A2A agonists/PDE inhibitor combinations induce apoptosis, increase cellular cAMP, and elevate PDE subtype expression. (A) The combination of A2A agonist CGS-21680 and PDE inhibitor trequinsin triggers the rapid induction of apoptosis of multiple myeloma cells. MM.1S cells were incubated with 50nM CGS-21680, 2μM trequinsin, or the combination of both agents. At 24 and 48 hours, cells were harvested and analyzed by FACS for annexin V–positive cells. (B) MM.1S cells were incubated with 10nM HE-NECA, 10μM trequinsin, 10 μM R-(-)-rolipram, or the combination of 10nM HE-NECA and 10μM trequinsin or 10nM HE-NECA and 10μM R-(-)-rolipram for 1 hour at 37°C followed by preparation of cell lysates and measurement of cAMP levels. (C) MM.1R cells were incubated with 1μM R-(-)-rolipram, 20nM HE-NECA or the combination of 1μM R-(-)-rolipram and 20nM HE-NECA for 4 hours at 37°C and then the cells were lysed to isolate total RNA. The RNA was used for RT-PCR quantitation of PDE RNA levels using subtype specific primers as described in “siRNA transfections.”