Browsing by Subject "RAS"
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Item Oncogenic roles of RAS in acute myeloid leukemia cooperated with Mll-AF9.(2008-07) Kim, Won-ilThree main sections are presented in this thesis. First, we investigated which hematopoietic cells express TRE -driven transgenes when combined with Vav-tTA , because mastocytosis was developed in Vav-tTA ; TRE-NRAS G12V transgenic mice without detectable other diseases. We assayed Vav-tTA -driven luciferase expression in hematopoietic cells including bone marrow-derived mast cells (BMMC) and CD34 positive hematopoietic progenitor cells (HPC) as well as myeloid and lymphoid lineages by live mouse imaging and relative light unit measurement before or after treating Vav-tTA ; TRE -luciferase co-transgenic mice with doxycycline (Dox). We found that B cells in the bone marrow and T cells in the thymus expresses Vav-tTA -driven luciferase at much higher levels than in myeloid cells, BMMC and CD34 positive HPC, which showed relatively low levels. Thus, we conclude that Vav-tTA -driven NRAS G12V expression is sufficient for mastocytosis development but not for myeloid leukemia, and lymphoid cells are resistant to NRAS G12V transformation despite high level of expression. Second, experiments were performed to study the oncogenic role of the NRAS oncogene ( NRAS G12V ) in the context of acute myeloid leukemia (AML). We transplanted AML, which was developed in Vav-tTA TRE-NRASG12V ; Mll-AF9 transgenic ( TRM -transgenic) mice, into recipient SCID mice. Conditional repression of NRAS G12V expression greatly reduced peripheral white blood cell (WBC) counts in leukemia recipient mice and induced apoptosis in the transplanted AML cells correlated with reduced Ras/Erk signaling. After marked decrease of AML blast cells, myeloproliferative disease (MPD)-like AML relapsed characterized by cells that did not express NRAS G12V . In comparison with primary AML, the MPD-like AML showed significantly reduced aggressiveness, reduced myelosuppression and a more differentiated phenotype. We conclude that, in AML induced by an Mll-AF9 transgene, NRAS G12V expression contributes to acute leukemia maintenance by suppressing apoptosis and reducing differentiation of leukemia cells. Moreover, NRAS G12V oncogene has a cell non-autonomous role in suppressing erythropoiesis that results in the MPD-like AML showed significantly reduced ability to induce anemia. Third, based on the results finding the relapse of NRAS G12V -independent AML, we tested a hypothesis that chemotherapeutic cytarabine (AraC) treatment in addition to the RAS oncogene suppression would prevent or delay the relapse of AML. After the establishment of full-blown AML. We treated recipient mice with Dox and/or AraC (50 mg/kg/day). Compared with recipient mice treated with either Dox or AraC, we found that co-treatment significantly postponed the relapse of resistant AML and increased the survival days of the TRM -transgenic AML recipient mice. These results suggest that oncogenic RAS-targeting therapy may increase the therapeutic potential against drug-resistant AML when combined with chemotherapeutic AraC treatments. Consequently, we conclude the oncogenic roles of NRAS G12V expression in AML induced in cooperation with Mll-AF9 are; (1) to induce proliferation of AML blast cells, (2) to induce cell non-autonomous myelosuppression, (3) to suppress apoptosis in AML blast cells, and (4) to inhibit differentiation of AML blast cells. In treatment of AML, oncogenic RAS suppression combined current chemotherapy may improve the therapeutic potential and achieve longer remission period.Item The role of renin in the adipose tissue renin-angiotensin system.(2009-07) Fowler, Jason DeanThe renin angiotensin system (RAS) has been implicated in a variety of adipose tissue functions including tissue growth, differentiation, metabolism, and inflammation. While expression of all components necessary for a locally derived adipose tissue RAS have been demonstrated within adipose tissue, independence of local adipose RAS component concentrations from corresponding plasma RAS fluctuations has not been addressed. To analyze this, we varied in vivo rat plasma concentrations of two RAS components, renin and angiotensinogen (AGT), to determine the influence of their plasma concentrations on adipose and cardiac tissue levels in both perfused (plasma removed) and nonperfused samples. Variation of plasma RAS components was accomplished by 4 treatment groups: Normal, DOCA-salt, Bilateral nephrectomy, and Losartan. Adipose and cardiac tissue AGT concentrations correlated positively with plasma values. Perfusion of adipose tissue decreased AGT concentrations by 11.1% indicating that adipose tissue AGT was in equilibrium with plasma. Cardiac tissue renin levels positively correlated with plasma renin concentration for all treatments. In contrast, adipose tissue renin levels did not correlate with plasma renin, with the exception of extremely high plasma renin concentrations achieved in the Losartan treated group. These results suggest that adipose tissue may control its own local renin concentration independently of plasma renin as a potential mechanism for maintaining a functional local adipose RAS. Whereas adipose tissue possesses a local renin-angiotensin system, the synthesis and regulated release of renin has not been addressed. To that end we utilized differentiating 3T3-L1 cells and analyzed renin expression and secretion. Renin mRNA expression and protein enzymatic activity were not detectable in preadipocytes. However, upon differentiation, renin mRNA and both intracellular and extracellular renin activity were up regulated. In differentiated adipocytes, forskolin treatment resulted in a 28-fold increase in renin mRNA while TNF alpha treatment decreased renin mRNA 4-fold. IL-6, insulin, and angiotensin II (Ang II) were without effect. In contrast, forskolin and TNF alpha each increased renin protein secretion by 12- and 7-fold, respectively. Although both forskolin and TNFalpha induce lipolysis in adipocytes, fatty acids, prostaglandin E2 or lipopolysaccharide had no effect on renin mRNA or secretion. To evaluate mechanism(s) by which forskolin and/or TNF alpha are able to regulate renin secretion, a general lipase inhibitor (E600) and PKA inhibitor (H89) were used. Both inhibitors attenuated forskolin induced renin release while having no effect on TNF alpha regulated secretion. In contrast, E600 potentiated forskolin-stimulated renin mRNA levels while H89 had no effect. Neither inhibitor had any influence on TNFalpha regulation of renin mRNA. Relative to lean controls, renin expression was reduced 78% in the epididymal adipose tissue of obese male C57Bl/6J mice, consistent with TNF alpha-mediated down regulation of renin mRNA in the culture system. In conclusion, the expression and secretion of renin are regulated under a complex series of hormonal and metabolic determinants in mature 3T3-L1 adipocytes.