Search

The complete 606 amino acid sequence of the human A-raf oncogene has been deduced from the 2453 nucleotide sequence of a human T cell cDNA. A cysteine-rich region located near the amino terminus, which is highly conserved in A-raf and c-raf, shows significant homology with protein kinase C. A 5' deleted fragment of the cDNA has been incorporated into a murine retrovirus which endows the virus with the ability to transform cells in vivo and in vitro. Functionally, human A-raf is similar to v-raf and v-mos in that transformation is independent of ras gene function.

Foci of transformed cells, produced by MSV(124), appeared to result only from the primary infection, since this virus stock yielded a virus-nonproducing infection. On the other hand, the majority of foci scored in MSV/MLV-infected cultures, were generated by multiple secondary infections with the progenies of the primary infection. Mouse interferon (IF) was highly inhibitory for cell transformation by both virus stocks. However, this inhibition was apparent in MSV(124) infected cultures only if IF was added at least 12 h before infection, whereas in MSV/MLV-infected cultures IF was highly effective even if added 24 h after infection. The inhibition of focus formation by MSV(124) was irreversible after removal of IF, suggesting that IF inhibited an early step before provirus integration into the host genome. By contrast, in MSV/MLV-infected cultures focus formation was almost completely restored after recovery from the IF effect. Nevertheless, examination of virus production after IF removal proved that in MSV/MLV infection, too IF exerted and inhibitory effect before provirus integration.

Mouse interferon (IFN) was found to inhibit murine sarcoma virus (MSV)-induced neoplastic transformation of normal rat kidney (NRK) cells. This effect was observed upon examining the formation of foci of morphologically altered cells and colonies of anchorage-independent cells. IFN had no cytotoxic effect on MSV-transformed NRK cells, nor on their focus or colony-forming ability. It was therefore apparent that its inhibitory effect was directed against the viral role in cell transformation. In attempts to define the mechanism of this effect, we found that IFN delayed the initiation of the cytoplasmic viral DNA synthesis. However, the amount of this DNA eventually formed in IFN-treated cells was the same as in the control cells. Furthermore, the transport of this DNA to the nucleus was slower in IFN-treated cells, although all of it was finally transferred. However, while most of the viral DNA integrated into the genome of the control cells, very little integration occurred in IFN-treated cells. The unintegrated viral DNA of these cells was slowly degraded. Therefore, if the cells recovered from the antiviral effect of IFN when intact viral DNA molecules still existed in their nucleus, they could resume viral DNA integration and cell transformation. IFN was found to block viral DNA supercoiling. Since supercoiled viral DNA is considered to be a precursor to integrated provirus, it seems that the inhibition of both integration and cell transformation is due to this impaired coiling.

Mouse interferon (IFN) inhibited retrovirus release by both mouse and rat cells with the same efficiency. However, the antiviral state developed more slowly in rat than in mouse cells, and after removal of IFN it also persisted for a longer time in rat than in mouse cells. Under conditions where IFN strongly inhibited virus production it had no effect on cell replication nor on cellular RNA or protein synthesis.

A 1.6-kilobase cDNA (A-raf) has been isolated from a murine spleen cDNA library which encodes part of a protein related to the raf oncogene. Its amino acid sequence has 85% homology to raf in a central portion of 100 amino acids. In contrast to raf, A-raf shows a highly restricted tissue distribution of expression, with highest levels observed in epididymis, followed by intestine. When incorporated into a retrovirus, the resulting gag-A-raf fusion gene causes transformation in vitro and induces tumors in newborn mice. Thus, A-raf represents a new proto-oncogene. Transformation by A-raf is independent of ras gene function, as is the case for raf and mos but not other oncogenes.

The present study demonstrates the advantages of a combination of concentration by polyethylene glycol-6000 and Sepharose Cl-4B chromatography as a rapid procedure for retroviruses purification. This procedure can be completed within 3 hours, providing a high degree of virus purification with minimal damage to its structural and biological properties. Using transmission electron microscopy we observed many intracellular type-C virions in cytoplasmic vacuoles of 3T3/NIH cells chronically infected with Moloney murine leukemia virus. There intracellular virions could be isolated from postmitochondrial cytoplasmic fractions prepared from the infected cells by a procedure which minimized its contamination by extracellular free or membrane-bound virions. SDS-polyacrylamide gel electrophoresis showed that the intracellular and extracellular virions contained similar protein composition.

