title: Suppression of granzyme B activity and caspase-3 activation in leukaemia cells constitutively expressing the protease inhibitor 9.
abstract: Immune surveillance against malignant cells is mediated by cytotoxic T-lymphocytes and NK-cells (CTL/NK) that induce apoptosis through the granzyme-B-dependent pathway. The serine protease inhibitor serpinB9/protease inhibitor-9 (PI-9) is a known inhibitor of granzyme B. Ectopic expression of PI-9 in tumour cells has been reported. However, the impact of PI-9 on granzyme-B-induced apoptosis in tumour cells remains unclear. The aim of this study was to investigate the influence of constitutive PI-9 expression in leukaemia cell lines on the activity of granzyme B and apoptosis induction. PI-9 negative (lymphoblastic Jurkat cells; myeloblastic U937 cells) and PI-9-expressing cell lines (myeloblastic K562 cells, EBV-transformed LCL-1 and LCL-2 B-cells, lymphoblastic Daudi cells, AML-R cells f leukaemia and the U937 subclone U937PI-9(+)). For accurate granzyme B activity determination a quantitative substrate (Ac-IEPD-pNA) cleavage assay was established and caspase-3 activation measured for apoptosis assessment. Cells were treated with a cytotoxic granule isolate that has previously been shown to induce apoptosis through granzyme B signalling. We found a robust correlation between constitutive PI-9 expression levels and the suppression of granzyme B activity. Further, inhibition of granzyme B translated into reduced caspase-3 activation. We conclude, suppression of granzyme B initiated apoptosis in PI-9-expressing cells could contribute to immune evasion and the measurement of granzyme B activity with our assay might be a useful predictive marker in immune-therapeutic approaches against cancer.
authors.affiliation: Dpt. of Hematology and Oncology, Albert Ludwigs-University Medical Center Freiburg, Hugstetter Str. 55, 79106, Freiburg, Germany.
authors.forename: Kristina
authors.initials: K
authors.lastname: Fritsch
authors.forename: Jürgen
authors.initials: J
authors.lastname: Finke
authors.forename: Carsten
authors.initials: C
authors.lastname: Grüllich
mesh_terms: D053148:Caspase 3 / Q000096:biosynthesis*; D004789:Enzyme Activation / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D053804:Granzymes / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D006801:Humans; D020014:K562 Cells; D007938:Leukemia / Q000201:enzymology*; D015843:Serpins / Q000096:biosynthesis*; D020298:U937 Cells
chemical_list: C102316:SERPINB9 protein, human; D015843:Serpins; D053804:Granzymes; D053148:Caspase 3