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

Nancy Ma, Jing Jin, Fred Lu, Jim Woodgett, Fei Fei Liu*

*Corresponding author for this work

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Purpose: 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 and Materials: 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°C × 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. Conclusion: 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.

Original languageEnglish (US)
Pages (from-to)1041-1050
Number of pages10
JournalInternational Journal of Radiation Oncology Biology Physics
Volume50
Issue number4
DOIs
StatePublished - Jul 15 2001

Fingerprint

Proto-Oncogene Proteins c-akt
cultured cells
breast
apoptosis
Hot Temperature
cancer
Breast Neoplasms
proteins
Cell Line
heat
Apoptosis
sensitivity
1-Phosphatidylinositol 4-Kinase
shock
MCF-7 Cells
Mutant Proteins
Heat-Shock Proteins
Survival
modulators
Phosphotransferases

Keywords

  • Apoptosis
  • Heat sensitivity
  • Human breast cancer cell line
  • PI(3)K
  • Protein kinase B

ASJC Scopus subject areas

  • Radiation
  • Oncology
  • Radiology Nuclear Medicine and imaging
  • Cancer Research

Cite this

@article{fcab890432aa4ecea63c61d38459cba1,
title = "The role of protein kinase B (PKB) in modulating heat sensitivity in a human breast cancer cell line",
abstract = "Purpose: 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 and Materials: 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°C × 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. Conclusion: 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.",
keywords = "Apoptosis, Heat sensitivity, Human breast cancer cell line, PI(3)K, Protein kinase B",
author = "Nancy Ma and Jing Jin and Fred Lu and Jim Woodgett and Liu, {Fei Fei}",
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The role of protein kinase B (PKB) in modulating heat sensitivity in a human breast cancer cell line. / Ma, Nancy; Jin, Jing; Lu, Fred; Woodgett, Jim; Liu, Fei Fei.

In: International Journal of Radiation Oncology Biology Physics, Vol. 50, No. 4, 15.07.2001, p. 1041-1050.

Research output: Contribution to journalArticle

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T1 - The role of protein kinase B (PKB) in modulating heat sensitivity in a human breast cancer cell line

AU - Ma, Nancy

AU - Jin, Jing

AU - Lu, Fred

AU - Woodgett, Jim

AU - Liu, Fei Fei

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N2 - Purpose: 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 and Materials: 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°C × 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. Conclusion: 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.

AB - Purpose: 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 and Materials: 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°C × 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. Conclusion: 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.

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