The role of protein kinase B (PKB) in modulating heat sensitivity in a human breast cancer cell line

Purpose: Protein kinase B (PKB) is a critical mediator of phosphoinositide 3-kinase-dependent survival signals in mammalian cells, Its activity is ind uced after heat shock, and is inhibited in cells undergoing apoptosis. We h ypothesized that PKB may be an important modulator for heat-induced apoptos is in human cancer cells. Methods and Materials: MCF-7 cells were transfected using four different pl asmids, encoding a kinase-dead mutant PKB-AAA, a constitutively activated m utant PKB-DD, wild-type PKB, and the neomycin-resistant selection gene. The se stable transfectants were subjected to heat shock, and assessed for PKB phosphorylation, PKB activity, and likelihood of undergoing apoptosis. Results: After heating to 45 degreesC x 30 mins, 25% of MCF-7/neo transfect ants underwent apoptosis, which increased to 38% in the presence of wortman nin (WT), an inhibitor of phosphoinositide 3-kinase. In contrast, 23% of th e constitutively activated MCF-7/DD transfectants underwent apoptosis, mini mally affected by WT. Heat-induced apoptosis occurred in 34% of the kinase- dead MCF-7/AAA transfectants, which increased further to 58% with the addit ion of WT. This in turn was associated with a two-fold reduction in clonoge nic survival compared to the MCF-7/neo transfectants. Conclusion: Heat shock activation of PKB in human MCF-7 cells appears to be a significant modulator of heat-induced apoptosis and survival. Further un derstanding of this important pathway may offer potential in developing nov el strategies in cancer therapy. (C) 2001 Elsevier Science Inc.