Investigation on the molecular mechanism of steroidogenesis increased by the activated mutant K-ras

• Main Authors: Wu, Chia-Hung, 吳佳鴻
• Other Authors: Lin, Shiu-Ru
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/05653653081315034634

博士 === 高雄醫學大學 === 醫學研究所 === 91 === Abstract In our previous study on the tumorigenesis of human functional adrenal tumors, we observed high frequency of K-ras gene point mutations in clinical adrenal tumors. Furthermore, we cloned the mutated K-ras gene from the tumors and inserted it into vectors to transfect normal bovine adrenocortical cells purchased from ATCC (American Tissue Culture Center) to express mutated K-ras gene. The results showed that the mRNA level of steroidogenic enzyme, such as Cholesterol side-chain cleavage enzyme (P450SCC), 17a-Hydroxylase/17, 20-lyase (P450c17) and 3b- Hydroxysteroid dehydrogenase (3bHSD) in the mutant K-ras stable transfected cells was elevated. To increase the credibility of our experiments, we had cultured the human adrenaocortical cell from the human adrenal cortex, the cortical tissues were separated from the medulla after the adrenal tissue was dissected from patients. The cultured human adrenocortical cells synthesize and secrete steroid hormones after stimulated with ACTH and Angiotensin II. Then, we transfected the cells with mutant K-ras expression plasmids constructed from human functional adrenal tumors. The results showed that cells after stable transfection grew faster than normal cells. Additionally, cell morphological change was observed in mutant K-ras transfected cells. Moreover, when analyzed the synthesis pathway of hormones, the mRNA of P450SCC, P450C17 and 3βHSD was found to have increased and the level of cortisol increased 16 to 22 times higher than control cells. Additionally, the increased steroid hormone production in mutant K-ras transfected cells was reversed by lovastatin, a pharmacological inhibitor of p21ras function. These results combined with our previous reports of steroidogensis in bovine adrenocortical cells suggest that the ability of K-ras oncogene indeed involved in steroidogenesis in human adrenocortical cells. Then we had transfected the pK568MRSV , an IPTG inducible mutated K-ras expression plasmid, into the cultured human adrenaocortical cell. Then, the stable transfectants were selected with G418 (400 ug/ml). Additionally, the results have shown that the increase of RasGTP levels in the pK568MRSV transfected cells was time-dependent manner after IPTG induction. Western blot analysis results revealed significant elevation in phosphorylation of c-Raf-1 and Mitogen-activated protein kinase (MAPK). The increase of mRNA amount in P450scc、P450c17 and 3βHSD and the elevation of cortisol level were inhibited with a pretreatment of PD098059, a specific extracellular signal -regulated kinase inhibitor. In our report, we proved that lovastatin, a pharmacological inhibitor of p21ras function, also reversed the increase of cortisol level in mutant K-ras stable transfectants. 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 The ever-increasing rate at which genomes are being sequenced is attracting attention to functional genomics ─ an area of genome research that is concerned with assigning biological function to DNA sequences. Therefore, we use the cDNA microarray, genes involved in cell cycles, signal transduction, apoptosis, tumorigenesis and some expressed sequence tags (ESTs), totally over 8000 unique human genes arrayed on glass slides, to examine the difference between the control cell and the K-ras transfected cell to understand the cross talks between the Ras-to-MAPK pathway and the regulation of the steroidogenesis process. For example, we interesting, human zinc finger protein 22, osteopontin, LIM domain kinase 2 (LIMK2), Homo sapiens dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2) and human syntaxin 3 were shown to be 28.5, 5.8, 3.3, 2.21 and 2.0- fold up-regulated, respectively, by microarray. Similarly, Retinoblastoma binding protein 1 (RBP1), Homo sapiens craniofacial development protein 1(CFDP1), DAP kinase-related-apoptosis-inducing protein kinase1 (DRAK1), SKI-interacting protein (SKIP) and human poly(A)-binding protein (PABP) gene were shown to be 4.0, 2.4, 2.3, 2.2 and 2.1-fold down-regulated, respectively. The roles of those targeted genes involve in cell proliferation, cell morphologic change, tumorigenesis and steroidogenesis still need to be confirmed and further investigation.