Elevation of cAMP Levels Inhibits Doxorubicin-Induced Apoptosis in Pre- B ALL NALM- 6 Cells Through Induction of BAD Phosphorylation and Inhibition of P53 Accumulation

Recognition of the molecular mechanisms of cAMP action against DNA damage-induced apoptosis can be
useful to improve the efficacy of DNA damaging therapeutic agents. Considering the critical role of bcl-2-
associated death promoter (BAD) and p53 proteins in DNA damage -induced apoptosis, the aim of this study
was to assess the effect of cAMP-elevating agents on these proteins in doxorubicin-treated pre-B acute
lymphoblastic leukemia (pre-B ALL) NALM-6 cells.The pre-B ALL cell line NALM-6 was cultured and treated
with doxorubicin in combination with or without cAMP-elevating agents forskolin and 3-isobutyl-1-
methylxanthine (IBMX). Cell viability was measured by trypan blue staining and MTT assay. For evaluation of
apoptosis, annexin-V staining by flow cytometry and caspase-3 activity assay were used. Protein expression of
p53, BAD and phoshorylated BAD was detected by western blotting analysis.cAMP-increasing agents
diminished the doxorubicin-mediated cytotoxicity in NALM-6 cells as indicated by the viability assays.
Annexin-V apoptosis assay showed that the cAMP-elevating agents decreased doxorubicin-induced apoptosis.
Moreover, doxorubicin-induced caspase-3 activity was attenuated in the presence of cAMP-increasing agents.
Western blot results revealed the reduced expression of p53 protein in cells treated with combination of cAMP-
elevating agents and doxorubicin in contrast to cells treated with doxorubicin alone. Expression of total BAD
protein was not affected by doxorubicin and cAMP-elevating agents. However, phosphorylation of BAD protein
was induced in the presence of cAMP-elevating agents. Our study suggests that elevated cAMP levels inhibit
doxorubicin-induced apoptosis in pre-B ALL cells through induction of BAD phosphorylation and abrogation of
p53 accumulation.