Rac is essential in the transformation of endothelial cells by polyoma middle T

Expression of the Polyoma Middle T (PyMT) antigen in endothelial cells results in single-step transformation to hemangioma producing malignant cells. To study the mechanism of PyMT transformation, we used the PyMT induced mouse brain endothelial cell line, bEND.3, expressing constitutively active and dominant negative mutants of the small GTPase Rac. The bEND.3 cell phenotype of tumorigenesis, loss of normal growth control and formation of cysts rather than capillary tubes in fibrin gels was reversed by expression of dominant negative Rac. The mechanism of N17 Rac action in blocking the endothelial cell transformant, PyMT, did not involve effects of Rac on the actin cytoskeleton since this component of the bEND.3 cell phenotype was not affected. Furthermore, the PyMT induced activation of the plasminogen activator (PA)/plasmin system was not affected by Rac inhibition. Inhibition of the downstream effectors of Rac, phosphatidylinositol 3-kinase (PI3-K) and p70S6k, which are known to be constitutively activated by PyMT transformation, inhibited bEND.3 cell proliferation and cyst formation in fibrin gels even in cells expressing V12 constitutively active Rac, but they did not restore capillary tube formation. These results demonstrate that middle T antigen induced endothelial cell transformation requires signal transduction by Rac. The downstream Rac effectors, P13-K and p70S6k, mediate PyMT/Rac effects on cell proliferation and cyst formation, but other unknown effectors of PyMT are required for the cytoskeletal changes and activation of the PA/plasmin system.     

Endothelioma cells expressing the polyoma middle T oncogene induce hemangiomas by host cell recruitment

Mouse endothelioma cells expressing the polyoma middle T oncogene induced hemangiomas in a variety of species such as mice, rats, chicks, and quails. In embryos and newborn mice the hemangiomas expanded within 10-18 hr of injection, disrupting the vasculature and causing the death of the animal. In contrast, the hemangiomas formed a stable structure reminiscent of benign human hemangiomas in adult mice within 5 days. Analysis of the cells comprising the hemangioma revealed that over 95% of the endothelial cells were host derived. No induction of host cell proliferation was detected, and no endothelial mitogens were secreted by the endothelioma cells in vitro. The maintenance of the hemangioma appeared to require the continuous presence of endothelioma cells. The results indicate that these endothelioma cells act as a potent stimulating agent in the rapid formation of hemangiomas by recruiting nonproliferating host endothelial cells.     

Embryonic lethalities and endothelial tumors in chimeric mice expressing polyoma virus middle T oncogene

The effect of the middle T oncogene of polyoma virus was studied in vivo using a replication-defective selectable retrovirus. Injection of virus into newborn and adult mice resulted in the rapid appearance of cavernous hemangiomas. Infection of embryos did not yield transgenic mice; therefore, embryonal stem (ES) cells were used as an alternative system. Several infected ES cell clones were established that constitutively expressed middle T and its associated tyrosine kinase activity. Chimeric embryos obtained by blastocyst injection of individual ES cell clones were specifically arrested at midgestation, when multiple hemangiomas disrupted blood vessel formation. From these tumors endothelial cell lines were established that retained expression of von Willebrand factor yet were tumorigenic in vivo. These results suggest that middle T acts in endothelial cells as a single-step oncogene and that ES cells provide a valuable system for the study of growth control during embryogenesis.     

The in vitro behavior of hemopoietic cells transformed by polyoma middle T antigen parallels that of primary human myeloid leukemic cells

A retrovirus encoding polyoma middle T antigen has been used to infect a murine hemopoietic cell line (FDC-P1) dependent on either granulocyte-macrophage colony-stimulating factor (GM-CSF) or multipotential colony-stimulating factor (Multi-CSF). A number of cell lines have been established on the basis of their initial ability to proliferate in the absence of added colony-stimulating factor (CSF). The transformed lines display one of three patterns of growth in vitro: those able to grow fully autonomously; those whose proliferation depends on cell density; and those displaying dependence on added CSF regardless cell density. This latter class of cells are reminiscent of the majority of primary myeloid leukemic cells. Unlike parental FDC-P1 cells, all three classes of transformed cells are leukemogenic in syngeneic mice; moreover, they produce variable amounts of GM-CSF which we believe underlies their neoplastic behavior.     