PEA1 (AP1) motif transcription enhancer activity was stimulated by v-raf and more efficiently by activated c-raf-1 or A-raf than by their normal counterparts, in agreement with a role for PEA1 in transformation by raf. Mutations in the ATP-binding site of v-raf prevented activation, suggesting that phosphorylation is somehow required.

The effect of interferon (IFN) on virus release and on assembly of intracellular virions in 3T3/NIH cells chronically infected with Moloney murine leukemia virus was studied by short labeling with 3H-leucine (viral proteins), 3H-glucosamine (viral glycoproteins) and 3H-uridine (vital RNA). With all of these labels, IFN pretreatment was found to strongly inhibit extracellular virus release. No difference was found between the extent of labeling of viral proteins and glycoproteins of intracellular virions. Incorporation of 3H-uridine into intracellular virions was strongly reduced by the IFN pretreatment. Since it is rather unlikely that encapsidated RNA is significantly degraded during the relatively short time of label incorporation, this finding suggests that IFN interferes with packaging of viral RNA. The effect of IFN on virus release and on 3H-uridine labeling of intracellular virion were found to develop with the same kinetics, though the maximal inhibition of virus release was stronger.

Expression of the c-myc gene is suppressed in NIH 3T3 mouse fibroblast cells infected with recombinant retroviruses expressing high levels of v-myc (10-fold greater than those of c-myc). Suppression of steady state levels of c-myc mRNA occurred at least in part at the level of transcription from c-myc promoters P1 and P2, and involved v-myc protein since cells infected with constructs containing frameshifts and deletions in v-myc had normal levels of c-myc mRNA and protein. Suppression of c-myc expression was also observed in fibroblasts transfected with a N-myc expression vector and in fibroblasts infected with a c-myc retrovirus. These findings establish that v-myc protein is involved either directly or indirectly in a regulatory circuit which represses c-myc proto-oncogene transcription. Feedback regulation of c-myc transcription may be relevant in establishing the lineage specific expression of myc family proto-oncogenes. Reduced steady state levels of c-myc mRNA were also observed in NIH 3T3 cells infected with 12S and 13S EIA recombinant retroviruses suggesting that the exogenous oncogene of adenovirus, EIA, can alleviate the requirement of myc for cell growth and may also share transcriptional target genes.

The effect of interferon (IFN) on the cytoplasmic synthesis of murine sarcoma virus DNA, its transport to the host nucleus, and its integration into the cellular genome were investigated. For this purpose, at various intervals after infection. DNA was extracted from the cytoplasmic fraction, nuclear Hirt supernatant, and chromosomal DNA pellet. The relative amount of viral DNA was estimated by C0t kinetics analysis of hybridization to murine sarcoma virus-specific [3H]cDNA. IFN was found to delay viral DNA synthesis by about 2.5 h, but the amount of viral DNA eventually formed in IFN-treated cells was comparable to that of the control. The transport of this DNA to the nucleus was delayed by IFN for 6 to 18 h, but once again, all the cytoplasmic viral DNA formed in IFN-treated cells was eventually transferred to their nucleus. However, although the main part of the viral DNA formed in control cells was finally integrated into the host genome, no significant integration was observed in IFN-treated cells. Alkaline sucrose gradient analysis revealed that IFN inhibited the accumulation of supercoiled viral DNA in the nucleus. Since supercoiled viral DNA is considered a precursor to integrated provirus, this observation may suggest that IFN inhibits viral DNA integration by blocking its supercoiling.

raf oncogenes were shown to act synergistically with myc in transformation. The contribution of myc was identified as that of a "second messenger" in signal transduction of at least some, competence inducing, growth factors. The role of raf appears to be that of a cytosolic ser/thr specific protein kinase which was placed downstream of ras in the signal transduction of serum growth factors by ras and raf antibody microinjection experiments. Because of the inability of raf to abrogate a cells need for myc inducing competence factors, as well as its synergistic effect with myc, raf was placed downstream of ras in the progression pathway of cellular growth control. We speculate that the basis for synergism with myc might be the ability of raf to activate competence factor induced myc protein or a myc induced protein by phosphorylation. The role of raf in lung tumors was examined by the development of a high incidence mouse model system using ethylnitrosourea as carcinogen and butylated hydroxytoluene as promoter. raf proteins of normal size were expressed at high levels, raf protein vaccination was apparently effective in eliminating the promoted phase of tumor induction.

