Date of Award

2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

College

College of Graduate Studies

Abstract

SMS is an enzyme belonging to the sphingolipid metabolic pathway. Since the discovery and initial characterizations of sphingolipids (SLs) in 1884, extensive research has established that these molecules not only are structural components of eukaryotic membranes but they are also critical bioactive lipids involved in fundamental cellular processes such as proliferation, differentiation, apoptosis, inflammation, migration, and autophagy. In particular, SMS produces sphingomyelin (SM) in mammalian cells by transfering the phosphorylcholine moiety from phosphatidylcholine (PC) onto ceramide forming SM and diacylglycerol (DAG). Both ceramide and DAG are bioactive lipids with opposing functions, and SMS produces sphingomyelin (SM) while consuming ceramide (negative regulator of cell proliferation) and forming diacylglycerol (DAG) (a mitogenic factor). Therefore enhanced SMS activity could favor cell proliferation. To examine if disregulated SMS contributes to leukemogenesis, we measured SMS activity in several leukemic cell lines and found that it is highly elevated in K562 chronic myelogenous leukemia (CML) cells. The increased SMS in K562 cells was caused by the presence of the BCR-ABL oncogene, a hallmark of CML, as stable expression of BCR-ABL elevated SMS activity in the BCR-ABL negative HL-60 cells while inhibition of the tyrosine kinase activity of BCR-ABL with Imatinib mesylate, decreased SMS activity in K562 cells. The increased SMS activity was the result of up­ regulation of the SMSI isoform. Inhibition of SMS activity with D609 (a pharmacological SMS inhibitor) or down-regulation of SMS1 expression by siRNA, selectively inhibited the proliferation of Bcr-abl positive cells. The inhibition was associated with an increased production of ceramide and a decreased production of DAG, conditions that antagonize cell proliferation. A similar change in lipid profile was also observed upon pharmacological inhibition of Bcr-abl (K526 cells) and siRNA-mediated down-regulation of BCR-ABL (HL-60/Bcr-abl cells). These findings indicate that SMS1 is a downstream target of Bcr-abl, involved in sustaining cell proliferation of Bcr-abl positive cells. The increased SMS1 activity in bcr-abl positive cells was associated with higher levels of the SMS1 protein due to enhanced transcription, as measured by quantitative real time PCR (QRT-PCR) of heteronuclear RNA (hnRNA). In particular, the highest increase of SMS1 transcript and mRNA was observed when probing down-stream of exon 6, suggesting the preferential utilization of an alternative transcription start site (TSS) in Bcr­ abl positive cells. Thus this study investigated the effect of Bcr-abl on SMS1 transcription. Indeed, RLM-RACE based analysis of Bcr-abl positive cells identified multiple SMS1 TSSs, including a TSS located within intron 6 in close proximity and upstream to exon 7 (TSS C). Notably, in silica analysis of the entire SMS1 locus, including large areas of its 5' and 3' UTR, revealed multiple putative promoter regions, including Promoter C located upstream to TSS C. Further promoter-based studies corroborated the possibility of Bcr-abl to mediate transcription of SMS1 through the putative Promoter C and the dependence of SMS1 transcription on Bcr-abl was further confirmed by demonstrating the inhibition of Promoter C activity upon inhibition of bcr-abl activity with Imatinib. A series of deletions from the 5' end of promoter C revealed a core promoter region from -110 to -113 from TSS C suggesting that the cis-element(s) needed for Bcr-abl mediated SMS1 regulation was located within this area. Overall, these results provide strong evidence of Bcr-abl 's regulation of SMS1 expression through enhanced transcription via a promoter switch.

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