Molecular assembly of mitogen-activated protein kinase module in ras-transformed NIH3T3 cell line

The ras, is a G-like protein that controls the mitogen-activated protein kinase (MAPK) pathway involved in control and differentiation of cell growth. MAPK is a key component of its signaling pathway and the aberrant activation may play an important role in the transformation process. To better understand roles of ras in the activation of MAPKs, we have established ras transformed NIH3T3 fibroblast cell line, and analyzed the MAPK module. The ras transformed cells formed numerous spikes at the edges of cells and showed loss of contact inhibition. The levels of ERK1/2 MAPKs as revealed by Western blot analysis were not significantly different between ras transformed and non-transformed cells. However, phosphorylation of ERK MAPKs and the level of MEK were significantly increased although the heavily expressed level of Raf-1, an upstream component of MAPK pathway was unchanged in ras transformed NIH3T3 cells. The sedimentation profile of the MAPK module kinases in a glycerol gradient showed the presence of a rather homogeneous species of multimeric forms of ERK1/2 and MEK as indicated by the narrow distribution peak areas. The broad sedimentation profile of the Raf-1 in a glycerol gradient may suggest possible heterologous protein complexes but the identification of interacting molecules still remains to be identified in order to understand the organization of the MAPK signal transduction pathway.

Expressions of c-fos and c-myc genes during 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB)-induced rat hepatocarcinoma

We investigated the expression of the growth-related nuclear proto-oncogenes, c-fos and c-myc, in early preneoplastic regions and tumor nodules of 3'-MeDAB induced rat hepatocarcinoma. To amplify the levels of these transcripts, we gave cycloheximide (100 mg/kg B.W. i.p.) to each group of rats. The elevated levels of the 2.2 kb c-fos and 2.4 kb c-myc transcripts appeared as early as the 2nd week after feeding on the 3'-MeDAB diet and lasted through the 4th; 6th weeks and tumor. Southern blot analysis indicated that gross amplification or rearrangements were not observed in DNA of the preneoplastic livers and hepatoma nodules. We also measured the rate of the incorporation of [3H] thymidine into hepatic DNA in order to monitor the rate of cell proliferation occurring at the early preneoplastic periods. We have found that the rate of [3H] thymidine incorporation corresponds to the elevated levels of c-fos and c-myc transcripts in the precancerous stages. This finding suggests that the elevated expressions of c-fos and c-myc may result from the continuous cell proliferative stimuli generated in the carcinogen altered cells, which is essential to the initiation and promotion of chemical hepatocarcinogenesis.