The role of protein kinase B (PKB) in modulating heat sensitivity in a human breast cancer cell line.
OBJECTIVE
Protein kinase B (PKB) is a critical mediator of phosphoinositide 3-kinase-dependent survival signals in mammalian cells. Its activity is induced after heat shock, and is inhibited in cells undergoing apoptosis. We hypothesized that PKB may be an important modulator for heat-induced apoptosis in human cancer cells.


METHODS
MCF-7 cells were transfected using four different plasmids, encoding a kinase-dead mutant PKB-AAA, a constitutively activated mutant PKB-DD, wild-type PKB, and the neomycin-resistant selection gene. These stable transfectants were subjected to heat shock, and assessed for PKB phosphorylation, PKB activity, and likelihood of undergoing apoptosis.


RESULTS
After heating to 45 degrees C x 30 mins, 25% of MCF-7/neo transfectants underwent apoptosis, which increased to 38% in the presence of wortmannin (WT), an inhibitor of phosphoinositide 3-kinase. In contrast, 23% of the constitutively activated MCF-7/DD transfectants underwent apoptosis, minimally affected by WT. Heat-induced apoptosis occurred in 34% of the kinase-dead MCF-7/AAA transfectants, which increased further to 58% with the addition of WT. This in turn was associated with a two-fold reduction in clonogenic survival compared to the MCF-7/neo transfectants.


CONCLUSIONS
Heat shock activation of PKB in human MCF-7 cells appears to be a significant modulator of heat-induced apoptosis and survival. Further understanding of this important pathway may offer potential in developing novel strategies in cancer therapy.
Ma
N
N
Department of Experimental Therapeutics, Princess Margaret Hospital, Ontario Cancer Institute, University Health Network, Toronto, Ontario, Canada.
Jin
J
J
Lu
F
F
Woodgett
J
J
Liu
F F
FF
D017209:Apoptosis / Q000502:physiology*; D001943:Breast Neoplasms / Q000201:enzymology / Q000503:physiopathology*; D002470:Cell Survival / Q000502:physiology; D004789:Enzyme Activation; D005260:Female; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D018869:Heat-Shock Response; D006358:Hot Temperature; D006801:Humans; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D013997:Time Factors; D014162:Transfection / Q000379:methods; D014407:Tumor Cells, Cultured
D006360:Heat-Shock Proteins; D011518:Proto-Oncogene Proteins; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt