The Role of Ras Oncogene in Differentiation of 3T3-L1 Preadipocytes

Ras is a proto-oncogene that codes for a small GTPase and is responsible for linking several extracellular signals to intracellular mechanisms that involve cell growth, differentiation and cell-programmed death in normal and diseased cells. In all these processes, Ras has been extensively investigated. However, the role of Ras GTPases is still poorly understood during the differentiation of 3T3-L1 preadipocytes. In this study I investigated the role of the H-Ras defective mutant, Ras:G12V on the differentiation of 3T3-L1 preadipocytes. Preadipocytes were differentiated in vitro to adipocytes (fat cells) by adding an induction medium containing several factors including glucose and insulin. The formation of fat cells evidenced by the visualization of lipid drops as well as by quantifying the accumulation of Oil red O into lipid drops. To examine the role of Ras:G12V mutant, several selective mutations were introduced in order to determine the signaling transduction pathways (i.e., PI3(K)kinase and MAP(K)Kinase) responsible for the Ras-dependent adipogenesis. Cells expressing Ras:G12V mutant stimulated 3T3-L1 preadipocyte differentiation without he need for induction media, suggesting that Ras activation is an essential factor required for 3T3-L1 preadipocyte differentiation. Introduction of a second mutation on Ras:G12V (i.e., Ras:G12V;E37G), which blocks the activation of the MAPKinase pathway, strongly inhibited the 3T3-L1 preadipocyte differentiation. It is also important to note Ras:G12V:E37G double mutant does not inhibit the activation of the PI3kinase pathway. Other Ras double mutants (Ras:G12V;S35T, and V12G;C40Y) showed a modest inhibition of the 3T3-L1 preadipocyte differentiation. Taken together, these observations indicate that Ras plays a selective role in 3T3-L1 preadipocyte differentiation. Thus, understanding which specific pathway Ras employs during preadipocyte differentiation could clarify some of the uncertainties surrounding fat production.

Alterations in Glutathione Levels and Apoptotic Regulators Are Associated with Acquisition of Arsenic Trioxide Resistance in Multiple Myeloma

Arsenic trioxide (ATO) has been tested in relapsed/refractory multiple myeloma with limited success. In order to better understand drug mechanism and resistance pathways in myeloma we generated an ATO-resistant cell line, 8226/S-ATOR05, with an IC50 that is 2–3-fold higher than control cell lines and significantly higher than clinically achievable concentrations. Interestingly we found two parallel pathways governing resistance to ATO in 8226/S-ATOR05, and the relevance of these pathways appears to be linked to the concentration of ATO used. We found changes in the expression of Bcl-2 family proteins Bfl-1 and Noxa as well as an increase in cellular glutathione (GSH) levels. At low, clinically achievable concentrations, resistance was primarily associated with an increase in expression of the anti-apoptotic protein Bfl-1 and a decrease in expression of the pro-apoptotic protein Noxa. However, as the concentration of ATO increased, elevated levels of intracellular GSH in 8226/S-ATOR05 became the primary mechanism of ATO resistance. Removal of arsenic selection resulted in a loss of the resistance phenotype, with cells becoming sensitive to high concentrations of ATO within 7 days following drug removal, indicating changes associated with high level resistance (elevated GSH) are dependent upon the presence of arsenic. Conversely, not until 50 days without arsenic did cells once again become sensitive to clinically relevant doses of ATO, coinciding with a decrease in the expression of Bfl-1. In addition we found cross-resistance to melphalan and doxorubicin in 8226/S-ATOR05, suggesting ATO-resistance pathways may also be involved in resistance to other chemotherapeutic agents used in the treatment of multiple myeloma.