Expression of c-myc gene in human ovary carcinoma cells treated with vanadate

The widely accepted hypothesis of vanadate action on cells postulates that this ion inhibits protein phosphatase(s) that dephosphorylates protein phosphotyrosine residues. This inhibition causes tyrosine hyperphosphorylation of cell proteins followed by changes in physiological action of phosphoproteins resulting in stimulation of cell proliferation, expression of protooncogenes, and transient cell transformation. We have found that treatment of human ovary carcinoma (CaOv) cells with vanadate causes the increase in total protein phosphorylation from 1.5- to 2.0-fold whereas the ratio between phosphoserine, phosphothreonine, and phosphotyrosine content remains unchanged. At the same time, enhancement of c-myc gene expression (not c-fos) was observed. Hence, the increase in the ratio of phosphotyrosine to phosphoserine and phosphothreonine is not an obligatory intermediate stage before vanadate-dependent activation of c-myc expression.     

Down-regulation of c-myc and c-Ha-ras gene expression by tiazofurin in rat hepatoma cells

There was an overexpression of the c-myc gene (11-fold) and of the c-Ha-ras gene (2-fold) in rat hepatoma 3924A cells compared to normal rat liver as measured by dot-blot analysis of total cytoplasmic RNA. The overexpression of c-myc was attributed to a 10- to 14-fold amplification and rearrangement of the c-myc sequences as determined by Southern blot analysis. The expression of the c-myc also was dependent upon the proliferative state of the hepatoma cells. Tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide; NSC 286193), an inhibitor of the activity of IMP dehydrogenase (EC 1.1.1.205), the rate-limiting enzyme of GTP biosynthesis, resulted in a rapid drop (less than 1 h) to 50% of control in the target enzyme activity in the hepatoma cells and in a subsequent marked decrease to 55% in GTP concentration. These events were followed at 12 h of tiazofurin treatment by a 3-fold reduction in the expression of the c-myc gene and a 9-fold decline in that of the c-Ha-ras gene. These results in the hepatoma cells provide evidence in support of the earlier demonstrated correlation in K562 cells between GTP concentration and expression of c-myc and c-ras genes (Olah et al., 1989). These genes might depend on GTP for their expression in hepatoma cells and they might cooperate in a signal pathway that controls cell proliferation.     

Expression of c-myc proto-oncogene in normal human intestinal epithelium

We studied the expression of the human c-myc proto-oncogene in normal human colon epithelium by both in situ hybridization and immunohistochemistry. c-myc was found to be expressed uniformly throughout the entire thickness of the colon epithelium. The present findings do not support the contention that the c-myc proto-oncogene is primarily expressed in proliferating intestinal epithelial cell compartments.     

Secretion of Mr 46,000 protein from human hepatoma cells treated with tumor promotors is regulated by c-myc gene expression

Previously an Mr 46,000 protein (named p46) was shown to be induced in the culture medium of human hepatoblastoma cells, Huh-6 Cl-5, treated with 12-O-tetradecanoyl phorbol-13-acetate (TPA). For further characterization of p46, Huh-7 Cl-4, another line of well differentiated liver cells, was incubated in the presence and absence of TPA, and proteins were labeled with [35S]methionine, and the proteins secreted into the medium were analyzed by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. A protein of Mr 46,000 was observed in the culture medium of Huh-7 Cl-4 cells treated by TPA or plated at low density, but only slightly in the culture medium of Huh-7 Cl-4 cells plated at high density. As these results suggested that the expression of p46 was regulated by a factor that was activated in the growth state and by TPA treatment, the effect of introduction of the competence gene c-myc into Huh-7 Cl-4 cells was examined. All 37 independent transfected colonies obtained showed enhanced expression of p46. As a control, the c-Ha-ras gene was introduced into Huh-7 Cl-4 cells and shown not to enhance expression of p46. These results strongly suggested that the expression of p46 was regulated by the competence gene c-myc.     

Methylation of c-myc gene changes in human lymphoproliferative diseases

The degree of methylation at the c-myc proto-oncogene was found to change in human lymphoproliferative diseases, when examined using a methylation-sensitive restriction enzyme. In peripheral blood mononuclear cells (PBMC) c-myc DNA showed hypomethylation in human lymphoproliferative diseases, in comparison to normal subjects matched in age and sex. In cases of chronic lymphocytic leukemia (CLL), the change was amplified in the crisis. When the DNA was examined at the actin gene, no significant change was observed. The results suggest that the change in c-myc proto-oncogene methylation might become an important clue in understanding the relationship between levels of gene expression and methylation in human lymphoproliferative diseases.     

Expression of haptoglobin receptors in human hepatoma cells.

