Inability of Rous sarcoma virus to cause sarcomas in the avian embryo

The injection of Rous sarcoma virus (RSV) into the wing web of newly hatched chicks causes a rapidly growing sarcomatous tumour which is palpable within 1 week of inoculation; and cultures of fibroblasts derived from chick embryos (CEF) and infected with RSV become rapidly transformed. Genetic studies have determined that expression of a single viral gene, designated v-src, is necessary for neoplastic transformation. This gene codes for a 60,000-molecular weight phosphoprotein termed pp60SPC , which possesses a protein kinase activity that phosphorylates polypeptides on tyrosine residues and is constitutively expressed in infected CEF cells. It has been suggested that transformation, and possibly tumorigenesis, may result solely from the consequences of this increase in tyrosine phosphorylations. The pathogenicity of RSV in chick embryos in ovo is less clear. Murphy and Rous suggested that RSV may have caused tumours in "various tissues" of "some embryos", but the subsequent studies of Milford and Duran - Reynals , as well as several other laboratories, failed to find any evidence of intraembryonic tumours in RSV-infected early embryos. The findings of Duran - Reynals , if correct, cannot be explained easily in view of our present understanding of RSV tumorigenicity. Thus, we have re-examined the interaction of RSV with the avian embryo and confirm here that RSV is nontumorigenic and non-teratogenic when microinjected into day 4 chicken embryos. In addition, we found that (1) the virus not only replicates in the embryo, but it also expresses an active src-specific protein kinase and (2) once the cells from the infected limbs are disrupted and placed in culture, they are capable of expressing the transformed phenotype after a 24-h delay.     

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.     

Three loci related to the src oncogene and tyrosine-specific protein kinase activity in Drosophila

Rous sarcoma virus (RSV) is an acutely oncogenic avian retrovirus which induces sarcomas in animals and transforms fibroblasts in cell culture. Genetic analysis indicates that the viral src gene (v-src) mediates neoplastic transformation. The product of v-src is a 60,000 molecular weight (MW) phosphoprotein (pp60v-src) possessing the enzymatic activity of a tyrosine-specific protein kinase. The viral src gene is derived from a cellular gene (c-src) which also encodes a 60,000 MW phosphoprotein (pp60c-src) with tyrosine-specific protein kinase activity. Both birds and mammals are known to possess c-src. Shilo and Weinberg have reported that the genome of the fruit fly, Drosophila melanogaster, contains nucleotide sequences that are homologous to v-src. We report here the molecular cloning and chromosomal mapping of three loci from the Drosophila genome that contain such sequences. We also show that Drosophila contain both phosphotyrosine and a tyrosine-specific protein kinase activity immunoprecipitated by antisera directed against pp60v-src. It should now be possible to identify the precise locus that encodes a src-specific protein kinase in Drosophila, and to explore the role of c-src in the growth and development of D. melanogaster.     

An N-terminal peptide from p60src can direct myristylation and plasma membrane localization when fused to heterologous proteins

The src gene product, p60src, of Rous sarcoma virus (RSV) is a tyrosine-specific protein kinase which is associated with the plasma membrane of infected cells. Myristic acid is bound in an amide linkage to glycine 2 of p60src. Of the N-terminal 30 kilodaltons of p60src, only amino acids 1-14 are required for myristylation, and myristylation of p60src may be required for its membrane association, and for cell transformation. To test the hypothesis that the first 14 amino acids of p60src contain a recognition sequence for myristylation, we have fused the DNA sequence coding for these amino acids to either the fps gene of the F36 derivative of Fujinami sarcoma virus (FSV), or to the chimpanzee alpha-globin gene. We report here that although the fusion proteins were myristylated, the parental proteins were not, and unlike the non-myristylated F36 p91fps which was not bound to the plasma membrane, the myristylated fusion protein was bound, like p60src. We conclude that the first 14 amino acids of p60src contain a sequence which is sufficient for myristylation, and which may direct proteins to the plasma membrane.     

A mutant protein kinase C that can transform fibroblasts

Expression of normal protein kinase C (PKC) isoenzymes in fibroblasts has been shown to alter growth regulation but has failed to induce complete transformation of the recipient cells. Here we report on a murine ultraviolet-induced fibrosarcoma cell line which has an unusual PKC subcellular distribution with 87% of the PKC activity associated with the membrane. We have cloned and sequenced the alpha-PKC complementary DNA from ultraviolet-induced-fibrosarcoma cells and from mouse Balb/c brain and found four point mutations in the fibrosarcoma PKC, of which three are in the highly conserved regulatory domain and one is in the conserved region of the catalytic domain. Expression of this mutant alpha-PKC gene in normal Balb/c 3T3 fibroblasts results in a fibrosarcoma-like PKC membrane localization and in cell transformation, as judged by their formation of dense foci, anchorage-independent growth and ability to induce solid tumours when inoculated into nude mice. By contast, transfectants expressing the normal alpha-PKC cDNA do not display a morphology typical of malignant transformed cells and fail to induce tumours in vivo. These findings demonstrate that point mutations in the primary structure of PKC modulate enzyme function and are responsible for inducing oncogenicity.     

