The role of nitric oxide on expression profile of genes altered by nitric oxide-donating aspirin in T-leukemia cells

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Introduction: Acute lymphoblastic leukemia (ALL) is the primary cause of cancer-related mortality in children. ß-catenin, a regulator of cell-cell adhesion and transcription, is expressed in T-acute lymphoblastic leukemia cells and tumor lines of hematopoietic origin and primary leukemia cells. Nitric oxide-donating aspirin (NO-ASA), comprising of a traditional ASA molecule covalently bound to an -ONO2 moiety via an aromatic spacer was reported to inhibit Jurkat T cell growth and degrade ß-catenin by a caspase-mediated pathway (BBRC 2005:326, 93-99). Here we investigated whether NO-ASA may alter the gene expression of other biological and signaling pathways that are potential targets in carcinogenesis. Further, in order to determine the role of NO, we compared the effects of NO-ASA and its denitrated analogue on differential gene expression.
 >Methods: Using Jurkat cells, the IC50 for cell growth inhibition for NO-ASA and its denitrated analogue was determined by an MTT assay (Roche). Following treatment of the Jukat cells with NO-ASA, or its denitrated analogue [0 µM (control cells), 10 µM or 20 µM for 18 hr], mRNA was isolated and labeled according to standard protocols and kits. The oligo arrays set from SuperArray (Bioscience) were used. The blots were hybridized and the ratios of the expression levels quantified and calculated. Potential candidate genes that were differentially expressed were validated by real time RT-PCR.
 >Results: Among the genes whose expression were altered, the following were induced in response to NO-ASA: HSPH1, HSPA1A, HSPA6, FMO4, and FOS. In particular, the gene expression of the heat shock proteins was increased up to 100-fold in response to 20 µM NO-ASA. Interestingly, the denitrated analogue of NO-ASA had the same effect in terms of fold-changes in gene expression for these candidate genes. This strongly suggests that the NO-donating group of NO-ASA may have a negligible role in modulating these gene expressions. Further studies are required to determine whether the aspirin moiety or the spacer are mediating these effects on gene expression. The characteristic of the genes induced or repressed and the relationship of their gene expression with the cell growth inhibition observed by the two compounds will be discussed.