American Society of Hematology

Fig. 4.

$Fig. 4. Effect of cell-permeable cAMP analogs in CLL cells. / Cell-permeable cAMP analogs mimic the ability of PDE4 inhibitors to induce accumulation of cytosolic cytochrome c and activation of caspases 3 and 9 in CLL cells. (A) CLL cells were cultured for 18 hours with media alone (CT), 10 μM rolipram (R), 40 μM forskolin (F), both (R/F), or the cell-permeable cAMP analogs 8-bromo-adenosine 3′,5′-cyclic monophosphate (Br), and N6-benzoyl-adenosine 3′,5′-cyclic monophosphate (Bz). Whole-cell lysates were separated into cytosolic and heavy-membrane fractions and immunoblotted for cytochrome c. Total cytochrome c, shown at the bottom, represents a sample in which equivalent portions of cytosolic and membrane fractions were combined. (B) CLL cells cultured as in panel A were immunoblotted for caspase-3. The caspase-3 cleavage product p18 is indicated with an arrow. (C) CLL cells cultured as in panel A were assayed for caspase-3–like (■) and caspase-9–like (░) enzymatic activity by means of a colorimetric assay with the substrates Ac-DEVD-pNA and Ac-LEHD-pNA, respectively. The SEM of triplicate samples is shown. Similar results were obtained in leukemic cells from 2 CLL patients for each of the assays shown.$

Effect of cell-permeable cAMP analogs in CLL cells.

Cell-permeable cAMP analogs mimic the ability of PDE4 inhibitors to induce accumulation of cytosolic cytochrome c and activation of caspases 3 and 9 in CLL cells. (A) CLL cells were cultured for 18 hours with media alone (CT), 10 μM rolipram (R), 40 μM forskolin (F), both (R/F), or the cell-permeable cAMP analogs 8-bromo-adenosine 3′,5′-cyclic monophosphate (Br), and N⁶-benzoyl-adenosine 3′,5′-cyclic monophosphate (Bz). Whole-cell lysates were separated into cytosolic and heavy-membrane fractions and immunoblotted for cytochrome c. Total cytochrome c, shown at the bottom, represents a sample in which equivalent portions of cytosolic and membrane fractions were combined. (B) CLL cells cultured as in panel A were immunoblotted for caspase-3. The caspase-3 cleavage product p18 is indicated with an arrow. (C) CLL cells cultured as in panel A were assayed for caspase-3–like (■) and caspase-9–like (░) enzymatic activity by means of a colorimetric assay with the substrates Ac-DEVD-pNA and Ac-LEHD-pNA, respectively. The SEM of triplicate samples is shown. Similar results were obtained in leukemic cells from 2 CLL patients for each of the assays shown.

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