The uptake of radio-labeled hemoglobin-haptoglobin complex (Hb-Hp) by human hepatoma PLC/PRF/5 and HepG2 cells was investigated in an attempt to characterize the uptake process and intracellular transport. Human hepatoma cells took up Hb-Hp in a receptor-mediated manner. Scatchard analysis of binding revealed that PLC/PRF/5 and HepG2 cells exhibited about 21,000 and 63,000 haptoglobin receptors/cell, with a dissociation constant (Kd) of 8.0 and 17 nM, respectively. Human hepatocytes in primary culture also expressed about 84,000 receptors/cells, with a Kd of 7.4 nM. The hemoglobin-haptoglobin complex was internalized and subsequently the internalized Hb-Hp was slowly degraded in the cells. Preincubation of the cells with Hb-Hp resulted in a decrease in binding of the radioactive Hb-Hp to the cell surface, and was accompanied with an accumulation of intracellular receptors. The uptake of Hb-Hp by the cells was not inhibited by 100 microM chloroquine or by 10 mM methylamine, but was inhibited by 50 microM monodansylcadaverine. Hemoglobin-heme taken up by the cells induced microsomal heme oxygenase. Thus, human hepatoma PLC/PRF/5 and HepG2 cells can take up Hb-Hp by haptoglobin receptor-mediated endocytosis and Hb-Hp probably causes translocation of the haptoglobin receptors from the cell surface to the cell interior where they can be degraded. The internalized heme-moiety of hemoglobin can regulate the expression of heme oxygenase.     

Expression of c-myc oncogene in human fibroblasts during in vitro senescence

Expression of the c-myc oncogene was determined in pre-confluent early and late passage human (IMR-90) fibroblast by dot blot analysis of cellular mRNA. Significant decreases in c-myc levels were found in late passage when compared to levels found in early passage cells. Cells restimulated with serum after serum restriction also showed reduced levels of c-myc in late passage. Confluent cells expressed levels of c-myc similar to that of pre-confluent cells, when serum stimulation was the same in both cases. Southern blots of Eco R1 digested DNA showed 2 fragments of similar size hybridizing to c-myc sequences in both early and late passage cells.     

Transactivation of the human c-myc gene by c-Myb.

We analyse the contribution of six Myb-binding sites in the upstream c-myc sequences to transactivation by co-transfection assays. Surprisingly, deletion of the six Myb-binding sites did not influence the transactivation of c-myc by c-Myb protein. Instead, the strongest transactivation was observed with a c-myc reporter plasmid which contains only 450 bp of exon 1 including the c-myc promoter P2. An exchange of the DNA binding domain of c-Myb by that of GAL4 led only to small transactivation effects indicating that the DNA binding domain of c-Myb is essential for transactivation of the c-myc gene. These results suggest either an indirect transactivation mechanism of the c-myc gene by c-Myb proteins or a role of the DNA binding domain for additional effects than DNA binding.     

Expression and characterization of the human c-myc DNA-binding protein.

In an effort to study in detail the nature of the protein product of the human protooncogene c-myc, we have expressed the gene at high levels in Escherichia coli. The c-myc coding region was taken from a full-length cDNA clone and inserted into a vector designed to express foreign gene products efficiently in E. coli. Pulse-labeling experiments indicated that the rate of expression of c-myc in this thermoinducible expression system is very efficient. The product was relatively stable and accumulated to approximately 10% of total cellular protein. A purification protocol was devised which allowed the c-myc protein to be readily purified in quantities sufficient for detailed biochemical and physical analyses. A high-titer polyclonal antiserum was raised against the pure protein and shown to immunoprecipitate the p110gag-myc fusion protein of MC-29-infected quail cells. This antiserum also selectively detects a protein with an apparent molecular weight of 64,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis from a Burkitt lymphoma cell line. We conclude that this 64-kilodalton protein is the human c-myc gene product since the E. coli-made protein exhibits an equivalent molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, even though its calculated molecular weight is 49,000. Furthermore, we demonstrate that the bacterially made human c-myc protein is a DNA-binding protein and that it exhibits a high nonspecific affinity for double-stranded DNA.     

c-myc gene expression in human cells is controlled by glucose

The c-myc oncogene is implicated in normal growth and differentiation processes. Human cell lines IM9 and HepG2 stably cultured at "low" glucose concentrations (5.5 mM) show c-myc mRNA levels 3-4 times higher than cells cultured at "high" glucose concentrations (25 nM). D-fructose (a metabolizable exose) substitutes for D-glucose in reducing c-myc expression while 3-ortho-methylglucose (a non metabolizable exose) is uneffective. c-myc expression is up-regulated (by PMA) or down-regulated (by dexamethasone and long-term exposure to FCS) in human cells cultured at "low" glucose but not in cells cultured at "high" glucose. We previously demonstrated that insulin receptor gene expression in human cell lines in enhanced by glucose. Therefore, glucose controls in an opposite way the expression of two genes important in the regulation of eukaryotic cell growth and differentiation.