5' portion of v-abl and of 54% between Dsrc and 3' portion of v-src. Comparison of the Drosophila and vertebrate sequences identifies amino acids that may be essential for the distinct functions of the c-src and the c-abl gene products and places the gene duplication event that has generated the two genes prior to the Chordate-Arthropod divergence.

Amino Acid Sequence↗
PubMed1988

Elevated expression of pp60c-src alters a selective morphogenetic property of epithelial cells in vitro without a mitogenic effect.

Madin-Darby canine kidney (MDCK) cells are highly differentiated and have retained the morphogenetic properties necessary to form polarized, multicellular epithelial structures (cysts) in vitro that resemble epithelial tissues in vivo. We introduced the c-src gene into MDCK cells to elevate the level of the plasma membrane-associated cellular tyrosine kinase, pp60c-src, to levels two- to ninefold higher than that expressed in parent MDCK cells. Our results revealed a highly discriminatory biological action of pp60c-src on the morphogenetic properties of MDCK cells. Elevated expression of pp60c-src conferred on MDCK cells the ability to undergo dramatic changes of cell shape that includes the formation of long cell processes (100 to 200 microns), never observed in control MDCK cells. The morphogenesis of multicellular epithelial cysts was altered by elevated levels of pp60c-src and led to predictable distortions of their three-dimensional architecture. However, these cells established morphologically normal cell polarity, formed adhesive epithelial cell-cell contacts indistinguishable from those of control MDCK cells, and exhibited neither focus-forming ability or anchorage-independent growth potential. Finally, we showed that MDCK cells expressing elevated levels of pp60c-src exhibit increased phosphorylation of a more limited number of phosphotyrosine-containing proteins than MDCK cells expressing pp60v-src. We suggest that a natural function of pp60c-src is to regulate the morphogenetic properties which determine the shape of differentiated cells and multicellular structures.

Animals↗
PubMed1995

Signalling by the p60c-src family of protein-tyrosine kinases.

The c-src gene family has nine known members (blk, c-fgr, fyn, hck, lck, lyn, c-src, c-yes and yrk), each encoding a cytoplasmic protein-tyrosine kinase (PTK) believed to be involved in signal transduction. The c-src PTKs contain three domains (SH1, SH2 and SH3) that are found in many other signalling proteins. The SH1 domain has PTK activity, whilst the SH2 and SH3 domains are involved in mediating protein-protein interactions by binding to phosphotyrosine-containing and proline-rich motifs, respectively. The expression patterns of the c-src PTKs suggest that they function in a broad range of biological situations, in many cases regulating the behaviour of terminally-differentiated, post-mitotic cell types. Targeted disruption of members of the c-src family in transgenic mice has confirmed important roles for p56lck and p59fym(T) in T-lymphocyte maturation and activation, but has also revealed unexpected roles for p60c-src in bone maintenance and for p59fym(B) in learning and memory. There is increasingly detailed information about the biochemical nature of the signalling pathways in which the c-src PTKs operate and about the other signalling proteins with which they interact. The c-src PTKs can associate with activated receptor PTKs, including the receptors for platelet-derived growth factor and epidermal growth factor, by means of SH2-phosphotyrosine binding. The c-src PTKs also associated with transmembrane proteins that lack PTK activity, frequently by means of interactions involving their unique amino-terminal sequences.

Animals↗
PubMed2002

Early phenotypic changes of in vitro transformed cells during in vivo progression: possible role of the host innate immunity.

We described the earliest in vivo changes of in vitro transformed cells of various origin manifested in highly-increased H(2)O(2)-catabolizing and PGE(2)-releasing activities. We designate it as [H(2)O(2)(CA) + PGE(S)]-phenotype. It provided tumor cells with resistance to cytotoxicity of Mph and NK cells and correlated with increased tumorigenicity. Used as a functional marker of the early step of in vivo tumor progression [H(2)O(2)(CA) + PGE(S)]-phenotype allowed to estimate the difference in the rates of natural selection of tumor cells grown locally and/or disseminated and its significant delay related to bcl-2 gene expression. The data suggests that in vivo sublethal damage of tumor cells and activation of src gene are relevant to acquisition of [H(2)O(2)(CA) + PGE(S)]-phenotype.

Animals↗
PubMed1989

A mutation in v-src that removes a single conserved residue in the SH-2 domain of pp60v-src restricts transformation in a host-dependent manner.