Abelson murine leukaemia virus transformation involves loss of epidermal growth factor-binding sites

Malignant transformation by mammalian RNA sarcoma viruses has previously been shown to involve a reduction in receptor sites for a well characterized 6,000-molecular weight (MW) growth-promoting substance, designated epidermal growth factor (EGF). Although Abelson murine leukaemia virus (AbLV) resembles sarcoma viruses in its ability to transform embryo fibroblasts in cell culture, AbLV induces a rapid B-cell lymphoid leukaemia rather than fibrosarcomas in vivo. The major translational product of AbLV is a highly phosphorylated polyprotein of MW 120,000 which exhibits an associated tyrosine-specific protein kinase activity and probable transforming function. We show here that AbLV transformation resembles transformation by RNA sarcoma viruses with respect to the abolition of EGF-binding sites. EGF binding is restored to control levels following loss of polyprotein expression in morphological revertants of AbLV-transformed clones and remains uninfluenced in cell lines infected with transformation-defective (td) AbLV mutants encoding polyproteins deficient in protein kinase activity. These findings indicate that AbLV transformation involves a polyprotein-associated, tyrosine-specific protein kinase activity which mediates its effect through a mechanism resulting directly or indirectly in the abolition of EGF-binding sites.     

Effect of cell history on response to helix-loop-helix family of myogenic regulators

In multinucleated heterokaryons formed from the fusion of differentiated muscle cells to either hepatocytes or fibroblasts, muscle-specific gene expression is activated, liver-specific gene expression is repressed, and there are changes in the location of the Golgi apparatus. An understanding of the regulatory mechanisms that underlie this plasticity is of particular interest given the stability of the differentiated state in vivo. We have now investigated whether MyoD or myogenin, regulators of muscle-specific gene expression that have a helix-loop-helix motif, can induce the phenotypic conversion observed in heterokaryons. When these regulators were stably or transiently introduced into fibroblasts or hepatocytes by microinjection, transfection or retroviral infection with complementary DNA in expression vectors, fibroblasts expressed muscle-specific genes, whereas hepatocytes did not. However, fusion of hepatocytes stably expressing MyoD to fibroblasts resulted in activation in the heterokaryon of muscle-specific genes of both cell types. These results imply that other regulators, present in fibroblasts but not in hepatocytes, are necessary for the activation of muscle-specific genes, and indicate that the differentiated state of a cell is dictated by its history and a dynamic interaction among the proteins that it contains.     

Direct evidence that oncogenic tyrosine kinases and cyclic AMP-dependent protein kinase have homologous ATP-binding sites

p60src, the transforming protein of Rous sarcoma virus (RSV), is a protein kinase that has a strict specificity for tyrosine. The phosphorylation of cellular proteins by p60src (ref. 4) results in transformation. Recently, Barker and Dayhoff discovered that residues 259-485 of p60src have 22% sequence identity with residues 33-258 of the catalytic subunit of cyclic AMP-dependent protein kinase, an enzyme that has a specificity for serine. Because it was necessary to introduce eight gaps to align the two proteins, the question remained as to whether this apparent homology reflected a common evolutionary origin. We demonstrate here that the ATP analogue p-fluorosulphonylbenzoyl 5'-adenosine (FSBA) inactivates the tyrosine protein kinase activity of p60src by reacting with lysine 295. When aligned for maximum sequence identity, lysine 295 of p60src and the lysine in the catalytic subunit which also reacts specifically with FSBA are superimposed precisely. This functional homology is strong evidence that the protein kinases, irrespective of amino acid substrate specificity, comprise a single divergent gene family.     

Possible positive autocrine feedback in the prereplicative phase of human fibroblasts

The growth of normal diploid fibroblasts is generally thought to be tightly controlled by exogenous growth factors such as platelet-derived growth factor (PDGF) and epidermal growth factor (EGF). Subversion of a growth factor pathway at a regulatory point is considered to be a key event in neoplastic transformation and tumorigenesis. Thus, simian sarcoma virus has acquired the gene encoding the B-chain of PDGF and there is direct experimental proof that SSV-transformation is mediated by a PDGF-like growth factor. There is accumulating evidence that PDGF-like molecules are also synthesized and released by certain normal cells, suggesting an important role of cellularly produced PDGF in development and tissue regeneration. We now present evidence that a transient expression of the gene encoding the PDGF A-chain, and the synthesis and release of functional A-chain homodimers, is an early event in the prereplicative phase of normal human foreskin fibroblasts exposed to PDGF or EGF. Since these cells are PDGF-responsive, the results imply the existence of a positive autocrine signal that may serve as an amplifier of the mitogenic response under certain conditions.     

