Immunodetection of histone epitopes correlates with early stages of apoptosis in activated human peripheral T lymphocytes.
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 amino 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.
Zunino
S J
SJ
Department of Pathology, University of Texas Southwestern Medical Center, Dallas 75235-9072, USA.
Singh
M K
MK
Bass
J
J
Picker
L J
LJ
D000595:Amino Acid Sequence; D017209:Apoptosis / Q000502:physiology*; D015415:Biomarkers; D015153:Blotting, Western; D002478:Cells, Cultured; D004249:DNA Damage / Q000502:physiology; D004253:DNA Nucleotidylexotransferase / Q000378:metabolism; D000939:Epitopes / Q000032:analysis*; D005434:Flow Cytometry / Q000379:methods; D006657:Histones / Q000032:analysis*; D006801:Humans; D008213:Lymphocyte Activation / Q000502:physiology; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D012016:Reference Values; D013601:T-Lymphocytes / Q000502:physiology*
D015415:Biomarkers; D000939:Epitopes; D006657:Histones; D004253:DNA Nucleotidylexotransferase