Secretion of RNA by normal and transformed cells

3T3 and SV-40 transformed 3T3 cells in culture secrete RNA into their culture media. This medium RNA is predominantly 5s in size as measured by sucrose gradient and Sephadex gel filtration. Medium RNA is metabolically stable and is heavily methylated in comparison with other major cytoplasmic species. Analysis of the radioactively labeled methylated bases of medium RNA by paper chromatography after formic acid hydrolysis shows a very simple pattern of two peaks in contrast to the very complex patterns seen in rRNA and tRNA. Mycoplasma and mouse leukemia virus contamination have been excluded. The source of this RNA is discussed.     

Phosphatidylinositol metabolism in cells transformed by polyomavirus middle T antigen.

Associated with the middle T antigen of polyomavirus is a novel phosphatidylinositol (PtdIns) kinase activity which phosphorylates PtdIns at the D-3 position of the inositol ring. We have undertaken an analysis of myo-[3H]inositol-containing compounds in a panel of NIH 3T3 cell lines stably transfected with transforming and nontransforming middle T antigen mutants. All cell lines from which PtdIns 3-kinase activity coprecipitated with middle T antigen exhibited modestly elevated levels of PtdIns(3)P and compounds with predicted PtdIns(3,4)P2 and PtdIns(3,4,5)P3 structures. Complex formation between middle T antigen and PtdIns 3-kinase correlated not with an increase in total inositol phosphate levels but rather with elevated levels of InsP2 and InsP4. A specific increase in the level of an InsP2 species which comigrated in high-pressure liquid chromatography analysis with Ins(3,4)P2 was observed. These results suggest that association of the polyomavirus middle T antigen with PtdIns 3-kinase activates a distinct inositol metabolic pathway.     

The sensitivity to growth factors of NIH 3T3 cells transformed by the v-myc oncogene

Transformation of NIH 3T3 cells, induced by v-myc oncogene, activates a proliferative potential of the cells cultivated in the serum-free medium, and reduces the ratio of 3H-Tdr incorporation into the cells grown in the presence of 10% fetal serum in comparison to those grown in the serum-free medium. The v-myc transformed cells (NIH 3T3-v-myc) as well as the untransformed ones are very responsive to insulin. On the other hand, the epidermal growth factor, able to stimulate proliferation of NIH 3T3 cells, exert no effects on the NIH 3T3-v-myc cells. The NIH 3T3-v-myc cells cultivated in the medium, containing 2.5% human plasma enriched with thrombocytes, have the same proliferative characteristics as cells grown in the thrombocyte-free plasma. It is concluded that transformation of NIH 3T3 cells induced by v-myc oncogene may reduce a requirement for thrombocyte-released growth factors and EGF but not for insulin.     

Endothelial cell tumors develop in transgenic mice carrying polyoma virus middle T oncogene

Inoculation of newborn mice with the murine polyoma (Py) virus leads to tumor formation in a wide range of tissues. In order to investigate viral oncogenesis, we generated transgenic mice carrying either the Py large T antigen (LT) gene or the Py middle T antigen (MT) gene linked to Py early region regulatory sequences. While Py LT mice exhibit no phenotype, Py MT mice develop multifocal tumors of the vascular endothelium. These hemangiomas are lethal to the animals and can be passaged in vivo. Transgene RNAs and protein are present in both hemangiomas and the testes of these mice, and the Py middle T protein in both tissues is complexed to a cellular tyrosine kinase. The expression of complexed middle T protein in both tumorigenic endothelial cells and unperturbed testes implies that endothelial cells may be particularly susceptible to the action of the middle T oncogene. These observations indicate that Py middle T disrupts the normal strict controls on vascular growth, and suggest that Py MT transgenic mice will provide a model for studying the control of angiogenesis.     

