Activation of the cAMP signaling pathway increases apoptosis in human B-precursor cells and is associated with downregulation of Mcl-1 expression.
During B- and T-cell ontogeny, extensive apoptosis occurs at distinct stages of development. Agents that increase intracellular levels of cAMP induce apoptosis in thymocytes and mature B cells, prompting us to investigate the role of cAMP signaling in human CD10+ B-precursor cells. We show for the first time that forskolin (which increases intracellular levels of cAMP) increases apoptosis in the CD10- cells in a dose-dependent manner (19%-94% with 0-1,000 microM forskolin after 48 hours incubation, IC50 = 150 microM). High levels of apoptosis were also obtained by exposing the cells to the cAMP analogue 8-chlorophenylthio-cAMP (8-CPT-cAMP). Specific involvement of cAMP-dependent protein kinase (PKA) was demonstrated by the ability of a cAMP antagonist, Rp-isomer of 8-bromo-adenosine- 3', 5'- monophosphorothioate (Rp-8-Br-cAMPS), to reverse the apoptosis increasing effect of the complementary cAMP agonist, Sp-8-Br-cAMPS. Furthermore, we investigated the expression of Bcl-2 family proteins. We found that treatment of the cells with forskolin or 8-CPT-cAMP for 48 hours resulted in a fourfold decline in the expression of Mcl-1 (n = 6, P = 0.002) compared to control cells. The expression of Bcl-2, Bcl-xL, or Bax was largely unaffected. Mature peripheral blood B cells showed a smaller increase in the percentage of apoptotic cells in response to 8-CPT-cAMP (1.3-fold, n = 6, P = 0.045) compared to B-precursor cells, and a smaller decrease in Mcl-1 levels (1.5-fold, n = 4, P = 0.014). Taken together, these findings show that cAMP is important in the regulation of apoptosis in B-progenitor and mature B cells and suggest that cAMP-increased apoptosis could be mediated, at least in part, by a decrease in Mcl-1 levels.
Myklebust
J H
JH
Department of Immunology, The Norwegian Radium Hospital, Oslo. junehm@ulrik.uio.no
Josefsen
D
D
Blomhoff
H K
HK
Levy
F O
FO
Naderi
S
S
Reed
J C
JC
Smeland
E B
EB
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000328:Adult; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001402:B-Lymphocytes / Q000737:chemistry / Q000166:cytology / Q000201:enzymology; D001854:Bone Marrow Cells / Q000737:chemistry / Q000166:cytology / Q000201:enzymology; D023201:CD40 Ligand; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D006412:Hematopoietic Stem Cells / Q000737:chemistry / Q000166:cytology* / Q000201:enzymology; D006801:Humans; D007085:Ilium / Q000166:cytology; D015847:Interleukin-4 / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000494:pharmacology; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009363:Neoplasm Proteins / Q000096:biosynthesis*; D015260:Neprilysin / Q000032:analysis; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013873:Thionucleotides / Q000494:pharmacology
C105593:8-bromoadenosine-3',5'-cyclic monophosphorothioate; D004791:Enzyme Inhibitors; D008562:Membrane Glycoproteins; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009363:Neoplasm Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D013873:Thionucleotides; D023201:CD40 Ligand; D005576:Colforsin; D015847:Interleukin-4; D015124:8-Bromo Cyclic Adenosine Monophosphate; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D015260:Neprilysin