Stable overexpression of specific segments of the P2P‐R protein in human MCF‐7 cells promotes camptothecin‐induced apoptosis

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Abstract
The stable overexpression of near full‐length P2P‐R protein in human Saos 2 cells restricts cell cycle progression by inducing mitotic arrest at prometaphase and mitotic apoptosis (Gao and Scott, 2002, J Cell Physiol 193: 199–207). Those effects of P2P‐R were observed in Saos‐2 cells that lack p53 and employ a caspase‐3‐dependent apoptotic signaling pathway. The current studies were performed to evaluate if overexpression of specific segments of the P2P‐R protein promote apoptosis in human MCF‐7 cells that contain p53 and employ a different apoptotic signaling pathway. Since segments of P2P‐R were found not to induce apoptosis independently, the ability of three different P2P‐R segments to promote camptothecin‐induced apoptosis was evaluated following their stable transfection and expression in MCF‐7 cells. Relative to full‐length P2P‐R (1–1560 aa), the three P2P‐R segments used in these studies included: P2P‐R‐2 (761–1560 aa), P2P‐R‐3 (1156–1560 aa), and P2P‐R‐4 (1314–1560 aa). The results document that overexpression of P2P‐R‐2 and P2P‐R‐3 promotes camptothecin‐induced apoptosis by three to fivefold when assayed by flow cytometric analysis of apoptotic sub 2n cell populations or by TUNEL assays. In contrast, P2P‐R‐4 had no effect on apoptosis. These results suggest that the ability of P2P‐R to promote camptothecin‐induced apoptosis in MCF‐7 cells involves a specific region (1156–1314 aa) that exists within P2P‐R. The data presented also show that the p53 binding domain of P2P‐R overlaps with the apoptosis‐associated region and previous studies documented that this region of P2P‐R also binds single‐strand nucleotides (Witte and Scott, 1997, Proc Natl Acad Sci USA 94: 1212–1217). Therefore, P2P‐R‐promoted apoptosis induced by camptothecin may be influenced by such interactions. J. Cell. Physiol. 197: 445–452, 2003
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