Local degradation of fibronectin at sites of expression of the transforming gene product pp60src

Local degradation of extracellular fibronectin, a major extracellular adhesive protein, is believed to play an important part in the migration of cells through the extracellular matrix during tumour invasion, morphogenetic movement and trophoblast implantation. Fibronectin is lost from the cell surface after transformation with Rous sarcoma virus (RSV). By using fluorescent and radiolabelled probes covalently coupled to the surface of substrata, we have recently identified a proteolytic activity that is expressed in RSV-transformed cells and is involved in the local degradation of fibronectin at cell-substratum contact sites. Here, we extend the relevance of these findings and gain some insight into the cellular functions of pp60src, the transforming gene product of RSV. We show that newly expressed viral pp60src is localized at the cytoplasmic surface of the cell membrane, corresponding to the cell contact sites where degradation of extracellular fibronectin occurs.     

Deregulated cyclin E induces chromosome instability.

Cyclin E, a regulatory subunit of cyclin-dependent kinase 2 (Cdk2), is an important regulator of entry into S phase in the mammalian cell cycle. In normal dividing cells, cyclin E accumulates at the G1/S-phase boundary and is degraded as cells progress through S phase. However, in many human tumours cyclin E is overexpressed and the levels of protein and kinase activity are often deregulated relative to the cell cycle. It is not understood how alterations in expression of cyclin E contribute to tumorigenesis. Here we show that constitutive cyclin-E overexpression in both immortalized rat embryo fibroblasts and human breast epithelial cells results in chromosome instability (CIN). In contrast, analogous expression of cyclin D1 or A does not increase the frequency of CIN. Cyclin-E-expressing cells that exhibit CIN have normal centrosome numbers. However, constitutive overexpression of cyclin E impairs S-phase progression, indicating that aberrant regulation of this process may be responsible for the CIN observed. These results indicate that downregulation of cyclin-E/Cdk2 kinase activity following the G1/S-phase transition may be necessary for the maintenance of karyotypic stability.     

Tumorigenic conversion of primary embryo fibroblasts requires at least two cooperating oncogenes

Transfection of embryo fibroblasts by a human ras oncogene does not convert them into tumour cells unless the fibroblasts are established and immortalized before transfection. The embryo fibroblasts become tumorigenic if a second oncogene such as a viral or cellular myc gene or the gene for the polyoma large-T antigen is introduced together with the ras gene.     

The src gene product of transformed and morphologically reverted ASV-infected mammalian cells.

Morphological revertants of avian sarcoma virus transformed vole cells contain the sarcoma gene product (pp60src) in an enzymatically active form, suggesting that the presence of pp60src protein kinase activity is infussicient to induce morphological transformation. Structural analyses of pp60src from infected vole cell clones suggest that in one of the revertant clones on alteration in pp60src may be responsible for morphological reversion while in a second clone, reversion may result from an alteration in a cell gene product with which pp60src must interact. As these morphological revertant cells are tumorigenic, different cell components are required to interact with pp60src to facilitate the two events.     

A neuronal clone derived from a Rous sarcoma virus-transformed quail embryo neuroretina established culture

Neuroretina (NR) is an evagination of the central nervous system (CNS) which is composed of photoreceptors, glial (Müller) cells and horizontal, bipolar, amacrine and ganglion neuronal cells. We describe here the usefulness of Rous sarcoma virus (RSV) in the establishment of a neuronal clone from quail embryo neuroretina. When primary cultures of chick and quail embryo neuroretina cells are transformed by RSV, neuronal markers such as ribbon synapses, choline acetyltransferase (CAT) and glutamic acid decarboxylase (GAD) specific activity are present. These RSV-transformed primary cultures can be established into permanent cell lines from which neuronal clones have been isolated. One of them, clone QNR/D, can generate tetrodotoxin(TTX)-inhibitable action potentials on electrical stimulation, has a high GAD activity and binds monoclonal antibodies raised against chick embryo neuroretina. The presence of these neuronal markers suggests that the QNR/D clone is derived from cells of the amacrine or ganglionic lineage. This is the first time that a neuronal cell clone of defined origin has been obtained from the CNS. The neuronal markers of the QNR/D clone are expressed at both the permissive and the non-permissive temperatures for transformation.     

