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8-Amino-Adenosine (8-NH2-Ado) is a ribonucleoside analog which is metabolized intracellularly to its triphosphate 8-amino-ATP (8-NH2-ATP). When the multiple myeloma cell line MM.1S was treated with 0.1, 0.3, 1, 3, and 10 μM 8-NH2-Ado for 2, 4, 6, and 8 hours, there was a rapid time and dose dependent accumulation of 8-NH2-ATP. Under these conditions, there was also a decrease in global RNA synthesis and the intracellular ATP pool, followed by cell death within 8 hours. In order to identify if a decrease in new transcript production occurred, a real-time RT-PCR assay was carried out to measure synthesis of new RNA transcripts. This was achieved by using a primer-probe set that would detect the pre-mRNA (unspliced) species of GAPDH. Total GAPDH mRNA was used as a control. The quantity of GAPDH pre-mRNA decreased by 50% within 6 hours of 10 μM 8-NH2-Ado treatment, suggesting there is an inhibition of new mRNA synthesis. Phosphorylation of serine 2 and serine 5 on the RNA polymerase II C-terminal domain (Pol II CTD) is required for transcription initiation and elongation. Immunoblot analysis was performed to determine the phosphorylation status of the Pol II CTD serine residues using specific antibodies. Total RNA Pol II was measured as a control. Upon treatment with 10 μM 8-NH2-Ado for 4 hours, the phosphorylation of serine 2 and serine 5 was reduced to 3-16% and 25-49% of the untreated control with a minor decrease in total RNA Pol II protein. These data demonstrate that 8-NH2-Ado inhibits phosphorylation of the Pol II CTD which leads to a decline in RNA transcription. The cyclin dependent kinases CDK9 and CDK7 phosphorylate serine 2 and serine 5 of the Pol II CTD respectively. To assess whether 8-NH2-Ado leads to decreased kinase levels, immunoblot analysis was performed. No changes in total CDK9 or CDK7 protein were detected. Since the relative levels of the kinases remain stable, we hypothesized that the kinase activity may be diminished due to 8-NH2-Ado treatment. In order to determine the effect of the metabolite 8-NH2-ATP on the activity of CDK9 and CDK7, in vitro kinase experiments were carried out by incubating recombinant CDK9/cyclin T or CDK7/cyclin H/MAT1 with recombinant GST-tagged RNA Pol II CTD and various concentrations of ATP and 8-NH2-ATP. The kinase reactions were stopped and the remaining ATP concentration was determined using a luminescence based assay. Preliminary experiments indicate that there is an inhibition of the kinase activity when 8-NH2-ATP is present; however, extended kinetic studies are needed to verify this inhibition. This study has demonstrated that 8-NH2-Ado treatment results in a decline in RNA synthesis which may due to a decrease in serine phosphorylation of the Pol II CTD. Future work will examine the effect of 8-NH2-ATP on the kinase activity with the purpose of understanding its mechanism of action.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA