N(alpha)-tosyl-L-phenylalanine chloromethyl ketone induces caspase-dependent apoptosis in transformed human B cell lines with transcriptional down-regulation of anti-apoptotic HS1-associated protein X-1

J Biol Chem. 2009 Oct 9;284(41):27827-27837. doi: 10.1074/jbc.M109.027912. Epub 2009 Aug 13.

Abstract

N(alpha)-tosyl-L-phenylalanine chloromethylketone (TPCK) has been widely used to investigate signal transduction pathways that are involved in gene expression and cell survival/cell death. However, contradictory effects of TPCK on apoptosis have been reported, and the underlying signaling events leading to TPCK-induced promotion or prevention of apoptosis are not fully understood. Here, we show that TPCK induces caspase-dependent apoptosis in Epstein-Barr virus (EBV)-transformed human B cell lines with release of pro-apoptotic proteins from mitochondria. TPCK treatment also results in down-regulation of the anti-apoptotic proteins, cIAP1, cIAP2, and HAX-1, and caspase-dependent cleavage of the anti-apoptotic proteins, Bcl-2 and XIAP. Quantitative PCR analysis confirmed that the TPCK-induced down-regulation of HAX-1 occurred at the transcriptional level, and experiments using the specific pharmacological inhibitor, Bay 11-7082, suggested that HAX-1 expression is subject to regulation by the transcription factor, NF-kappaB. B cell lines derived from patients with homozygous HAX1 mutations were more sensitive to TPCK-induced apoptosis when compared with normal donor cell lines. Furthermore, N-acetylcysteine effectively blocked TPCK-induced apoptosis in EBV-transformed B cell lines and prevented the down-regulation or cleavage of anti-apoptotic proteins. Taken together, our studies demonstrate that TPCK induces apoptosis in human B cell lines and exerts multiple effects on pro- and anti-apoptotic factors.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylcysteine / metabolism
  • Adaptor Proteins, Signal Transducing
  • Antioxidants / metabolism
  • Apoptosis / drug effects*
  • Apoptosis / physiology
  • B-Lymphocytes / cytology
  • B-Lymphocytes / drug effects*
  • B-Lymphocytes / metabolism
  • Baculoviral IAP Repeat-Containing 3 Protein
  • Caspases / metabolism*
  • Cell Line, Transformed
  • Cell Transformation, Neoplastic
  • Coumarins / metabolism
  • Cysteine / metabolism
  • Dose-Response Relationship, Drug
  • Down-Regulation / drug effects
  • Fluorescent Dyes / metabolism
  • Herpesvirus 4, Human / genetics
  • Herpesvirus 4, Human / metabolism
  • Humans
  • Inhibitor of Apoptosis Proteins / genetics
  • Inhibitor of Apoptosis Proteins / metabolism
  • Jurkat Cells
  • Mitochondria / drug effects
  • Mitochondria / metabolism
  • NF-kappa B / antagonists & inhibitors
  • NF-kappa B / metabolism
  • Oligopeptides / metabolism
  • Poly(ADP-ribose) Polymerases / genetics
  • Poly(ADP-ribose) Polymerases / metabolism
  • Protein Synthesis Inhibitors / pharmacology*
  • Proteins / genetics
  • Proteins / metabolism*
  • Proto-Oncogene Proteins c-bcl-2 / metabolism
  • Reactive Oxygen Species / metabolism
  • Tosylphenylalanyl Chloromethyl Ketone / pharmacology*
  • Transcription, Genetic / drug effects*
  • Ubiquitin-Protein Ligases
  • X-Linked Inhibitor of Apoptosis Protein / genetics
  • X-Linked Inhibitor of Apoptosis Protein / metabolism

Substances

  • Adaptor Proteins, Signal Transducing
  • Antioxidants
  • Coumarins
  • Fluorescent Dyes
  • HAX1 protein, human
  • Inhibitor of Apoptosis Proteins
  • NF-kappa B
  • Oligopeptides
  • Protein Synthesis Inhibitors
  • Proteins
  • Proto-Oncogene Proteins c-bcl-2
  • Reactive Oxygen Species
  • X-Linked Inhibitor of Apoptosis Protein
  • XIAP protein, human
  • acetyl-aspartyl-glutamyl-valyl-aspartyl-amino-4-methylcoumarin
  • Tosylphenylalanyl Chloromethyl Ketone
  • BIRC2 protein, human
  • BIRC3 protein, human
  • Baculoviral IAP Repeat-Containing 3 Protein
  • Ubiquitin-Protein Ligases
  • Poly(ADP-ribose) Polymerases
  • Caspases
  • Cysteine
  • Acetylcysteine