Procaspase-3 and its active large subunit localized in both cytoplasm and nucleus are activated following application of apoptotic stimulus in Ramos-Burkitt lymphoma B cells.
Ramos-Burkitt lymphoma (Ramos-BL) B cell line is a neoplastic model of normal B cell selection by apoptosis at the germinal center site during maturation of the humoral immune response and can be triggered into apoptosis by cross-linking their surface antigen receptor with antibodies directed against immunoglobulin (Ig)M (anti-IgM) or by treating with the calcium ionophore ionomycin. We have recently demonstrated that anti-IgM and ionomycin trigger significant activation of caspase-3, -7 and -8 and for cleavage of the resident nuclear proteins poly(ADP-ribose) polymerase (PARP) and lamin B1 in Ramos-BL B cells, suggesting that these caspases may be localized to the nucleus as well as to the cytoplasm of Ramos-BL B cells. In order to examine this hypothesis further, we fractionated Ramos-BL B cells into their cytosolic and nuclear components and examined for expression of the endogenous proform and active large subunit of caspase-3; procaspase-3 and its active p17 large subunit were identified in both the cytosolic and nuclear fractions of Ramos-BL B cells. Immunofluorescence staining together with ordinary and confocal microscopy confirmed the observations that procaspase-3 immunoreactivity was clearly identified in the cytoplasm and nucleus while Fas ligand staining was localized to the cell surface and PARP immunoactivity to the nucleus, which were used as controls; procaspase-3 exhibited granular nuclear immunoreactivity whereas PARP displayed diffuse nuclear immunoreactivity; both of which was more intense in the internucleolar regions. Taken together, we now present evidence that procaspases and their active large subunits are found in both the cytoplasm and the nucleus and that procaspases localized not only in the cytoplasm but also in the nucleus are activated following application of apoptotic stimulus in Ramos-BL B cells.
An
Sungkwan
S
Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK.
Park
Myung-Jin
MJ
Park
In-Chul
IC
Hong
Seok-Il
SI
Knox
Kirstine
K
D000888:Antibodies, Anti-Idiotypic / Q000378:metabolism; D017209:Apoptosis / Q000502:physiology*; D001402:B-Lymphocytes / Q000187:drug effects / Q000378:metabolism*; D002051:Burkitt Lymphoma / Q000378:metabolism*; D053148:Caspase 3; D020169:Caspases / Q000187:drug effects / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D002675:Child, Preschool; D017637:Clonal Deletion / Q000502:physiology; D003593:Cytoplasm / Q000378:metabolism; D004792:Enzyme Precursors / Q000187:drug effects / Q000378:metabolism*; D006657:Histones / Q000378:metabolism; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D034921:Lamin Type B / Q000187:drug effects / Q000378:metabolism; D008297:Male; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D011506:Proteins / Q000187:drug effects / Q000378:metabolism
D000888:Antibodies, Anti-Idiotypic; D004792:Enzyme Precursors; D006657:Histones; D007476:Ionophores; D034921:Lamin Type B; D011506:Proteins; C044203:anti-IgM; D015759:Ionomycin; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases