Factors which disorganize microtubules or microfilaments increase the frequency of cell transformation by polyoma virus.

Griseofulvin, 12-O-tetradecanoyl phorbol-13-acetate, melittin, epidermal growth factor, vinblastine, cytochalasin B, podophyllotoxin, colcemid, and colchicine were unable to transform cells but could increase from 8- to 40-fold the frequency of cell transformation by polyoma virus. The 3T3-like cells were resting at confluence and were exposed to the drug only during the 1st week after viral infection. Griseofulvin, a tumor promoter, reduced or increased the frequency of transformation depending on the dose with which the infected cells were treated. The antitumor activity of tumor promoters is discussed.     

Mutation causing premature termination of the polyoma virus medium T antigen blocks cell transformation

We used site-specific mutagenesis to introduce a termination codon, TGA, into the reading frame for the polyoma virus medium T antigen. We induced this mutation in a region of the polyoma genome in which the overlapping coding regions for the large and medium TE antigens are translated in different reading frames. Therefore, the mutation terminated translation of the medium T antigen, but it caused only a single amino acid substitution in the large T antigen and did not affect the small T antigen. Cells infected by the mutant virus produced normal-size small and large T antigens. The infected cells produced a 28,000-dalton fragment of the 48,000-dalton medium T antigen, whose size and tryptic peptide map were consistent with its being a truncated N-terminal fragment terminating at the new termination codon of the mutant. Immunoprecipitates of mutant-infected cell extracts did not show medium-T-antigen-associated protein kinase activity. The mutant virus replicated normally in mouse 3T6 cells and induced cellular DNA synthesis in resting mouse 3T3 cells, but it failed to transform rat or hamster cells, as judged by focus formation and growth in agar. The mutant complemented a tsA mutant which affects the large T antigen for transformation, implying that the mutant defect for transformation was in the medium T antigen. These results imply that the small T antigen and the large T antigen together are insufficient to cause transformation and support the conclusion that the medium T antigen is essential for cell transformation by polyoma virus.     

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.     

Phenotypic and karyotypic transitions in the spontaneous transformation of a rat cell line

After 20-50 transfers, a rat myofibroblast line, Hmf-n, 'spontaneously' transforms to an established (immortalized) line of smaller, rapidly cycling fibroblastoid cells (tHmf-f). From these 1 degree transformants, colonies of larger, slower growing anchorage-independent (tHmf-e) cells of epithelioid phenotype emerge. Both transformants grow in low serum and low calcium media, but the tHmf-f cells are highly tumorigenic in nude mice, have diminished substrate adhesivity, and limited anchorage independence, whereas tHmf-e are less tumorigenic, firmly substrate adherent, and markedly anchorage independent. Most tHmf-f are trisomic; most tHmf-e transformants are hypodiploid, a third are tetraploid, and all have chromosomal abnormalities, but no trisomy. Hmf-n cells have polar stress fiber arrays terminating in vinculin adhesion plaques, colinear extracellular fibronectin matrices, and linear non-coincident deposits of fodrin. Microtubules (mt) and vimentin-intermediate filaments (IF) parallel the actin cables. Stress fibers of the tHmf-f are moderately reduced, their vinculin adhesion plaques and fibronectin matrices intact; fodrin is diffuse. Mts and IFs are normal and axial. Most epithelioid tHmf-e have no stress fibers, adhesion plaques, or extracellular fibronectin; instead, dense actin microfilament meshworks are attached to plasma membrane, as is fodrin. Mt and IF are radial. Both transformed phenotypes are stable over greater than 300 continuous passages. The differentiation-inducing agents DMSO, cyclic AMP, 5-azacytidine, and mezerein, were ineffective in normalizing shape or cytoskeleton of transformed Hmf, and butyrate was selectively toxic to 50% of tHmf-e. But hydrocortisone induced striking polarization, and increase in number, and alignment of stress fibers of both tHmf-f and tHmf-e. Growth, anchorage, cytoskeletal arrangements, and tumorigenic potential are not closely correlated in these stable, spontaneously transformed lines of distinct pheno- and karyotype originating from the same normal parental cell, suggesting independent acquisition of properties associated with transformation.     

N-terminal amino acid sequences of the polyoma middle-size T antigen are important for protein kinase activity and cell transformation.

We constructed deletion mutations which removed N-terminal coding sequences of various lengths from a cloned polyoma middle-size T antigen (MT antigen) gene. We introduced the MT antigen genes into a simian virus 40 expression vector so that they were expressed at high levels under the control of the simian virus 40 late promoter in COS-1 cells. The deletion mutant genes synthesized truncated MT antigens whose size was consistent with the deletion of either 70 or 106 amino acids from N termini, owing to initiation of translation at internal methionine codons in the MT antigen-coding region. The truncated MT antigens were found in cell membrane fractions but failed to show MT antigen-associated protein kinase activity. The cloned deletion mutant DNAs failed to transform rat F2408 or mouse NIH 3T3 cells. Therefore, N-terminal amino acid sequences of the polyoma MT antigen, as well as C-terminal sequences, are important for protein kinase activity and cell transformation.     

Cell transformation mediated by chromosomal deoxyribonucleic acid of polyoma virus-transformed cells.

