Role of Phosphatidylinositol 3′- Kinase Pathway in Primary Effusion Lymphoma Survival.

Phosphatidylinositol 3′-kinase (PI3′-kinase) is a key player in cell growth signaling and has been shown to be activated by the K1 protein of Kaposi sarcoma associated herpes virus (KSHV/HHV8). However, the exact role of PI3′-kinase activation in KSHV-associated PEL has not been elucidated. Therefore, we have studied the PI3′-kinase pathway and apoptosis in five PEL cell lines (BC1, BC3, BCBL1, BCP1 and HBL6). Our data show that inhibition of PI3′-kinase by a specific inhibitor, LY294002, induced apoptosis as detected by Annexin V/Propidium Iodide dual staining in the majority of PEL cell lines, including BC1 (43.5+9%), BC3 (62.7+2.4%), BCBL1 (75+5.2%) and HBL6 (36+4.7%). In contrast, BCP1 was resistant to LY294002-induced apoptosis (2%+0.5). We then dissected the PI3′-kinase pathway by analyses of downstream targets of phosphorylation by Western blot. We found that AKT/PKB was constitutively phosphorylated, and thus activated, in all PEL cell lines including BCP1. Interestingly, 24 hours after LY294002 treatment, AKT was completely de-phosphorylated in all cell lines except BCP1, in which a residual phosphorylation level was detected. The downstream elements of AKT, ForkHead (FKHR) and GSK3 were also constitutively phosphorylated in all PEL cell lines. Similarly, treatment with LY294002 prevented this phenomenon in all the cell lines regardless of their final apoptotic endpoint. To confirm specificity of LY294002 treatment on the PI3′-kinase pathway, we tested an unrelated signaling cascade (p38/MAPK) and no changes were observed. Since FKHR was previously shown to upregulate Fas-L in a variety of cells, we analyzed the Fas/Fas-L system in sensitive PEL cell lines following treatment with LY294002. We have previously shown surface expression of CD95 in these cell lines. We now observed that neutralization of Fas/CD95 by the ZB4 antibody did not influence LY294002 apoptosis. Furthermore, co-treatment with LY294002 and CH11 had an additive apoptotic effect. Inhibition of PI3′-kinase activity further downstream induced cleavage of Bid in all PEL cells. However, cytochrome C was only released from mitochondria in LY294002- sensitive BC1 cells and not in the resistant BCP1 cells. The release of cytochrome C in the sensitive BC1 cell line led to activation of Caspase-9 and 3 and cleavage of PARP, none of which occured in the LY294002 resistant BCP1 cell line. Similarly, the expression of the inhibitor of apoptosis, XIAP, which is also a downstream target of AKT, was compromised in the sensitive cell lines following LY294002 treatment. Our data demonstrate that the PI3′-kinase pathway plays a major role in growth and survival of PEL cells since blocking PI3′-kinase activity induces apoptosis. Although this LY294002 induced apoptosis does not appear to involve Fas/Fas-L, it is caspase dependent and compromises XIAP expression. The residual AKT activity in the LY294002 resistant BCP1 cell line may be protecting this cell line from apoptosis. Altogether, these results suggest that blocking the PI3′-kinase pathway may be a potential target for therapeutic intervention in most primary effusion lymphomas.