Characterization of the interleukin-1 beta-converting enzyme/Ced-3-family protease, caspase-3/CPP32, in Hodgkin's disease - Lack of caspase-3 expression in modular lymphocyte predominance Hodgkin 's disease

Apoptosis (programmed cell death) serves an important role in the normal mo rphogenesis, immunoregulation, and homeostatic mechanisms in both normal an d neoplastic cells. Caspase-3/CPP32, a member of the ICE/Ced-3-family of cy steine proteases, is an important downstream mediator of several complex pr oteolytic cascades that result in apoptosis Ln both hematopoietic and nonhe matopoietic cells. Previous studies have demonstrated that caspase-3 is com monly expressed in classical Hodgkin's disease (CHD); however, the biologic al significance of its expression in Hodgkin's disease is unknown, In this report, the expression of caspase-3 in nodular lymphocyte predominance Hodg kin's disease (NLPHD) was evaluated by immunohistochemistry; in addition, w e investigated the role of caspase-3 in CD95 (Fas)mediated apoptosis in thr ee CHD cell lines. Formalin-fixed, paraffin-embedded tissue sections from 1 1 cases of NLPHD were immunostained for caspase-3 using a polyclonal rabbit antibody that detects both the 32-kd zymogen and the 20-kd active subunit of the caspase-3 protease, Only 1/11 cases of NLPHD demonstrated caspase-3 immunopositivity in lymphocytic/histiocytic cells. Caspase-3 expression was also evaluated in three CHD cell Lines, HS445, L428, and KMH2, Whereas cas pase-3 expression was detected in HS445 and L428 cell lines, no expression was found in KMH2 cells by immunohistochemical staining. Treatment of HS445 and L428 cell lines for 72 hours with agonistic CD95 monoclonal antibody i nduced marked apoptosis that was significantly inhibited by pretreatment wi th the caspase-3 inhibitor, DEVD-FMK, as determined by terminal deoxynucleo tidyl transferase-mediated dUTP nick end-labeling assay and flow cytometric analysis of 7-amino-actinomycin D staining. In addition, a significant inc rease in caspase-3 activity as determined by an enzyme colorimetric assay w as detected in HS445 and L428 cells after 48 hours of CD95 stimulation, In marked contrast, treatment of caspase-3-deficient KMH2 cells with anti-CD95 mAb did not demonstrate an increase in caspase-3 activity or induce apopto sis, These data demonstrate caspase-3 is important for CD95-mediated apopto sis in CHD cell lines. In addition, the majority of NLPHD cases examined in this study failed to express detectable levels of caspase-3, suggesting th ese tumor cells may be resistant to apoptotic stimuli dependent on caspase- 3 activity. Furthermore, these data suggest the differential expression of caspase-3 noted between NLPHD and CHD may provide additional evidence that each is a unique disease entity.