The v-src oncogene of Rous sarcoma virus (RSV) is able to transform both avian and mammalian cells, but the mutant allele v-src-L displays a host range dependence for transformation, transforming chicken but not rat cells with wild-type efficiency. This host range restriction can be detected by measuring growth in low serum, saturation density, and anchorage independent growth. In addition, rat cells expressing v-src-L do not form tumors in syngeneic rats or nude mice, but RSV carrying the mutant allele causes tumors in chicks, although at a reduced efficiency and with increased latency. To determine the lesion responsible for this phenotype, we sequenced the entire v-src gene from the parental B77 strain of RSV, as well as the mutant allele. v-src-L is missing 3 nucleotides present in the wild-type parent, RSV B31, eliminating Phe-172, an invariant residue in a conserved region of src-related proteins known as SH-2. The kinase activity of pp60v-src-L was indistinguishable from that of the wild type in chicken cells but was significantly reduced in rat cells as assayed by an in vitro immune complex assay; in vivo phosphorylation of one specific substrate, p36 (calpactin I heavy chain); and total phosphotyrosine-containing proteins. In addition, the pattern of phosphotyrosine-containing proteins in rat cells was qualitatively different when cells containing pp60v-src-L were compared with cells with wild-type pp60v-src, even though both pp60v-src proteins were membrane associated. The data are consistent with a role for the SH-2 region in substrate specificity.

Alleles↗
PubMed1986

Altered stem cell (CFU-S) function following infection of hematopoietic cells with a virus carrying V-src.

Long-term murine bone marrow cultures were used to support the growth and development of hematopoietic cells. After hematopoiesis was established, the cultures were infected with a recombinant murine amphotropic virus carrying the avian sarcoma virus src gene and the CFU-S kinetics were examined. The CFU-S from the src-infected cultures displayed a reduced seeding efficiency in the standard spleen colony assay. The self-renewal capacity of these CFU-S was tested by their ability to reestablish hematopoiesis when serially transplanted on irradiated bone marrow cultures and by serial passage in spleens of irradiated mice. In both tests, cells from the src-infected cultures exhibited an enhanced ability to sustain a high level of self-renewal. The other property of stem cells which may be measured is the probability of self-renewal at each cell division which dictates the distribution between stem cells and differentiated type progeny. CFU-S from the src-infected cultures had higher average probabilities of self-renewal and therefore reduced differentiation. These differences suggest that expression of src had indirectly or directly altered the normal differentiation program of the stem cells.

Animals↗
PubMed1979

The in vitro of Rous sarcoma virus RNA and function of the viral protein during the viral replication.

The gag gene and pol gene of the Rous sarcoma virus are translated in vitro from the 35S viral RNA. The env gene cannot be translated in vitro from the 35S RNA. For the in vitro translation of the src gene. 3' end fragments of the viral RNA are used. The gag protein p15 has a proteolytic activity and specifically processes its own protein precursor pr76. The gag protein p19 suppresses the in vitro translation of the pol gene.

Animals↗
PubMed1982

Inhibition of tyrosine protein kinases by halomethyl ketones.

A chloromethyl ketone derivative of lactic acid was shown to inhibit protein phosphorylation in plasma membranes of Ehrlich ascites tumor cells [Johnson, H. J., Zimniak, A., & Racker, E. (1982) Biochemistry 21, 2984-2989]. We now show that this inhibitor as well as three halomethyl ketone derivatives of amino acids and peptides specifically inhibits tyrosine protein kinase activity in intact plasma membranes and Triton extracts of plasma membrane of A-431 tumor cells. The most effective inhibitor is a bromomethyl ketone derivative of leucine that inhibits the phosphorylation of a protein that migrates to the same position as the EGF receptor in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Inhibition of phosphorylation took place in the presence or absence of added EGF, and the inhibitor did not interfere with the binding of EGF to the receptor nor with the dephosphorylation of the EGF-stimulated phosphoprotein. EGF-dependent phosphorylation in a Triton extract of plasma membranes from normal placenta was considerably less sensitive to the bromomethyl ketone derivative of leucine. The tyrosine protein kinase activity of the transformation gene product of Fujinami virus was particularly sensitive to the bromomethyl ketone derivative of leucine, while the src gene product of Rous sarcoma virus was comparatively less sensitive. The bromomethyl ketone inhibitor interfered with the phosphorylation of the EGF receptor by [gamma-32P]-8-azido-ATP but much less with the light-sensitive binding. This observation and the lack of interference with EGF binding suggest that the inhibitor interacts with the protein kinase portion of the receptor complex.

