Specific inhibition of Stat5a/b promotes apoptosis of IL-2-responsive primary and tumor-derived lymphoid cells.
Stat5a/b exhibits 96% homology and are required for normal immune function. The present studies examined Stat5a/b function in lymphoid cells by specific and simultaneous disruption of both proteins using novel phosphorothioate-2'-O-methoxyethyl antisense oligodeoxynucleotides (asODN). Efficient delivery was confirmed by the presence of fluorescent TAMRA-labeled ODN in >or=55 and 95% in human primary and tumor cell lines, respectively. Acute asODN administration reduced levels of Stat5a (90%) in 6 h, whereas Stat5b required nearly 48 h to attain the same inhibition, suggesting that the apparent turnover rate for Stat5a was 8-fold higher than that for Stat5b. Expression of the closely related Stat3 protein was unchanged after asODN treatment, however. Molecular ablation of Stat5a/b promoted apoptotic cell death in a significant population of primary PHA-activated T cells (72%) and lymphoid tumor cell line (e.g., YT; 74%) within 24 h, as assessed by 1) visualization of karyolytic nuclear degeneration and other generalized cytoarchitectural alterations, 2) enzymatic detection of TdT-positive DNA degradation, and 3) automated cytometric detection of annexin V translocation. Contrary to findings from Stat5a/b-null mice, cell cycle progression did not appear to be significantly affected. Interestingly, IL-2-insensitive and unprimed T cells and Jurkat cells remained mostly unaffected. Finally, evidence is provided that the cytotoxicity associated with Stat5a/b ablation may derive from activation of caspase-8, an initiator protease that contributes to apoptotic cell commitment. We propose that in lymphoid cells competent to activate Stat5a and Stat5b, both proteins preferentially mediate an antiapoptotic survival influence.
Behbod
Fariba
F
Department of Integrative Biology and Pharmacology, University of Texas Medical School, Houston, TX 77030, USA.
Nagy
Zsuzsanna S
ZS
Stepkowski
Stanislaw M
SM
Karras
James
J
Johnson
Charlene R
CR
Jarvis
W David
WD
Kirken
Robert A
RA
D017209:Apoptosis / Q000235:genetics / Q000276:immunology*; D053181:Caspase 8; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000235:genetics / Q000276:immunology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000235:genetics / Q000276:immunology; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D004789:Enzyme Activation / Q000235:genetics / Q000276:immunology; D004792:Enzyme Precursors / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D007376:Interleukin-2 / Q000502:physiology*; D019169:Jurkat Cells; D007700:Kinetics; D016131:Lymphocyte Subsets / Q000166:cytology / Q000276:immunology* / Q000473:pathology*; D008894:Milk Proteins; D016376:Oligonucleotides, Antisense / Q000378:metabolism; D050799:STAT5 Transcription Factor; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology; D015534:Trans-Activators / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D014162:Transfection; D025521:Tumor Suppressor Proteins
D061945:Caspase Inhibitors; D004268:DNA-Binding Proteins; D004792:Enzyme Precursors; D007376:Interleukin-2; D008894:Milk Proteins; D016376:Oligonucleotides, Antisense; D050799:STAT5 Transcription Factor; C494092:STAT5A protein, human; C494095:STAT5B protein, human; D015534:Trans-Activators; D025521:Tumor Suppressor Proteins; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases