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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.

Fatemi A, Kazemi A, Kashiri M, Safa M - Int J Mol Cell Med (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology, School of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Effect of cAMP levels on BAD expression and phosphorylation. NALM-6 cells were treated with forskolin and IBMX. After 30 min, the pretreated cells were incubated with doxorubicin for 4 h and then protein expression was assessed using western blot analysis. Elevated cAMP increased BAD phosphorylation at ser 99 and ser 118, but no ser 75. There was no difference in expression of total BAD proteins
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Figure 6: Effect of cAMP levels on BAD expression and phosphorylation. NALM-6 cells were treated with forskolin and IBMX. After 30 min, the pretreated cells were incubated with doxorubicin for 4 h and then protein expression was assessed using western blot analysis. Elevated cAMP increased BAD phosphorylation at ser 99 and ser 118, but no ser 75. There was no difference in expression of total BAD proteins

Mentions: To investigate the effect of cAMP increasing agents on phosphorylation of BAD after doxorubicin- induced DNA damage, three serine residues (Ser75, 99 and 118) were evaluated after treatment of NALM-6 cells with doxorubicin in the presence or absence of cAMP-increasing agents. Significantly, increased phosphorylation of BAD on 99 and 118 serine sites were seen in the cells pretreated with forskolin/IBMX (see fig. 6). Expression of total BAD proteins and phosphorylation of BAD on Ser75 were not affected by doxorubicin and cAMP- elevating agents.


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.

Fatemi A, Kazemi A, Kashiri M, Safa M - Int J Mol Cell Med (2015)

Effect of cAMP levels on BAD expression and phosphorylation. NALM-6 cells were treated with forskolin and IBMX. After 30 min, the pretreated cells were incubated with doxorubicin for 4 h and then protein expression was assessed using western blot analysis. Elevated cAMP increased BAD phosphorylation at ser 99 and ser 118, but no ser 75. There was no difference in expression of total BAD proteins
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4499571&req=5

Figure 6: Effect of cAMP levels on BAD expression and phosphorylation. NALM-6 cells were treated with forskolin and IBMX. After 30 min, the pretreated cells were incubated with doxorubicin for 4 h and then protein expression was assessed using western blot analysis. Elevated cAMP increased BAD phosphorylation at ser 99 and ser 118, but no ser 75. There was no difference in expression of total BAD proteins
Mentions: To investigate the effect of cAMP increasing agents on phosphorylation of BAD after doxorubicin- induced DNA damage, three serine residues (Ser75, 99 and 118) were evaluated after treatment of NALM-6 cells with doxorubicin in the presence or absence of cAMP-increasing agents. Significantly, increased phosphorylation of BAD on 99 and 118 serine sites were seen in the cells pretreated with forskolin/IBMX (see fig. 6). Expression of total BAD proteins and phosphorylation of BAD on Ser75 were not affected by doxorubicin and cAMP- elevating agents.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology, School of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus