Mos overexpression in Swiss 3T3 cells induces meiotic-like alterations of the mitotic spindle.

High levels of mos protooncogene product are expressed during oocyte meiotic maturation and Mos has been implicated in formation of the spindle and spindle pole. Here, we show that in Swiss 3T3 cells with 4N DNA content, high levels of Mos lead to the production of binucleated cells. The Swiss 3T3 cells in mitosis, before binucleation occurs, are anastral and the spindle poles are juxtaposed to the cell membrane. These phenotypes may be related to the meiotic process of attachment of the spindle pole to the oocyte membrane during polar body formation. The production of binucleated somatic cells could result from attachment of the altered mitotic spindle pole to the cell membrane that interferes with cytokinesis but not karyokinesis. This can explain at least one form of genetic instability that leads to altered DNA content in tumor cells.

Infection with Moloney murine sarcoma virus inhibits myogenesis and alters the myogenic-associated (2'-5')oligoadenylate synthetase expression and activity.

Infection of rat skeletal muscle cultures on the first or second day in vitro with Moloney murine sarcoma virus (MSV) led to the arrest of myotube formation and to inhibition of both the synthesis of the muscle-specific proteins acetylcholine receptors and creatine kinase and the expression of the myosin light chain-2. Mos-specific RNA transcripts were readily detected at 1 day after infection indicating that viral genes were expressed in infected cells. In parallel, the expression of the cell growth-associated gene--c-myc--in uninfected muscle cultures was drastically reduced with time, while in MSV-infected myoblasts, the amount of c-myc-specific RNA transcripts gradually increased with time after infection. Under these conditions we could demonstrate that the interferon-induced gene (2'-5')oligoadenylate synthetase (2-5A synthetase) was transiently activated in uninfected muscle culture reaching a peak activity on the third day. Infection of myoblasts with murine leukemia virus did not alter the pattern of 2-5 synthetase activity observed in uninfected cells. However, infection with MSV on the second day led to a slight reduction in activity followed by a significant increase on the sixth and seventh day. Similarly, 2-5A synthetase gene expression was down-regulated with time in culture in uninfected myoblasts while re-expressed between the fourth and seventh days in MSV-infected cultures.

Elevated expression of v-mos is correlated with altered differentiation of carcinoma cells.

Permanent alterations of the epithelial differentiation pattern were investigated after infection of HH-16 cl. 4 adenocarcinoma cells with Moloney murine sarcoma virus (MoMuSV). Transformed cell clones with fibroblastoid morphology were isolated and compared with clones of unchanged epithelioid phenotype. Southern blot analyses showed intact MoMuSV proviral genomes in copy numbers between 4 and 9 in the DNA of the morphologically transformed cell clones as well as in the clones with unaltered morphology. The fibroblastoid cells produced sarcomas after inoculation of newborn rats, whereas MoMuSV-infected cell clones with unaltered epithelioid morphology yielded adenocarcinomas. Immunocytochemical analyses revealed that morphological transformation into the fibroblastoid phenotype was accompanied by loss of cytokeratin expression and appearance of the mesenchymal marker protein vimentin. Proviral DNA was transcribed in the infected cell clones irrespective of their phenotype; however, transcription was significantly higher in cells with fibroblastoid morphology than in epithelioid cells.

Synthesis and biological evaluation of dinucleoside methylphosphonates of 3'-azido-3'-deoxythymidine and 2', 3'-dideoxycytidine.

The 5'----5' dinucleoside methylphosphonates of 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxycytidine (DDC) were prepared and evaluated for their inhibitory properties against different viruses, including human immunodeficiency virus (HIV). The synthesis of the compounds was achieved by reaction of AZT or N4-(4-monomethoxytrityl)-2',3'-dideoxycytidine with in situ prepared methylphosphonic bis (triazolide), followed in the latter case by an acidic treatment. The two title compounds showed in vitro anti-HIV activity, that was 200- to 450-fold less pronounced that that shown by the corresponding monomeric nucleosides AZT and DDC. The decreased antiviral activity may be ascribed to nuclease resistance of the methylphosphonate linkage.

Regulation of RNA splicing in gag-deficient mutants of Moloney murine sarcoma virus MuSVts110.