Avian Sarcoma Viruses↗
PubMed1986

DNA methylation and transcriptional controls of proviral DNA in avian sarcoma virus-transformed mammalian cells.

Restriction mapping has been used to study the integration state of the single provirus present in the DNA of two subclones, RS2/3 and RS2/6, of hamster cells transformed in vitro by Rous sarcoma virus, but differing markedly in their level of proviral transcription which was higher in RS2/3 cells. It was observed that both proviruses are complete and located at the same integration site in each DNA. However, the RS2/6 provirus and its flanking cellular sequences were found to be hypermethylated, although a very short region was hypomethylated at about 1 kb upstream of the src gene. A low level of methylation was observed in RS2/3 cells, in the proviral region. Northern analysis of viral RNA detected only the src mRNA in RS2/6 cells, whereas the two other viral mRNA were found in RS2/3 cells, however their levels were very low compared to that of the src mRNA. These findings suggest a correlation between the methylation state and the transcriptional control of the proviral genes. Sequences responsible for such a control by methylation should lie within both the provirus and its 5' flanking cellular sequences.

Animals↗
PubMed1989

Transgenic mice and host cell mutants resistant to transformation as model systems for identifying multiple components in oncogenesis.

Tumorigenesis appears to be a multistep process involving mutations of conventional, dominantly acting proto-oncogenes, mutations of other genes that may act in a recessive manner, and interactions (or a lack of interactions) between the products of mutant and wild-type genes. Our laboratory is using a few well-established, dominant oncogenes to pose experimental questions that could lead to a better understanding of the more elusive genetic interactions which occur during tumour development. Two such situations are described: (1) We have created a line of transgenic mice that carry the int-1 proto-oncogene under the control of the enhancer element in the mouse mammary tumour virus long terminal repeat. Such mice express the transgene in mammary glands, salivary glands and male reproductive tract; mammary glands from both male and female animals are grossly hyperplastic, yet tumours arise rarely in the males and sporadically in the females (80% of female mice have one or a few tumours by six months of age). Thus expression of int-1 in these mice appears to place a large number of mammary cells at risk for secondary events that lead to carcinogenesis, providing a provocative experimental context for identifying such secondary events. (2) We have isolated a rat cell line that lacks most of the characteristics of transformed cells, despite the expression of two wild-type copies of the v-src gene of Rous sarcoma virus. This line harbours what appears to be a dominant mutation in an unidentified gene that renders the cell resistant to transformation by v-src and several other oncogenes. Isolation of the mutant gene responsible for suppressing transformation in this line should provide new insights into the interactions between oncoproteins and other cellular proteins.

Animals↗
PubMed1994

Aberrant protein phosphorylation at tyrosine is responsible for the growth-inhibitory action of pp60v-src expressed in the yeast Saccharomyces cerevisiae.

Expression of pp60v-src, the transforming protein of Rous sarcoma virus, arrests the growth of the yeast Saccharomyces cerevisiae. To determine the basis of this growth arrest, yeast strains were constructed that expressed either wild-type v-src or various mutant v-src genes under the control of the galactose-inducible, glucose repressible GAL1 promoter. When shifted to galactose medium, cells expressing wild-type v-src ceased growth immediately and lost viability, whereas cells expressing a catalytically inactive mutant (K295M) continued to grow normally, indicating that the kinase activity of pp60v-src is required for its growth inhibitory effect. Mutants of v-src altered in the SH2/SH3 domain (XD4, XD6, SPX1, and SHX13) and a mutant lacking a functional N-terminal myristoylation signal (MM4) caused only a partial inhibition of growth, indicating that complete growth inhibition requires either targeting of the active kinase or binding of the kinase to phosphorylated substrates, or both. Cells arrested by v-src expression displayed aberrant microtubule structures, alterations in DNA content and elevated p34CDC28 kinase activity. Immunoblotting with antiphosphotyrosine antibody showed that many yeast proteins, including the p34CDC28 kinase, became phosphorylated at tyrosine in cells expressing v-src. Both the growth inhibition and the tyrosine-specific protein phosphorylation observed following v-src expression were reversed by co-expression of a mammalian phosphotyrosine-specific phosphoprotein phosphatase (PTP1B). However a v-src mutant with a small insertion in the catalytic domain (SRX5) had the same lethal effect as wild-type v-src, yet induced only very low levels of protein-tyrosine phosphorylation. These results indicate that inappropriate phosphorylation at tyrosine is the primary cause of the lethal effect of pp60v-src expression but suggest that only a limited subset of the phosphorylated proteins are involved in this effect.

