Suppression of granzyme B activity and caspase-3 activation in leukaemia cells constitutively expressing the protease in
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ORIGINAL ARTICLE
Suppression of granzyme B activity and caspase-3 activation in leukaemia cells constitutively expressing the protease inhibitor 9 Kristina Fritsch & Jürgen Finke & Carsten Grüllich
Received: 1 February 2013 / Accepted: 9 July 2013 # Springer-Verlag Berlin Heidelberg 2013
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 Bcells, lymphoblastic Daudi cells, AML-R cells f leukaemia and the U937 subclone U937PI-9+). For accurate granzyme B activity determination a quantitative substrate (Ac-IEPDpNA) 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
K. Fritsch : J. Finke : C. Grüllich Dpt. of Hematology and Oncology, Albert Ludwigs-University Medical Center Freiburg, Hugstetter Str. 55, 79106 Freiburg, Germany C. Grüllich (*) National Center for Tumor Diseases, Dpt. of Medical Oncology, Heidelberg University Medical Center, Im Neuenheimer Feld 460, 69120 Heidelberg, Germany e-mail: [email protected]
might be a useful predictive marker in immune-therapeutic approaches against cancer. Keywords Leukaemia . Granzyme B . Apoptosis . Serpin
Introduction It has been proposed for decades that the immune system is able to destroy cancer cells. This primary defence against malignancy is known as immune surveillance. Its main effectors are cytotoxic T lymphocytes and natural killer cells (CTL/NK) that recognize and kill cancer cells. Models explaining the failure of the immune system to kill malignant cells include insufficient immune recognition caused by insufficient tumour antigen presentation or the creation of a tolerogenic microenviroment within the tumour [1, 2]. A further mechanism for failure may be the resistance of malignant cells to apoptosis induced by CTL/NK cells. The predominant effector mechanism of CTL/NK cell-induced apoptosis is the
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