To study the mechanism of deoxyribonucleic acid (DNA)-mediated gene transfer, normal rat cells were transfected with total cellular DNA extracted from polyoma virus-transformed cells. This resulted in the appearance of the transformed phenotype in 1 X 10(-6) to 3 X 10(-6) of the transfected cells. Transformation was invariably associated with the acquisition of integrated viral DNA sequences characteristic of the donor DNA. This was caused not by the integration of free DNA molecules, but by the transfer of large DNA fragments (10 to 20 kilobases) containing linked cellular and viral sequences. Although Southern blot analysis showed that integration did not appear to occur in a homologous region of the recipient chromosome, the frequency of transformation was rather high when compared with that of purified polyoma DNA, perhaps due to "position" effects or to the high efficiency of recombination of large DNA fragments.     

Mechanisms of transformation by polyoma virus middle T antigen

This review addresses a fundamental question of polyoma virus biology: What is the molecular mechanism by which the polyoma virus middle T antigen (MTAg) transforms cells in culture? Since MTAg has no known intrinsic biochemical activity, it is believed to act by modulating the properties of the host cell's proteins (see review by Courtneidge [26]). Experiments to date have largely focused on the interaction of MTAg with the cellular tyrosine kinase, pp60c-src. However, recent data from a number of laboratories have demonstrated the importance of other MTAg-associating cellular proteins in MTAg-mediated transformation, including pp62c-yes and a phosphatidylinositol kinase. In this review, we will summarize what is presently known about the proteins interacting with MTAg. The extent to which the currently known details of the biochemistry of MTAg and its associated proteins can explain the transforming properties of the various mutant alleles of MTAg will be assessed.     

Electrochemical modification of vesicular stomatitis virus glycoprotein by host cell transformation

Electrochemical properties of the glycoprotein of vesicular stomatitis virus (VSV) grown in Rous sarcoma virus (RSV)-transformed cells was compared with that of its counterpart grown in nontransformed cells. In DEAE-Sephadex column chromatography, the glycoproteins of VSV derived from transformed cells appeared more heterogeneous and had a tendency to elute with higher concentrations of NaCl than those from nontransformed cells. In isoelectric focussing, the glycoproteins of VSVs derived from transformed and nontransformed cells appeared as multiple components differing in the isoelectric point, and the glycoproteins from virus from transformed cells had isoelectric points that were more acidic than their counterparts from nontransformed cells. These results show that the glycoprotein of VSV consists of populations of molecules differing in charge and their isoelectric points were shifted to the acidic side by host cell transformation.     

Analysis of functional domains of the host cell factor involved in VP16 complex formation.

We present biochemical analyses of the regions of the host cell factor (HCF) involved in VP16 complex formation and in the association between the N- and C-terminal domains of HCF itself. We show that the kelch repeat region of HCF (residues 1-380) is sufficient for VP16 complex formation, but that residues C-terminal to the repeats (positions 381-450) interfere with this activity. However, these latter residues are required for the interaction between the N- and C-terminal regions of HCF. The extreme C-terminal region of HCF, corresponding to an area of strong conservation with a Caenorhabditis elegans homologue, is sufficient for interaction with the N-terminal region. These results are discussed with respect to possible differences in the roles of HCF in VP16 activity versus its normal cellular function.     

Overexpression of virD1 and virD2 genes in Agrobacterium tumefaciens enhances T-complex formation and plant transformation.

The VirD1 and VirD2 proteins encoded by an inducible locus of the virulence (vir) region of the Agrobacterium tumefaciens Ti plasmid are required for site-specific nicking at T-DNA border sites. We have determined the nucleotide sequence of a 3.6-kilobase-pair fragment carrying the virD locus from nopaline Ti plasmid pTiC58. In contrast to the previous report (Hagiya et al., Proc. Natl. Acad. Sci. USA 82:2669-2673, 1985), we found that the first three open reading frames were capable of encoding polypeptides of 16.1, 49.7, and 21.4 kilodaltons. Deletion analysis demonstrated that the N-terminal conserved domain of VirD2 was absolutely essential for its endonuclease activity. When extra copies of the virD1 and virD2 genes were present in an A. tumefaciens strain carrying a Ti plasmid, increased amounts of T-strand and nicked molecules could be detected at early stages of vir induction. Such strains possessed the ability to transform plants with higher efficiency.     

Modulation of host glutamine anabolism enhances the sensitivity of small cell lung cancer to chemotherapy

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Vesicular stomatitis virus envelope glycoprotein alterations induced by host cell transformation

Vesicular stomatitis virus (VSV) contains a single structural glycoprotein in which the sugar sequences are largely host specified. We have used VSV as a probe to study the changes in cell glycoprotein metabolism induced by virus transformation. Analysis of purified VSV grown in baby hamster kidney (BHK) or polyoma transformed BHK cells showed that the virus glycoproteins have identical apparent molecular weights. The glycopeptides derived from the glycoproteins by extensive pronase digestion have an identical molecular weight distribution.On the basis of labeling experiments with fucose, mannose, and glucosamine, the oligosaccharide moieties of the VSV glycoprotein were different in virus from the two cell lines. The VSV glycopeptides from transformed cells showed an increased resistance to cleavage by an endoglycosidase, indicating structural changes in the core region of the oligosaccharides. They also showed an increased ratio of sialic acid to N-acetylglucosamine.VSV grows in a wide variety of cell types, and the carbohydrate structures of its single glycoprotein are amenable to analysis with specific glycosidases. The virus thus provides an excellent tool with which to study alterations induced by cell transformation in the glycosylation of membrane proteins.