Figure 1

Activation of PKA-I, but not Epac or PKA-II, induces CRE-dependent IPC cell apoptosis counteracted by Bcl2. (a) The upper row show that IPC_WT AML cells underwent apoptosis with cell fragmentation and chromatin condensation after 5 h incubation with either 200 μM of the cAMP analog 8-CPT-cAMP, which activates both PKA and Epac or 700 μM of the PKA-specific analog N⁶-MB-cAMP. The specific Epac activator 8-CPT-2′-O-Me-cAMP (700 μM) did not induce any apoptosis. Rows 2,3 show that the Bcl2 overexpressing IPC cells (IPC_BCL2) or IPC cells with enforced expression of the CREB antagonist ICER (IPC_ICER) were protected against apoptosis by the same cAMP analogs. (b) The PKA activity was determined (at saturating cAMP concentration) in extracts of IPC_WT and four cAMP-resistant IPC clones stably transduced with shRNAi against the catalytic (Cα) subunit of PKA. (c) The panel shows the new cAMP analog 2-Cl-8-AHA-cAMP, highly selective for site BI of PKA isozyme I (Supplementary Table S2), to be an inefficient inducer of IPC_WT apoptosis alone (○). When combined with the new site AI-specific analog N⁶-Bnz-8-Pip-cAMP (300 μM), its efficiency was enhanced >20-fold (•). This analog pair is expected to synergize strongly for PKA-I activation (inset), but not for PKA-II (Supplementary Table S3). (d and e) The analogs Sp-5,6-DCl-cBIMPS and N⁶-MB-cAMP (○) were, respectively, moderately (d) and only marginally (e) more efficient when combined with N⁶-Bnz-8-Pip-cAMP (•). The insets show their expected PKA-I synergy (from Supplementary Table S3). Note that N⁶-Bnz-8-Pip-cAMP (300 μM) had only little effect (5% increase of the IPC cell death) when present alone (c–e). Error bars indicate S.E.M., n=3–8