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.

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.

Factors Influencing the Pathogenicity of Entamoeba Histolytica

In summarizing the results of the experimental studies up to the present, it is conjectured that the pathogenicity of Entamoeba histolytica or establishment of amoebiasis is not unique but differs by strain and age of Entamoeba histolytica and the age of the host. A non-virulent strain is more likely adapt to as low a temperature as 32 . This is not so in the strains which originated form clinical cases. Iso-enzyme patterns may roughly characterize pathogenic strains from non-pathogenic, Red blood cells may contribute as nutrients for growth of Entamoeba histolytica only after they have been hemolysed, but they are toxic to the amoebae as long as they remains intact. A low protein diet and stress may facilitate the establishment of amoebiasis; male sex hormones or previous infection by enteric bacteria provide a more advantageous condition than the female; and hepatotoxic agents will accelerate amoebic hepatitis.

Characterization of Tumor Specific Antigens on the Plasma Membrane Surface of Rat Hepatomas lnduced by 3'-Me DAB and ldentification of the Common Tumor Specific Antigens from Rat Hepatomas lnduced by Different Chemical Hepatocarcinogens

Three different chemical carcinogens, 2-acetylaminofluorene (AAF), diethylnitrosamine(DENA), and 3'-methyl-4dimethylaminoazobenzene (3'-Me DAB) were used to induce hepatomas in rats. Plasma membrane surface proteins of normal rat liver cells and rat hepatomas were extracted with 3M KCI. From the analysis of the proteins of normal rat liver and rat hepatoma induced by 3'-Me DAB by discontinuous polyacrylamide gel electrophoresis(Disc-PAGE), under nonreducing and nondenaturing conditions polyacrylamide gel electrophoresis in the presence of SDS and 2-mercaptoethanol (SDS-PAGE), Sephadex G-200 gel permeation chromatography, DEAE-A50 ion-exchange chromatography and two-dimensional gel electrophoresis, at least three tumor specific antigens were identified. One had a molecular weigh of 66,000 (pl=6.79) while the other two had the same molecular weight 73,000 but differed in their isoelectric points (7.58 and 7.81). For immunological analysis of tumor specific antigens, the absorbed antiserum was prepared. Plasma membrane surface proteins of rat hepatoma induced by 3'-Me DAB were used to obtain New Zealand White male rabbit antiserum. Rabbit antiserum was then reacted with the proteins isolated from the plasma membrane surface of normal rat liver and the absorbed antiserum reacting specifically with the tumor specific antigens derived by 3'-Me DAB was obtained. Using the absorbed antiserum, the immunoreactivities of plasma membrane surface proteins isolated from rat hepatomas induced by 3'-Me DAB, AAF, and DENA were compared by Ouchterlony double immunodiffusion analysis and immunoelectrophoresis. To characterize the proteins reacting to the absorbed antiserum, immunoglobulin G was separated from the absorbed antiserum and coupled to cyanogen bromide-activated Sepharose CL-4B. The isolated proteins from the plasma membrane surface proteins of 3'-Me DAB-induced hepatoma using this immunoaffinity chromatography had molecular weights of 66,000 and 73,000. The localization of these proteins on surface plasma membranes of rat hepatomas induced by 3'-Me DAB was confirmed by an immunofluorescence technique. The experimental results revealed the existence of cross-reacting common antigens on the plasma membrane surface of rat hepatomas induced by different hepatocarcinogens.

A Study of the Regulation of the Glucose Transporter in the Plasma Membranes of Hepatoma Cells Induced by 3'-Me DAB

5'-nucelotidase and glucose-6-phosphatase are liver plasma and microsomal membranes markers and their respective activities were determined. In the liver homogenate, the activities of 5'-nucleotidase were 0.58 +/- 0.08 and 0.29 +/- 0.07 micromols/mg protein/10min in the control and 3'-methyl-4-dimethyl aminoazobenzene (3'-Me DAB) groups respectively. The enzyme activities m the partially purified plasma membranes were 2.15 +/- 0.25 and 1.31 +/- 0.23 micromols/mg protein/10min in the control and 3'-Me DAB groups respectively. The glucose-6-phosphatase activities in the homogenates of the control and 3'-Me DAB groups were 0.23 +/- 0.10, and 0.45 +/- 0.25 micromols/mg protein/10min, and in the microsomal fraction, 1.14 +/- 0.32, and 0.63 +/- 0.11 micromols/mg protein/10min, respectively, The concentrations of glucose carrier in the plasma membranes from the control and 3'-Me DAB group were 25, and 35 pmols/mg membrane protein, respectively, and the Ka values for cytochalsin B in each group were 5.20 X 109. and 5.14 X 109ml/mol, respectively. However in the microsomal fraction, no significant differences of glucose carrier were found between the control and 3'-Me DAB groups from the DEAE Sephadex A-50 ion exchange chromatography, fractions I and ll were obtained. Electrophoretic analysis of fraction I revealed a major protein band with a molecular weight of 45,000 and minor bands with MWs of 50,000, 55,000 and 15,000. Following AcA 34 gel filtration, a major protein band with a MW of 45,000 was obtained. From these results, it can be concluded that the glucose carrier protein was increased on plasma membrane of hepatoma induced by 3'-Me DAB, and the carrier protein showed similar molecular weight to other glucose carrier found in the RBC, muscle cells and adipocyte.

Surface Properties of Cell Membrane in Early Stage of Transformed Cell -I. Early Detection of Transformed Cell by Concanavalin A; II. Properties of Plasma Membrane of Transformed Rat Liver Cell Induced by 3'-Me DAB

The present study was designed in order to investigate the lectin induced cytoagglutination properties of normal and transformed cells and surface alterations in the early stage of the transformed cells by characterizing the structural changes on the hepatoma surface membrane. Rat and rabbit erythrocytes and Sarcoma 180 ascites tumor cells were used for the lectin-induced cytoagglutination. Plotting % agglutination versus concanavalin A(Con A) concentration, sigmoid curves appeared in all cases. alpha-methyl-D-mannopyranoside(alphaMM) inhibited Con A induced cytoagglutination and the degrees of inhibition depended on the cell types and species. When rats were fed a diet containing 0.06% 3'-methyl-4dimethylaminoazobenzene(3'-Me DAB) for 12 weeks, almost all of the rats had solid liver tumors. Polyacrylamide gel electrophoresis of surface membrane proteins of these rat livers and of transplanted tumor cells showed three distinct protein bands, of which two were absent in normal rat livers. The molecular weights of these proteins were 73,000, 66,000, and 57,000 daltons. Antiserum against primary hepatocarcinoma surface proteins precipitated with three membrane proteins obtained from primary hepatocarcinoma cells as well as transplanted hepatocarcinoma cells, suggesting the presence of specific tumor antigens in these cells.