Effect of inhibitors of proteolytic enzymes on the growth of normal and polyoma transformed BHK cells

This paper reports the effect of tosyl-phenylalanyl-chloromethyl ketone, Pepstatin, Antipain, Leupeptin and Trasylol, all inhibitors of proteolytic enzymes, on the growth and morphology of normal and polyoma transformed baby hamster kidney cells in culture. Above 1 μM, tosyl phenylalanyl chloromethyl ketone inhibited the growth of both normal and transformed cells to the same extent, and also inhibited the incorporation of radioactive leucine into protein. Pepstatin and Antipain also had no differential effect on the growth of normal or transformed cells, while Leupeptin and Trasylol inhibited the growth of normal cells more than transformed cells.     

Antibody to the nonapeptide Glu-Glu-Glu-Glu-Tyr-Met-Pro-Met-Glu is specific for polyoma middle T antigen and inhibits in vitro kinase activity

Middle T antigen of polyoma virus has an associated tyrosine kinase activity which phosphorylates tyrosine residue 315 on middle T in immunoprecipitates. A peptide representing the sequence of middle T from residue 311 to 319 has been synthesized. This peptide acts as a weak inhibitor of the kinase reaction. An antiserum has been raised against this peptide after conjugation to bovine serum albumin. The antibody is middle T-specific. Middle T antigen precipitated by this serum is largely inactive in the kinase reaction. Dissociation of the immune complex with peptide releases middle T in a kinase-active form.     

Secretion of a TGF-beta-like growth inhibitor by normal rat mammary epithelial cells in vitro

We have examined conditioned medium (CM) from cultures of normal rat mammary epithelial (RME) cells for growth factor activity on fresh RME cell cultures. RME cell-derived CM contained potent growth inhibitory activity toward fresh RME cell cultures when the medium was acidified by dialysis against 1% acetic acid prior to concentration. Dialysis of the CM at neutral pH resulted in CM that had growth stimulatory activity and no inhibitory activity. The acid-activated growth inhibitor was heat and acid stable, protease sensitive, and eluted from a Bio-Gel p60 column with a peak of activity in the 28 kDa range. Incubation of the acidified-concentrated CM with neutralizing antiserum (affinity purified IgG) against transforming growth factor (TGF)-beta completely abolished the inhibitory activity of the CM. Furthermore, RME cell growth in the presence of the growth inhibitor plus TGF-beta antiserum was greater than that observed in growth medium alone. Subsequent experiments demonstrated that addition of TGF-beta antiserum alone to serum-free medium enhanced RME cell growth, whereas addition of nonimmune IgG was without effect even at 25-fold higher concentrations. Zymographic analysis of RME-CM revealed the presence of plasminogen activator proteases that may mediate the partial activation of the latent growth factor. These results indicate that normal RME cells secrete a latent TGF-beta-like growth factor into conditioned medium. Furthermore, the results indicate that some of the latent growth factor is activated in situ and contributes to the growth potential of the cells in primary culture in an autocrine manner.     

Protein kinase activity associated with polyoma virus middle T antigen in vitro.

A protein kinase activity can be detected in immunoprecipitates of extracts from polyoma virus (Py)-infected cells using antiserum raised against Py-transformed cells (anti-T serum). The activity is not detected in uninfected cells or when using control serum. Using rat anti-T serum both Py middle T and the heavy chain of rat IgG are phosphorylated, whereas using hamster anti-T serum only Py middle T is phosphorylated. Experiments using a number of different mutants of Py indicate that the kinase activity detected is under viral control and is associated with Py middle T. Consistent with this the kinase, like middle T, can be detected in purified preparations of plasma membranes. The kinase can also be detected in a large number of Py-transformed cells, but not in untransformed cells or in cells transformed by other viruses. Some of the Pytransformed cells which contain kinase activity lack full sized Py large T but all contain middle T. Kinase activity is not detected in a cell line (18.37) which contains integrated viral DNA of a nontransforming hr-t deletion mutant and which contains Py large T but not middle T or small t. These results show that Py middle T or a protein which specifically binds to it has protein kinase activity in vitro. Although these results raise the possibility that protein kinases play an essential role in Py-induced transformation, however, thus far we have no data which show unequivocally that the results are physiologically significant.     

