Immunofluorescent detection of activation of initiator caspases-8 and -9 during pharmacologically induced apoptosis of cultured HeLa and endothelial cells

Suresh G. Joshi, Sanjeev Sahni

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Caspases, a group of cysteine-activated aspartate-directed proteases, play an integral role in the execution of programmed cell death or apoptosis. In the cellular caspase cascade, the processing of native proenzymes into activated forms of downstream, effector caspases is dependent on the activation of initiator caspases-8 and -9. We describe a staining procedure for immunofluorescence-based analysis of activation of caspase-8 and -9 during pharmacologically induced apoptosis in primary cultures of human umbilical vein-derived endothelial cells and in an established line of HeLa cells. Using cleavage site-directed antibodies, specific intracellular detection for cleaved fragments of caspase-8 and -9 was accomplished during apoptosis induced by staurosporine and etoposide. The population of cells displaying morphological signs of apoptosis, evidence for DNA strand breaks by TUNEL analysis, and positive staining for active forms of caspase-8 and caspase-9 increased with the duration of treatment, suggesting activation of initiator caspases in correlation with the onset and progression of apoptosis. The application of immunocytochemical staining procedures for quick and specific in situ detection may effectively aid the identification of participating upstream caspases and elucidation of complex apoptosis signaling mechanisms.

Original languageEnglish (US)
Pages (from-to)463-468
Number of pages6
JournalHistochemistry and Cell Biology
Volume119
Issue number6
DOIs
StatePublished - Jun 1 2003
Externally publishedYes

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Initiator Caspases
Caspase 9
Caspase 8
Endothelial cells
apoptosis
Cell death
initiators
HeLa Cells
Cultured Cells
Endothelial Cells
Chemical activation
activation
Apoptosis
Caspases
staining
Staining and Labeling
Effector Caspases
Activation Analysis
Enzyme Precursors
DNA Breaks

Keywords

  • Apoptosis
  • Capsase-9
  • Caspase-8
  • Endothelial cells
  • HeLa cells
  • TUNEL (terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling)

ASJC Scopus subject areas

  • Cell Biology
  • Instrumentation

Cite this

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abstract = "Caspases, a group of cysteine-activated aspartate-directed proteases, play an integral role in the execution of programmed cell death or apoptosis. In the cellular caspase cascade, the processing of native proenzymes into activated forms of downstream, effector caspases is dependent on the activation of initiator caspases-8 and -9. We describe a staining procedure for immunofluorescence-based analysis of activation of caspase-8 and -9 during pharmacologically induced apoptosis in primary cultures of human umbilical vein-derived endothelial cells and in an established line of HeLa cells. Using cleavage site-directed antibodies, specific intracellular detection for cleaved fragments of caspase-8 and -9 was accomplished during apoptosis induced by staurosporine and etoposide. The population of cells displaying morphological signs of apoptosis, evidence for DNA strand breaks by TUNEL analysis, and positive staining for active forms of caspase-8 and caspase-9 increased with the duration of treatment, suggesting activation of initiator caspases in correlation with the onset and progression of apoptosis. The application of immunocytochemical staining procedures for quick and specific in situ detection may effectively aid the identification of participating upstream caspases and elucidation of complex apoptosis signaling mechanisms.",
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AU - Joshi, Suresh G.

AU - Sahni, Sanjeev

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AB - Caspases, a group of cysteine-activated aspartate-directed proteases, play an integral role in the execution of programmed cell death or apoptosis. In the cellular caspase cascade, the processing of native proenzymes into activated forms of downstream, effector caspases is dependent on the activation of initiator caspases-8 and -9. We describe a staining procedure for immunofluorescence-based analysis of activation of caspase-8 and -9 during pharmacologically induced apoptosis in primary cultures of human umbilical vein-derived endothelial cells and in an established line of HeLa cells. Using cleavage site-directed antibodies, specific intracellular detection for cleaved fragments of caspase-8 and -9 was accomplished during apoptosis induced by staurosporine and etoposide. The population of cells displaying morphological signs of apoptosis, evidence for DNA strand breaks by TUNEL analysis, and positive staining for active forms of caspase-8 and caspase-9 increased with the duration of treatment, suggesting activation of initiator caspases in correlation with the onset and progression of apoptosis. The application of immunocytochemical staining procedures for quick and specific in situ detection may effectively aid the identification of participating upstream caspases and elucidation of complex apoptosis signaling mechanisms.

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