Expression of granzyme B sensitizes ALK+ ALCL tumour cells to apoptosis-inducing drugs.

Joel D Pearson,Robert J Ingham,Jingxi Zhang,Julinor Tc Bacani,Zuoqiao Wu,Kayla D Thew,Katelynn J Rowe

doi:10.1186/1476-4598-13-199

Abstract

BackgroundThe serine protease Granzyme B (GzB) is primarily expressed by cytotoxic T lymphocytes and natural killer cells, and functions in allowing these cells to induce apoptosis in virally-infected or transformed cells. Cancers of both lymphoid and non-lymphoid origin also express GzB, and in some cases this expression has been linked to pathogenesis or sensitizing tumour cells to cell death. For example, GzB expression in urothelial carcinoma was implicated in promoting tumour cell invasion, whereas its expression in nasal-type NK/T lymphomas was found to correlate with increased apoptosis. GzB expression is also a hallmark of the non-Hodgkin lymphoma, anaplastic lymphoma kinase-positive, anaplastic large cell lymphoma (ALK+ ALCL). Given the fact that ALK+ ALCL exhibits high levels of apoptosis and is typically responsive to conventional chemotherapy, we examined whether GzB expression might play a role in sensitizing ALK+ ALCL tumour cells to apoptosis.MethodsALK+ ALCL cell lines stably expressing GzB or non-targeting (control) shRNA were generated and apoptosis was examined by anti-PARP western blotting and terminal deoxynucleotidyl transferase dUTP nick end labelling. Both spontaneous apoptosis and apoptosis in response to treatment with staurosporine or doxorubicin were investigated. In order to assess whether additional granzymes might be important in promoting cell death in ALK+ ALCL, we examined whether other human granzymes were expressed in ALK+ ALCL cell lines using reverse-transcriptase PCR and western blotting.ResultsExpression of several GzB shRNAs in multiple ALK+ ALCL cell lines resulted in a significant decrease in GzB levels and activity. While spontaneous apoptosis was similar in ALK+ ALCL cell lines expressing either GzB or control shRNA, GzB shRNA-expressing cells were less sensitive to staurosporine or doxorubicin-induced apoptosis as evidenced by reduced PARP cleavage and decreased DNA fragmentation. Furthermore, we found that GzB is the only granzyme that is expressed at significant levels in ALK+ ALCL cell lines.ConclusionsOur findings are the first to demonstrate that GzB expression sensitizes ALK+ ALCL cell lines to drug-induced apoptosis. This suggests that GzB expression may be a factor contributing to the favourable response of this lymphoma to treatment.

Highlights

The serine protease Granzyme B (GzB) is primarily expressed by cytotoxic T lymphocytes and natural killer cells, and functions in allowing these cells to induce apoptosis in virally-infected or transformed cells
GzB protein levels and activity are significantly reduced in ALK+ Anaplastic lymphoma kinase-positive anaplastic large cell lymphoma (ALCL) cell lines treated with GzB short-hairpin RNA (shRNA) In order to examine whether GzB sensitizes ALK+ ALCL to apoptosis, we generated ALK+ ALCL cell lines where GzB expression had been stably knocked-down with shRNA
To confirm our findings that GzB knock-down reduces the sensitivity of ALK+ ALCL cell lines to staurosporineinduced apoptosis and to better quantify this difference, we examined the effect of GzB knock-down on the degree of terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)-positive staining in ALK+ ALCL cell lines treated with staurosporine

Summary

Introduction

The serine protease Granzyme B (GzB) is primarily expressed by cytotoxic T lymphocytes and natural killer cells, and functions in allowing these cells to induce apoptosis in virally-infected or transformed cells. Granzyme B (GzB) is a serine protease found in the cytoplasmic granules of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells It is released along with other granule proteins such as the pore-forming protein perforin, into the immunological synapse where it is translocated into virally-infected or transformed cells to initiate apoptosis [1,2]. GzB has substrates outside the cell, and may gain access to the extracellular space as a result of leakage from the immunological synapse or by direct secretion from cytotoxic cells [3,4] These substrates include extracellular matrix (ECM) proteins such as vitronectin, laminin, fibronectin, decorin, and biglycan [5,6,7]. Cleavage of these proteins by GzB has been implicated in detaching adherent cells bound to these proteins [5], promoting the migration of cells through matrigel [6], and releasing matrix-bound cytokines [7]

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