Immunodetection of histone epitopes correlates with early stages of apoptosis in activated human peripheral T lymphocytes

Susan J. Zunino, Manoj K. Singh, Julia Bass, Louis J. Picker

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

By coupling intracellular staining with terminal deoxynucleotidyl transferase (TdT)-mediated labeling of internucleosomal DNA strand breaks in a flow cytometric assay, we observed a strong correlation between apoptosis- associated DNA strand breaks and immunoreactivity with the monoclonal antibody (MAb) B-F6 in activated human peripheral blood T lymphocytes (PBTs). Although MAb B-F6 has been reported to be specific for the cytokine interleukin-6, Western blot analysis of activated PBT lysates revealed that the predominant protein band detected by this MAb was 17 kd (p17), distinct from the 23-kd core protein and 26- to 30-kd mature glycosylated forms of interleukin-6. Immunoaffinity isolation and amino-terminal amine acid sequence analysis of p17 revealed identity with the histone H2B, a finding confirmed by Western blot analysis of purified histones and by similar staining of activated PBTs with an unrelated anti-histone MAb. Neither histone staining nor DNA strand breakage was observed in freshly isolated PBTs; however, after T cell activation, histone immunoreactivity appeared to precede the appearance of DNA strand breaks, with both increasing to a maximal level by day 3 after activation. Two-parameter confocal immunofluorescence microscopy of histone and DNA staining confirmed a lack of histone immunoreactivity in viable cells and demonstrated co-localization of histone epitopes with abnormally clumped chromatin in apoptotic cells. These data indicate that alteration of histone epitope accessibility is a marker of early apoptosis and suggest that multiparameter flow cytometric analysis of intracellular epitopes may be a powerful tool in the elucidation of intracellular mechanisms of apoptosis.

Original languageEnglish (US)
Pages (from-to)653-663
Number of pages11
JournalAmerican Journal of Pathology
Volume149
Issue number2
StatePublished - Aug 1996
Externally publishedYes

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Histones
Epitopes
Apoptosis
T-Lymphocytes
DNA Breaks
Monoclonal Antibodies
Staining and Labeling
Interleukin-6
Western Blotting
DNA Nucleotidylexotransferase
DNA
Fluorescence Microscopy
Confocal Microscopy
Chromatin
Amines
Sequence Analysis
Proteins
Cytokines
Acids

ASJC Scopus subject areas

  • Pathology and Forensic Medicine

Cite this

Immunodetection of histone epitopes correlates with early stages of apoptosis in activated human peripheral T lymphocytes. / Zunino, Susan J.; Singh, Manoj K.; Bass, Julia; Picker, Louis J.

In: American Journal of Pathology, Vol. 149, No. 2, 08.1996, p. 653-663.

Research output: Contribution to journalArticle

Zunino, Susan J. ; Singh, Manoj K. ; Bass, Julia ; Picker, Louis J. / Immunodetection of histone epitopes correlates with early stages of apoptosis in activated human peripheral T lymphocytes. In: American Journal of Pathology. 1996 ; Vol. 149, No. 2. pp. 653-663.
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