Avian Sarcoma Viruses↗
PubMed1996

c-Cbl is downstream of c-Src in a signalling pathway necessary for bone resorption.

The primary defect in mice lacking the c-src gene is osteopetrosis, a deficiency in bone resorption by osteoclasts. Osteoclasts express high levels of the c-Src protein and the defect responsible for the osteopetrotic phenotype of the c-src-deficient (src-) mouse is cell-autonomous and occurs in mature osteoclasts. However, the specific signalling pathways that require c-Src expression for normal osteoclast activity have not been elucidated. We report here that the proto-oncogene product c-Cbl is tyrosine-phosphorylated in a Src-dependent manner in osteoclasts, where the two proteins colocalize on some vesicular structures. In vitro bone resorption by osteoclast-like cells (OCLs) is inhibited by both c-src and c-cbl antisense oligonucleotides. Furthermore, tyrosine phosphorylation of c-Cbl and the localization of c-Cbl-containing structures to the peripheral cytoskeleton are impaired in resorption-deficient c-src- OCLs, as well as in wild-type OCLs that have been treated with c-src antisense oligonucleotides. These results indicate that c-Cbl may act downstream of c-Src in a signalling pathway that is required for bone resorption.

3T3 Cells↗
PubMed1994

Endothelin peptides and compensatory growth of renal cells.

Endothelins are paracrine or autocrine peptides that regulate diverse aspects of renal function. In addition to their potent vasoconstrictor activity, recent evidence suggests that endothelin-1 is a growth factor for renal cells. Different forms of renal injury markedly upregulate endothelin-1 secretion, which is postulated to contribute to compensatory renal growth. Similar roles have been hypothesized for other vasoactive peptides, such as angiotensin II and arginine vasopressin. New information has recently emerged regarding pathways of mitogenic signaling linking activation of endothelin receptors to changes in gene expression. ETA receptor subtypes activate downstream effectors, such as protein kinase C, protein tyrosine kinases of the src gene family, and mitogen-activated protein kinases. These cytosolic effectors in turn lead to altered programs of gene expression by activating, among others, AP-1 and serum response factor transcription factors. In addition, recent studies in organisms amenable to genetic analysis, such as Drosophila, Dictyostelium, and yeast, are providing important clues to effector mechanisms employed by vasoactive peptide receptors in higher organisms. Information on the molecular mechanisms for mitogenic signaling by endothelin receptors might be used to gain insight into the pathogenesis of compensatory renal growth and the development of novel therapeutic strategies.

Animals↗
PubMed1988

Temperature-sensitive cellular mutant for expression of mRNA from murine retrovirus.

The cellular mutant B812 isolated from a Fisher rat cell line shows temperature sensitivity of focus formation induced by various retroviruses such as recombinant murine retrovirus containing the middle T gene of polyomavirus (PyMLV), Kirsten murine sarcoma virus, Moloney murine sarcoma virus, and recombinant murine retrovirus containing the src gene of Rous sarcoma virus. B812 cells, however, show normal ability to proliferate and synthesize protein at the nonpermissive temperature, suggesting that their mutation is in a gene specifically concerned with the process of transformation by retroviruses. In this work, experiments with hybrids of mutant and wild-type cells showed that the temperature-dependent defect of this mutant was complemented by wild-type cells. To determine the step of transformation that is restricted at the nonpermissive temperature in B812, we examined the expressions of the oncogene (middle T antigen) in no. 7 (wild-type cells) and B812 cultures infected with PyMLV (the chimeric retrovirus containing the middle T gene of polyomavirus) at the permissive and nonpermissive temperatures. Middle T-associated protein kinase activity, the expression of middle T antigen, and PyMLV-specific mRNA were reduced at the nonpermissive temperature in B812 cultures infected with PyMLV. However, integration of PyMLV into the chromosomal DNA of the mutant was not affected at the nonpermissive temperature. These results suggest that B812 cells have a mutation affecting the expression of viral mRNAs from integrated proviral DNA at the nonpermissive temperature.

Animals↗
PubMed2001

The hunting of the Src.

The non-receptor tyrosine kinase Src is important for many aspects of cell physiology. The viral src gene was the first retroviral oncogene to be identified, and its cellular counterpart was the first proto-oncogene to be discovered in the vertebrate genome. Src has been important, not only as an object of study in itself, but also as an entry point into the molecular genetics of cancer.

Animals↗