Forced expression of the H11 heat shock protein can be regulated by DNA methylation and trigger apoptosis in human cells.
H11, the eukaryotic homologue of a herpes simplex virus protein, has the crystallin motif of heat shock proteins (Hsp), but it differs from canonical family members in that mRNA and protein levels were reduced in various tumor tissues and cell lines (viz. melanoma, prostate cancer and sarcoma) relative to their normal counterparts. In these cells, expression was not restored by heat shock, but rather by the demethylating agent 5-aza-2'-deoxycytidine (Aza-C). Forced H11 expression by Aza-C treatment, transient transfection with H11 expression vectors, or retrovirus-mediated delivery of H11 under the control of a tetracycline-sensitive promoter triggered apoptosis. This is evidenced by a significant (p < 0.001) increase in the percentage of cells positive for terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) and for activation of caspase-3 and p38MAPK and by the co-localization of TUNEL+ nuclei with increased H11 levels. Apoptosis was partially inhibited by the pancaspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone or the p38MAPK inhibitor SB203580. It was abrogated by co-treatment with both inhibitors, suggesting that H11-triggered apoptosis is both caspase- and p38MAPK-dependent. A single site mutant (H11-W51C) had cytoprotective activity related to MEK/ERK activation, and it blocked H11-induced apoptosis in co-transfected and Aza-C-treated cells, indicating that it is a dominant negative mutant. This is the first report of a heat shock protein with proapoptotic activity.
Gober
Michael D
MD
Department of Pharmacology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Smith
Cynthia C
CC
Ueda
Kaori
K
Toretsky
Jeffrey A
JA
Aurelian
Laure
L
D017209:Apoptosis; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D020169:Caspases / Q000502:physiology; D045744:Cell Line, Tumor; D019175:DNA Methylation; D000077209:Decitabine; D006360:Heat-Shock Proteins; D006358:Hot Temperature; D006801:Humans; D048728:MAP Kinase Kinase Kinase 1; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology; D018832:Molecular Chaperones; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000502:physiology*; D048051:p38 Mitogen-Activated Protein Kinases
C413214:HSPB8 protein, human; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D000077209:Decitabine; D017346:Protein Serine-Threonine Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048728:MAP Kinase Kinase Kinase 1; C482576:MAP3K1 protein, human; D020169:Caspases; D001374:Azacitidine