Neurokinin A induces c-fos, c-jun, and c-myc expression in L6 rat myoblasts

Neurokinin A (NKA), a neuropeptide belonging to the tachykinin family, induced c-fos proto-oncogene mRNA expression in serum-deprived L6J1 rat skeletal myoblasts in vitro. The marked increase reached maximal levels after 15 to 30 min. In contrast to this, c-jun and c-myc proto-oncogene expression were only slightly induced, with peak levels after 30 min. NKA did not stimulate DNA synthesis or cell proliferation in serum-deprived L6J1 myoblasts. We demonstrate a relationship between NKA treatment and induction of c-fos, c-jun and c-myc mRNA expression in serum-deprived L6J1 rat myoblasts. The results on DNA synthesis and cell proliferation indicate that the induced proto-oncogene expression alone is not enough to induce a cellular response to NKA. Possible mechanisms of action are discussed.     

Use of a multiple S1 nuclease protection assay to monitor changes in RNA levels for type 1 phosphatase and several proto-oncogenes in response to insulin.

Changes in insulin-regulated gene expression occur in a time- and tissue-dependent fashion. To monitor these changes we have adapted the S1 nuclease protection assay to allow simultaneous estimation of multiple RNA species in a single sample by using synthetic oligonucleotides of various lengths as probes for specific RNA species, which can then be resolved by electrophoresis. The multiple S1 nuclease protection assay was used to assess the influence of insulin on the RNA concentrations of 12 different genes in human skeletal muscle. Estimates obtained by this assay were comparable with those obtained by Northern analysis. RNA levels for proto-oncogene c-src displayed a transient 4-fold increase, whereas RNA levels for type 1 protein phosphatase were suppressed by 50% during the same time period. RNAs corresponding to known insulin-responsive genes such as c-fos, c-myc, c-Ha-ras, and c-src displayed rapid and transient 2-4-fold increases between 30 and 60 min as detected by either Northern analysis or the multiple S1 nuclease protection assay. In addition, RNA levels for the insulin receptor, Glut-4, Glut-3, and c-jun were apparently unaffected by exposure of the cells to insulin.     

Dsi-1, a region with frequent proviral insertions in Moloney murine leukemia virus-induced rat thymomas.

Dsi-1 is a region of chromosomal DNA that underwent proviral insertion in 3 of 24 Moloney murine leukemia virus-induced rat thymomas. In one of these tumors, a provirus is also integrated adjacent to the proto-oncogene c-myc. The proviruses in Dsi-1 have been characterized and appear to be complete. The proviruses were located within a 2-kilobase region that contained four prominent DNase I-hypersensitive sites. These hypersensitive sites were observed in Moloney murine leukemia virus-induced thymomas but not in NRK cells. The region of Dsi-1 immediately 3' to the insertions cross-hybridized with human and chicken DNA, indicating that it contains highly conserved sequences. No evidence could be found for the expression of this highly conserved region. Dsi-1 was mapped to mouse chromosome 4. This location demonstrates that Dsi-1 is different from 16 of the known proto-oncogenes (c-abl, c-erbA c-erbB, c-ets-1, c-ets-2, c-fes, c-fos, c-myb, c-myc, c-raf, A-raf, c-Ha-ras, c-Ki-ras, N-ras, c-sis, and c-src) and 12 cellular regions of tumor-associated integrations in retrovirus-induced tumors (c-erbB, Fis-1, int-1, int-2, Mis-1/pvt-1, Mlvi-1, Mlvi-2, c-mos, c-myb, c-myc, Pim-1, and c-Ha-ras). Hybridization experiments indicated that Dsi-1 is probably different from five additional proto-oncogenes (c-fgr, c-fms, c-mos, neu, and c-yes) and from two additional frequent integration regions (lck and Mlvi-3).     

Inhibition of myogenic differentiation by fibroblast growth factor or type beta transforming growth factor does not require persistent c-myc expression

Skeletal muscle differentiation is accompanied by accumulation of the mRNA encoding the muscle isoenzyme of creatine kinase (MCK) and can be suppressed by serum components, fibroblast growth factor (FGF), or type beta transforming growth factor (TGF beta). Using the nonfusing myogenic cell line, BC3H1, the potential involvement of c-myc in growth factor-dependent inhibition of myogenesis was examined. Withdrawal of undifferentiated myoblasts from the cell cycle in medium with 0.5% serum was associated with a precipitous decline in expression of c-myc mRNA followed by induction of MCK mRNA. In 0.5% serum containing TGF beta, c-myc mRNA declined to a level identical to that in differentiated cells; however, MCK mRNA was not expressed. Exposure of quiescent differentiated cells to FGF or TGF beta caused disappearance of muscle-specific gene products and was accompanied by only transient low level induction of c-myc mRNA. These data indicate that persistent c-myc expression is not required for growth factor-mediated inhibition of myogenic differentiation.     

Regulation of proliferation in a murine colony-stimulating factor-dependent myeloid cell line: superinduction of c-fos by the growth inhibitor 8-Br-cyclic adenosine 3':5' monophosphate

We have investigated the effect of 8-Br-cyclic adenosine 3':5' monophosphate (cAMP), a pharmacological activator of cAMP-dependent protein kinase, on the proliferation and the nuclear proto-oncogene induction in a murine granulocyte macrophage colony-stimulating factor (GM-CSF)-dependent myeloid cell line. Cells were growth arrested by granulocyte macrophage colony-stimulating factor and serum deprivation and were allowed to proceed in the cell cycle by addition of the lymphokine in the presence or absence of 8-Br-cAMP. 3H-thymidine incorporation assays showed that addition of 8-Br-cAMP inhibited the entry of cells into S phase and the subsequent proliferation. Northern analysis showed that 8-Br-cAMP had opposite effects on c-fos and c-myc mRNA induction. 8-Br-cAMP induced c-fos in the absence of any GM-CSF. In the presence of GM-CSF, c-fos mRNA was superinduced (30-fold induction compared to four- to fivefold by each signal alone). On the contrary, 8-Br-cAMP was not able to induce c-myc in the absence of growth factor and hardly interfered with the induction of c-myc by GM-CSF. Phorbol myristate acetate (PMA), a pharmacological activator of the lipid and CA++-dependent protein kinase C, was shown to induce nuclear proto-oncogene mRNA in the GM-CSF-dependent cell line. We investigated the effect of 8-Br-cAMP on PMA-induced c-fos and c-myc mRNA levels. When both cAMP dependent and lipid-dependent kinase systems were co-stimulated in the absence of GM-CSF, c-fos message was again superinduced (60-fold induction). On the contrary, c-myc message induction by PMA was inhibited by 80% by coactivation of cAMP-dependent protein kinase with 8-Br-cAMP. Our data indicate that an antiproliferative signal induces or even superinduces c-fos message and hardly interferes with c-myc induction, suggesting that the intracellular pathways resulting in c-fos and c-myc induction may be distinct and that two different pathways can lead to c-fos induction, with synergistic effects when both are activated.