Phosphorylation and activation of epidermal growth factor receptors in cells transformed by the src oncogene.

Because functionally significant substrates for the tyrosyl protein kinase activity of pp60v-src are likely to include membrane-associated proteins involved in normal growth control, we have tested the hypothesis that pp60v-src could phosphorylate and alter the signaling activity of transmembrane growth factor receptors. We have found that the epidermal growth factor (EGF) receptor becomes constitutively phosphorylated on tyrosine in cells transformed by the src oncogene and in addition displays elevated levels of phosphoserine and phosphothreonine. High-performance liquid chromatography phosphopeptide mapping revealed two predominant sites of tyrosine phosphorylation, both of which differed from the major sites of receptor autophosphorylation; thus, the src-induced phosphorylation is unlikely to occur via an autocrine mechanism. To determine whether pp60v-src altered the signaling activity of the EGF receptor, we analyzed the tyrosine phosphorylation of phospholipase C-gamma, since phosphorylation of this enzyme occurs in response to activation of the EGF receptor but not in response to pp60v-src alone. We found that in cells coexpressing pp60v-src and the EGF receptor, phospholipase C-gamma was constitutively phosphorylated, a result we interpret as indicating that the signaling activity of the EGF receptor was altered in the src-transformed cells. These findings suggest that pp60v-src-induced alterations in phosphorylation and function of growth regulatory receptors could play an important role in generating the phenotypic changes associated with malignant transformation.     

Malignant transformation of mouse BALB/3T3 cells by polyoma middle T antigen requires epidermal growth factor receptor expression.

The mouse cell line MO-5, which is defective in receptor-binding activity of epidermal growth factor (EGF), is very poorly transformed by polyoma middle T antigen or v-src gene, but activated c-H-ras and v-mos gene can induce the transformation (M. Ono, M. Yakushinji, K. Segawa, and M. Kuwano, Mol. Cell. Biol., 8: 4190-4196, 1988). We established clones of MO-5 expressing a functional EGF receptor (EGF-R) after introduction of the human EGF-R complementary DNA into MO-5 (MNER23 and MNER31), and we also established a clone (BNER4) expressing human EGF-R from the parental cell line, BALB/3T3. MNER23, MNER31, and BNER4 expressed EGF-R activity at about 2- to 6-fold higher levels than did control BALB/3T3 cells. A marked increase in DNA synthesis in response to EGF was observed in these BNER4, MNER23, and MNER31 cell lines compared to BALB/3T3 cells; however, there was little if any increase in DNA synthesis of MO-5 in the presence of EGF. Introduction of the polyoma middle T antigen gene into BALB/3T3, BNER4, MNER23, and MNER31 resulted in the appearance of transformation foci, but MO-5 again showed little response. We purified clones B4-mT-2, M23-mT-1, M23-mT-2, M23-mT-3, and M31-mT-13 from transformation foci of BNER4, MNER23, and MNER31 cells, which were respectively transfected with the middle T antigen. All of the middle T antigen-positive transfectants demonstrated abilities to form both colonies in soft agar and tumors in nude mice. The presence of EGF-R appears to be indispensable for malignant transformation by polyoma middle T antigen.     

Integrated polyoma genomes in inducible permissive transformed cells.

Using the approach described by Botchan, Topp, and Sambrook (Cell 9:269-287, 1976), we analyzed the organization of the integrated viral sequences in five clonal isolates from the same permissive, inducible cell line (Cyp line) transformed by the tsP155 mutant of polyoma virus. In all five clones, viral sequences were found that could be assigned to a common integration site, as they were joined to the cellular DNA in the same fashion in every instance. However, the sequences comprised between these points differed markedly from clone to clone, as if cell propagation had been accompanied by amplification or recombination or both within the viral insertion. When the clones were compared, no correlation could be found between the abundance, or the organization, of the integrated viral sequences and the amount, or the nature, of the free viral DNA molecules produced during induction. Altogether, our findings suggest that specific events, occurring during either the excision or the subsequent replication of the integrated viral sequences, are responsible for the predominant production of nondefective viral DNA molecules by permissive transformed cells, such as Cyp cells.