The Effect of Elevated cAMP Levels on Phosphorylation of Bcl-۲-Associated Death Promoter Protein in Pre-B Acute Lymphoblastic Leukemia Cell Line (NALM-۶) Treated with Doxorubicin

Kashiri M۱, Safa M۲, Kazemi A۳ ۱Dept. of Hematology, Allied Medical School, Tehran University of Medical Sciences, Tehran, Iran ۲Cellular and Molecular Research Center, Dept. of Hematology and Blood Banking, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran ۳Dept. of Hematology & Blood Banking, School of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran Corresponding Author: Safa M, Cellular and Molecular Research Center, Dept. of Hematology and Blood Banking, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran E-mail: [e-mail] Received: ۸ Feb ۲۰۱۴ Accepted: ۲۲ Aug ۲۰۱۴ Background and Objective: BAD (Bcl-۲-associated death promoter) is a proapoptotic protein which in phosphorylated state is inactivated in terms of its apoptotic function while in dephosphorylated status causes proceeding of apoptosis via inhibition of BCL-XL. cAMP elevating agents attenuate Doxorubicin induced-apoptosis. The aim of this study was to assess the effects of cAMP elevating agents via Forskolin and IBMX on phosphorylation of BAD in Pre-B acute lymphoblastic leukemia (pre-B ALL) cells. Materials and Methods: NALM-۶ cells were incubated with cAMP increasing agents along with two concentrations of Doxorubicin ۲۵۰ and ۵۰۰ for ۲۴ hours. Assessment of cell death was performed by Trypan blue staining and apoptosis was assessed by Annexin V via flowcytometry. Western blot was used for evaluating the expression of BAD protein and phosphorylation changes. Data was analyzed by Paired t-test. Results: The flowcytometry analysis of Annexin V revealed that cAMP increasing agents in combination with Doxorubicin lead to decreased rate of apoptosis in comparison to Doxorubicin alone. Analysis of western blotting showed that cAMP increasing agents cause phosphorylate serine residues of BAD. The study also indicated that Doxorubicin alone is unable to alter the phosphorylation status of BAD. 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Keywords: cAMP , Acute lymphoblastic Leukemia , Doxorubicin , Forskolin , IBMX