Mutant K-ras oncogene regulates steroidogenesis of normal human adrenocortical cells by the RAF-MEK-MAPK pathway.

The result of our previous study has shown that the K-ras mutant (pK568MRSV) transfected human adrenocortical cells can significantly increase cortisol production and independently cause cell transformation. The aim of this study is to investigate the effect of the active K-ras oncogene on the cortisol production in normal human adrenocortical cells. First we used isopropyl thiogalactoside to induce the inducible mutant K-ras expression plasmid, pK568MRSV, in the stable transfected human adrenocortical cells. The result showed that the increase of RasGTP levels in transfected cells was time-dependent after isopropyl thiogalactoside induction. Additionally, results from Western blot analysis revealed significant elevation in phosphorylation of c-Raf-1 and Mitogen-activated protein kinase. We also detected the levels of mRNA encoding Cholesterol side-chain cleavage enzyme (P450(SCC)), 17alpha-Hydroxylase/17,20-lyase (P450(c17)) and 3beta-Hydroxysteroid dehydrogenase (3betaHSD) were increased in human adrenocortical cells transfected with mutant K-ras after IPTG treatment. The increase of mRNA amount in P450(scc) P450(c17) and 3betaHSD and the elevation of cortisol level were inhibited with a pretreatment of PD098059, a specific extracellular signal-regulated kinase inhibitor. In our previous report, we proved that lovastatin, a pharmacological inhibitor of p21(ras) function, also reversed the increase of cortisol level in mutant K-ras stably transfected human adrenocortical cells. Taken together, these findings proved that the active mutant Ras enhanced not only cell proliferation but also steroidogenesis in steroidogenic phenotype cells by activating Raf-MEK-MAPK related signal transduction pathway. Therefore, we believe that K-ras mutants influence regulation of steroidogenesis in adrenocortical cells through RAF-MEK-MAPK pathway.

Overexpression of the c-met protooncogene in human gastric carcinoma--correlation to clinical features.

Overexpression of hepatocyte growth factor receptor (c-met) has been detected in many human tumors. To investigate the possible involvement of c-met in human gastric carcinogenesis, we examined c-met expression in 45 patients with gastric carcinoma using Northern blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), and immunohistochemical staining. The c-met mRNA expression was increased twofold and sevenfold in gastric carcinoma tissues compared to the adjacent normal tissues by Northern blot analysis and RT-PCR, respectively. In the immunohistochemical study, c-met protein was detected in 32 of 45 (71.1%) patients, with marked overexpression in gastric carcinoma compared with matched normal gastric tissues. The c-met-positive immunoreactivities were more frequently encountered in serosa-exposed and serosa-infiltrating gastric cancer (p = 0.003). In addition, tumor stage was another statistically significant parameter that was observed between the two groups (p = 0.02). Multivariate analyses revealed that the tumor stage (p = 0.014) and c-met overexpression (p = 0.031) were independently correlated with survival. These data suggest that overexpression of c-met may play a part in gastric carcinogenesis and may represent a prognostic factor for gastric cancer.

The p53 codon 249 mutant--derived from human functional adrenal tumors--can modify the cell shape of normal adrenocortical transfected cells.

Our previous study has indicated that p53 gene mutation occurred in 73% of human functional adrenal tumors, and the mutation hot spots were focused on codons 100, 102 (exon 4), and 249 (exon 7). Furthermore, a transcriptional activity study revealed that the mutant p53 protein derived from human functional adrenal tumors lost 90% transcriptional activity and the ability to bind with the p53 sequence. In order to investigate the influence of the mutant protein extracted from adrenocortical tumors on normal adrenal cells, we first cloned p53 cDNA from the human primary aldosteronism and constituted it with isopropyl thiogalactoside (IPTG) inducible expression vector as recombinant plasmid. The recombinant plasmid was then transfected to normal bovine adrenocortical cells through electroporation. The results showed that the p53 protein mutations at codons 100 and 102 could neither affect the cell morphology nor enable cell growth on the soft agar. In addition, no significant difference was found in cortisol level between the p53 transfected and the control cells. On the other hand, cell morphological changes and cell proliferation rate increase were observed when we used IPTG to induce the expression of the p53 protein, which mutated at codon 249, in adrenocortical cells. The cell morphology changes included less flattened and decreased elongation when compared to non-transfected cells. However, the cortisol level in transfected cells was not affected by the p53 mutants. Taken together, we concluded that the mutant p53 protein indeed participates in adrenal carcinogenesis; however, it has no influence on hormone production and secretion.

Significantly increased cortisol secretion in normal adrenocortical cells transfected with K-ras mutants derived from human functional adrenocortical tumors.

Our previous study showed that the mutation hotspots of the K-ras proto-oncogene in human functional adrenocortical tumors are in codons 15, 16, 18, and 31, thus differing from the sites in other tumors. In addition, analyzing the K-Ras protein by a recombinant DNA technique showed that the activity of endogenic GTPase and the GTPase-activating protein (GAP)-binding ability were significantly decreased in patients with these tumors. The aim of this study was to understand whether those K-ras mutants, which were found only in human adrenocortical tumors, play an important role in these tumors. Thus, the mutant K-ras cDNA was constructed with mammalian expression vectors and transfected into normal adrenocortical cells. The amount of cortisol secreted by the transfected cells was 20 to 30 times that of normal cells. Furthermore, Northern blot analysis revealed that the expression of the three steroidogenesis-related genes P450(scc) (cholesterol side-chain cleavage enzyme), P450(C17) (17alpha-hydroxylase/17, 20-lyase), and P450(C21) (steroid 21-hydroxylase) gene increased in the transfected cells. The K-ras oncogene significantly increases cortisol secretion by normal adrenocortical cells.