title: Expression of granzyme B sensitizes ALK+ ALCL tumour cells to apoptosis-inducing drugs.
abstract: BACKGROUND
  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. 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.
  
  
  METHODS
  ALK+ 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.
  
  
  RESULTS
  Expression 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.
  
  
  CONCLUSIONS
  Our 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.
authors.forename: Joel D
authors.initials: JD
authors.lastname: Pearson
authors.forename: Jingxi
authors.initials: J
authors.lastname: Zhang
authors.forename: Zuoqiao
authors.initials: Z
authors.lastname: Wu
authors.forename: Kayla D
authors.initials: KD
authors.lastname: Thew
authors.forename: Katelynn J
authors.initials: KJ
authors.lastname: Rowe
authors.forename: Julinor T C
authors.initials: JT
authors.lastname: Bacani
authors.affiliation: Department of Medical Microbiology and Immunology and Li Ka Shing Institute of Virology, University of Alberta, Katz Group Centre for Pharmacy and Health Research, University of Alberta, Edmonton AB T6G 2E1, Canada. ringham@ualberta.ca.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Ingham
mesh_terms: D000077548:Anaplastic Lymphoma Kinase; D017209:Apoptosis; D045744:Cell Line, Tumor; D004317:Doxorubicin / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D055785:Gene Knockdown Techniques; D053804:Granzymes / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D017728:Lymphoma, Large-Cell, Anaplastic / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology*
chemical_list: D034741:RNA, Small Interfering; D004317:Doxorubicin; D011065:Poly(ADP-ribose) Polymerases; C000626173:ALK protein, human; D000077548:Anaplastic Lymphoma Kinase; D020794:Receptor Protein-Tyrosine Kinases; C509213:GZMB protein, human; D053804:Granzymes; D019311:Staurosporine