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.

The deoxyribonucleic acid regions involved in the hormonal regulation of thyroglobulin gene expression.

Transcription of the thyroglobulin (TG) gene is stimulated by TSH via cAMP. We have characterized the sequence elements responsible for the hormone-dependent expression of TG gene in rat thyroid FRTL-5 cells using internal deletion and linker-scanning mutants of the minimal TG promoter (-170 basepairs) fused with the bacterial chloramphenicol acetyltransferase reporter gene. The TG gene is regulated by at least two regions located between -165 and -140 bp (TG-III) and between -95 and -65 bp (TG-I) from the transcription initiation site. The intervening region can be deleted without significant effect on the promoter activity. Either of the two regions alone does not promote hormone-dependent transcription. A DNase footprinting assay showed that TG-I and TG-III are the principal protein-binding sites and that the proteins interacting with these two regions are induced by TSH or cAMP. These results suggest that the hormone-dependent expression of TG gene may be achieved by cooperative interaction of the proteins bound to TG-I and TG-III.

A study on the regulation of translocation of glucose transporters during hepatocarcinogenesis induced by 3'-Me DAB.

The mechanism of glucose transported (GT) expression on the plasma membranes of hepatoma cells in rats induced by 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) was studied. Cytochalasin B binding to plasma membrane fractions from control and 3'-MeDAB group in the absence of cold cytochalasin B showed 9,825 +/- 925 and 30,165 +/- 625 dpm/mg membrane protein. Scatchard plot analysis showed that the GTs present on the plasma membrane fractions in control and 3'-Me DAB groups were 5.0 and 16.0 pmol/mg membrane protein and their Kd values were 151 and 157 nM, respectively. These results suggest that the numbers of GTs in plasma membrane were increased in the 3'-Me DAB group compared to the control group. In contrast, the amounts of GTs in low density microsomal (LDM) fractions measured by a photoaffinity labeling technique using [3H]-cytochalasin B were 31,207 and 11,702 dpm/mg protein in the control and 3'-Me DAB group, respectively. These results suggest that GTs were translocated from LDM to plasma membranes during carcinogenesis. To confirm these results by an independent method 10% SDS-polyacrylamide gel electrophoresis was carried out. Gel slice No. 13 corresponding to MW of 45 kDa from plasma membrane fractions showed increased radioactivities in the 3'-Me DAB group compared to the control group. However, LDM fractions of the 3'-Me DAB group showed decreased radioactivities compared to the control group. Western blot analysis using anti-human RBC GT antibody present in the plasma membranes and LDM fractions from control and 3'-Me DAB groups did not show any significant difference, indicating low cross-reactivity between them. These results indicate that increased glucose transport seems to be more likely due to reciprocal redistribution of GTs between plasma membrane and LDM fractions.

Similar nuclear factors mediate stimulation of rat thyroglobulin gene transcription by thyrotropin and insulin-like growth factor-I.

Transcription of the thyroglobulin (TG) gene in rat thyroid FRTL-5 cells is stimulated by two hormones, TSH and insulin-like growth factor-I (IGF-I). The effect of TSH is mimicked by cAMP. Promoter regions of the rat TG gene responsible for hormonal action as well as the nuclear regulatory proteins that interact with these regions were characterized. Minimal promoter that responds to both hormones has been found to be up to -171 basepairs from the transcription initiation site. In DNase-I footprinting analysis, nuclear extracts from cells treated with either of these hormones protected the same two major regions within the minimal promoter. Mutations in these two regions abolished basal, TSH-stimulated, as well as IGF-I-stimulated expression of the fused reporter gene chloramphenicol acetyltransferase. DNA mobility shift assay revealed that cAMP and IGF-I induce binding of similar nuclear proteins to these promoter regions. These results suggest that rat TG gene transcription is regulated by the convergent action of two distinct signaling pathways, possibly involving similar DNA-binding nuclear proteins and regulatory sequences of the TG gene promoter.

Potent reversible anticholinesterase agents. Bis- and mono-N-substituted benzoquinolinium halides.

A number of bis- and mono-N-substituted benzoquinolinium salts and their analogues were prepared and evaluated as inhibitors of acetylcholinesterase (AcChE) and butyrylcholinesterase (BuChE). These compounds were also used to help identify some of the morphologic characteristics of the surface at or near the active sites of the cholinesterases. The shape, size, configuration, and conformation of the onium moieties of the quaternary ammonium compounds were found to be the important factors in their anticholinesterase activity. A high concentration of the positive charge of the quaternary ammonium compound is not a critical factor for the cholinesterase inhibitory activity. The order of decreasing potency of cholinesterase inhibition of the benzoquinolinium compounds was found to be acridinium greater than phenanthridinium greater than 5,6-benzoquinolinium greater than 7,8-benzoquinolinium. The inhibitory activity of the monobenzoquinolinium halides against cholinesterases is influenced by the N-substituent. A bis-quaternary ammonium compound with a flexible bridge that links the two nitrogen atoms was found to be more potent in inhibiting AcChE and less potent in inhibiting BuChE than a bis-quaternary ammonium compound with a rigid bridge. The acridinium and phenanthridinium derivatives of the benzoquinolinium compounds are very potent reversible inhibitors against both AcChE and BuChE.