Type I phosphatidylinositol kinase makes a novel inositol phospholipid, phosphatidylinositol-3-phosphate

The generation of second messengers from the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PtdInsP2) by phosphoinositidase C has been implicated in the mediation of cellular responses to a variety of growth factors and oncogene products. The first step in the production of PtdInsP2 from phosphatidylinositol (PtdIns) is catalysed by PtdIns kinase. A PtdIns kinase activity has been found to associate specifically with several oncogene products, as well as with the platelet-derived growth factor (PDGF) receptor. We have previously identified two biochemically distinct PtdIns kinases in fibroblasts, and have found that only one of these, designated type I, specifically associates with activated tyrosine kinases. We have now characterized the site on the inositol ring phosphorylated by type I PtdIns kinase, and find that this kinase specifically phosphorylates the D-3 ring position to generate a novel phospholipid, phosphatidylinositol-3-phosphate (PtdIns(3)P). In contrast, the main PtdIns kinase in fibroblasts, designated type II, specifically phosphorylates the D-4 position to produce phosphatidylinositol-4-phosphate (PtdIns(4)P), previously considered to be the only form of PtdInsP. We have also tentatively identified PtdIns(3)P as a minor component of total PtdInsP in intact fibroblasts. We propose that type I PtdIns kinase is responsible for the generation of PtdIns(3)P in intact cells, and that this novel phosphoinositide could be important in the transduction of mitogenic and oncogenic signals.     

Increased pH and tumorigenicity of fibroblasts expressing a yeast proton pump

A common early response of eukaryotic cells to stimuli which activate their proliferation is an increase in intracellular pH (ref. 1). In animal cells this is caused by the activation of an Na+/H+ exchange system; in fungi and plants an H+-pumping ATPase is involved. The critical question is whether this intracellular alkalinization is merely coincident with the activation of cell proliferation or whether it is a regulatory signal. To increase intracellular pH bypassing the usual physiological stimuli (growth factors, hormones etc.) alkaline media or ammonia have been used in the past. Both approaches suffer from long-term toxicity effects and cannot be used in tumorigenic assays with whole organisms. We introduce here a more specific approach which involves expressing the gene for the yeast plasma membrane H+-ATPase in fibroblasts. The resulting cells have an elevated intracellular pH and acquire tumorigenic properties, suggesting that the yeast ATPase gene behaves as an oncogene in mammalian cells. These experiments support a crucial role of intracellular pH in the growth control of animal cells.     

Elevated levels of diacylglycerol and decreased phorbol ester sensitivity in ras-transformed fibroblasts

Diacylglycerol (DG) plays a central role in phospholipid metabolism and is an endogenous activator of protein kinase C. We have suggested that constitutive activation of this kinase is one mechanism by which oncogenes transform cells. The ras-encoded proteins are similar to regulatory G-proteins and are candidates for the unknown G-protein that modulates phosphatidylinositol (PI) turnover. Differences in polyphosphoinositide metabolism have been reported for ras-transformed cells. But because these experiments were performed on confluent cultures of established cell lines, the differences are difficult to attribute to ras transformation. Here we show that exponentially growing NIH 3T3 fibroblasts recently transformed by Ha-ras or Ki-ras possess elevated DG concentrations without significant alterations in the levels of other polyphosphoinositide metabolites. The basal phosphorylation of protein kinase C substrate of relative molecular mass (Mr) 80,000 (80K) is significantly increased in all the ras-transformed cell lines. Surprisingly, however, further phosphorylation of this protein on addition of phorbol ester was greatly reduced. Ha-ras cells also show less binding of phorbol ester than control cells, suggesting that elevation of DG causes partial down-regulation in addition to activation of protein kinase C.     

Cooperation between gene encoding p53 tumour antigen and ras in cellular transformation

The protein p53 is highly expressed in a large variety of transformed cell types originating from diverse species. These include cells transformed by Simian virus 40 (SV40), adenovirus and Abelson virus, as well as a variety of chemically transformed cells. Substantial amounts of p53 are also present in certain non-transformed cells, for example, some embryonic tissues. The protein may be localized in different cellular compartments in normal and transformed cells. The strong correlation between tumorigenicity and high levels of p53 suggests an important role of p53 in tumorigenesis. We report here experiments in which we have co-transfected the murine cellular gene encoding for p53 with a ras gene into primary rat embryo fibroblasts. Our results indicate that the p53-encoding gene can play a causal role in the conversion of normal fibroblasts into tumorigenic cells.     

Mutation of protein kinase A causes heterochronic development of Dictyostelium.

In heterochronic mutants the relative timing of developmental events is altered compared with the wild type. This generally results in a disordered embryo, though heterochronic mutations may also be an important source of evolutionary variation. In the rapidly developing (rde) mutants of Dictyostelium, stalk and spore cells differentiate before morphogenesis is complete. We have traced the lesion in one class of these mutants to the regulatory subunit of cyclic AMP-dependent protein kinase (pk-A). Inactivation of this protein results in the unrestrained activity of the catalytic subunit, so prematurely triggering terminal cell differentiation.