We investigated whether the MuSVts110 gag gene product (P58gag) can regulate the novel growth temperature dependence of MuSVts110 RNA splicing. MuSVts110 mutants with either frameshifts or deletions in the gag gene were tested for their ability to maintain the MuSVts110 splicing phenotype. Only small decreases in splicing efficiency and no changes in the thermosensitivity of viral RNA splicing were observed in MuSVts110 gag gene frameshift mutants. Deletions within the gag gene, however, variably decreased MuSVts110 splicing efficiency but had no effect on its thermosensitivity. Another class of MuSVts110 splicing mutants generated by treatment of MuSVts110-infected cells with NiCl2 was also examined. In these "nickel revertants," P58gag is made, but splicing of the viral transcript is nearly complete at all growth temperatures. The splicing of "tagged" viral RNA transcribed from a modified MuSVts110 DNA introduced into nickel revertant cells remained thermosensitive, arguing against trans effects of viral gene products on splicing efficiency. These experiments indicated that neither the MuSVts110 P58gag protein nor any other viral gene product acts in trans to regulate MuSVts110 splicing.

Reversion of thermosensitive splicing defect of Moloney murine sarcoma virus ts110 by oversplicing of viral RNA.

Moloney murine sarcoma virus ts110 possesses a thermosensitive splicing defect. By continuously growing nonproducer cells at the nonpermissive temperature, a new class of revertant cells, termed 6m3, that had lost the thermosensitive splicing defect was produced, and six distinct clones were selected. These cell clones were transformed at either permissive or restrictive temperatures. Unlike parental 6m2 cells, which contain two virus-specific RNA species of 4.0 and 3.5 kilobases (kb) at temperatures permissive for transformation, the 3.5-kb RNA was the only virus-specific RNA species detected in 6m3 clones. No new v-mos-containing DNA fragment was observed in Southern blot analysis of these cell clones compared with parental 6m2 cells, indicating that the 3.5-kb RNA was a splicing product rather than a direct transcript. Moreover, these cells expressed P85gag-mos but not P58gag at any temperature. The reversion of the phenotype in 6m3 cell clones appears to be the result of a selective loss of the temperature sensitivity of the splicing reaction, without affecting the thermosensitivity of the protein kinase activity. This change also appears to alter the mechanism regulating the efficiency of the genomic RNA-splicing reaction.

Stable expression of a selectable myeloproliferative sarcoma virus in murine T lymphocyte and monocyte cell lines.

We have investigated whether a retroviral vector based on the myeloproliferative sarcoma virus (MPSV) can be expressed in murine T cells and macrophages. This vector (neoR MPSV) carries the dominant selection marker for neomycin resistance (neoR) and the mos oncogene. The murine T cell line BW5147 and the monocytic cell line P388D1 were either transfected with neoR MPSV DNA or infected with neoR MPSV virus. From both lines, neoR cell clones could be established by retroviral infection, but not by calcium-phosphate precipitation-mediated DNA transfection. The efficiency of infection could be increased 60- to 200-fold upon cocultivation of target cells with irradiated neoR MPSV virus-producing cells. All neoR clones showed neoR MPSV specific sequences as revealed by dot blot and Southern blot analysis. The integration and expression of neoR MPSV was stable over a period of now more than 4 months, even in the absence of selection for neomycin resistance. Northern blot analysis showed that neoR clones express full length neoR MPSV. Further, clones of both T cell and monocyte origin were capable to produce infectious virus particles as revealed by focus formation on fibroblasts and conversion of neomycin sensitive fibroblasts to a neomycin resistant phenotype.

Monoclonal antibody study of the subcellular localization and DNA-stimulating activity of murine sarcoma virus-activated transformation-associated proteins.

Previously, we reported the monoclonal antibody detection of transformation-associated proteins (TAP) in ts110 murine sarcoma virus-transformed normal rat kidney (6M2) cells (Chan et al., 1986). In this study, we used the same monoclonal antibody to investigate the subcellular localization, the fate and the mitogenic activity of TAP, as well as the correlationship between TAP synthesis and the expression of transformation properties of 6M2 cells. It was found that TAP were localized in the cytoplasm (probably the Golgi apparatus) of 6M2 cells. TAP were found as three intracellular polypeptides (mol wt of 66K, 63K, and 60K, respectively), and were rapidly released into extracellular medium. Upon release, TAP changed to two extracellular polypeptides (mol wt of 68K and 64K, respectively). Furthermore, the synthesis of TAP was temperature sensitive and correlated closely with the expression of transformation properties of the 6M2 cells. TAP have been purified by monoclonal antibody-affinity column chromatography and found to have a synergistic effect with insulin in stimulating the DNA synthesis of normal rat kidney cells.

Temperature-dependent cytocidal effects of Moloney murine sarcoma virus.

Infection of L6E9 rat myoblasts with Moloney murine sarcoma virus (Mo-MuSV) at 37 degrees C resulted in the killing of target cells in less than one week. However, cells infected at 37 degrees C and shifted to 40 degrees C were not killed but underwent morphological transformation and had reduced levels of virus-specific RNA. A marked decrease in a cellular 55 kDa protein, as judged by immunoblotting, correlated with increased expression of Mo-MuSV RNA in infected cells.