An accessory cell line, designated line A, was generated from bone marrow stem cells which differentiated in vitro in response to colony-stimulating factor (CSF) in substratum cultures. The cells were found to constitutively secrete large amounts of CSF, the activity of which was neutralized by anti-CSF-1 antibodies. Cells of line A and its supernatants potentiate the suboptimal response of thymocytes to PHA, manifesting an interleukin-1 (IL-1)-like activity. Culture fluids of this line also reconstitute the response to T cell mitogens of spleen cells depleted of adherent accessory cells. It was also found that cells of line A bear low levels of surface Ia, and they efficiently present soluble antigen to proliferating memory T cells. Constitutive prostaglandin secretion, which sometimes masks antigen-presenting capacity, was also demonstrated. Cells of line A are poorly phagocytic, do not secrete lysozyme, and lack Fc and complement receptors. However, they manifest strong cytoplasmic nonspecific esterase staining and an ectoenzyme profile resembling that of elicited inflammatory macrophages. In addition, the cell surface antigen Mac-2 was demonstrated, while stainings with anti-Mac-1 and anti-Mac-3 were negative. Thus, line A may represent a unique subpopulation of immunoregulatory accessory cells, the features of which are discussed.

Normal rat kidney (NRK) cell were found to be resistant to neoplastic transformation by diverse carcinogenic chemicals. To study chemical-retroviral co-carcinogenesis in this cells they were infected with a low multiplicity of Moloney murine leukemia virus (M-MLV). Using a single cell cloning procedure, a virus-producing clone was isolated from the infected cells, which was shown to carry only one integrated M-MLV provirus per cell. It was found that this single provirus was sufficient to render the clone susceptible to transformation by 3-methylcholanthrene (3-MC). However this clone responded differently to the carcinogen at different passages after infection. When exposed to 3-MC at a low passage postinfection (passage 5), cell transformation was evident only after 11 subsequent subcultures. On the other hand when it was chemically treated at a high passage postinfection (passage 29), cell transformation could clearly be detected already at the next subculture after the chemical treatment. It is suggested that an M-MLV-mediated cumulative effect is necessary to complement the action of the carcinogen in order to complete the carcinogenic process in these cells. This cumulative viral effect appeared to be associated with a change in the control of the virus expression, since 3-MC was found to stimulate virus replication in this clone also only at the high passage postinfection. Indeed virus release by cells of isolated transformed foci, produced by the chemical-M-MLV co-carcinogenesis, was extremely higher than by untransformed cells.

Moloney murine sarcoma virus (MSV) clone 124 was found to induce rapid syncytium formation upon infection of 3T3/NIH at a high multiplicity of infection. This effect became apparent, by light microscopy, within 1 to 2 hours, whereas by scanning electron microscopy, clusters of 4 to 25 cells were seen in their initial steps of syncytium formation within 20 to 30 minutes after virus addition. It appeared that the cell fusion was initiated by connection between the cells through virus bound to their surface. After a few more hours several neighbouring syncytia fused into giant cells containing over a hundred nuclei. Though MSV (124) stocks contained also some MLV it appeared that the syncytium inducing activity was related to the MSV particles. MLV particles were not only incapable to induce syncytium formation, they even interfered with this activity of MSV. The MSV-induced cell fusion required the adsorption of intact virions, but was independent on protein synthesis. Mouse interferon remarkably reduced the rate of syncytium development.

The present study was carried out with a normal rat kidney cell clone that was initiated from a single cell infected by only one infectious particle of the non-transforming retrovirus, Moloney murine leukemia virus. Although the parental cells were highly resistant to chemical carcinogenesis, this infected clone was rendered susceptible to transformation by chemical carcinogens. However, when it was exposed to the tested carcinogen at a low subculture passage post-infection, cell transformation was detectable only after many subsequent passages. On the other hand, if it was exposed to the carcinogen at a high passage post-infection, cell transformation was detectable sometimes even without further passage, or in other cases after the first subsequent passage. Mouse interferon could inhibit the transformation by carcinogen employed at the low but not at the high passage post-infection. This inhibitory effect was reversible; if interferon was removed, even after the cells had been passaged many times in its presence, cell transformation became visible at passage 11 after interferon removal, although no treatment with the carcinogen was repeated. Interferon had no effect on the replication or the focus-forming capacity of cells transformed by such chemical-retroviral co-carcinogenesis. The possibility that this carcinogenic process depends on amplification of the integrated provirus DNA, and that this amplification can be inhibited by interferon is discussed.