Crucial roles for protein kinase C isoforms in tumor-specific killing by apoptin.
The chicken anemia virus-derived protein apoptin induces apoptosis in a variety of human malignant and transformed cells but not in normal cells. However, the mechanisms through which apoptin achieves its selective killing effects are not well understood. We developed a lentiviral vector encoding a green fluorescent protein-apoptin fusion gene (LV-GFP-AP) that can efficiently deliver apoptin into hematopoietic cells. Apoptin selectively killed the human multiple myeloma cell lines MM1.R and MM1.S, and the leukemia cell lines K562, HL60, U937, KG1, and NB4. In contrast, normal CD34(+) cells were not killed and maintained their differentiation potential in multilineage colony formation assays. In addition, dexamethasone-resistant MM1.R cells were found to be more susceptible to apoptin-induced cell death than the parental matched MM1.S cells. Death susceptibility correlated with increased phosphorylation and activation of the apoptin protein in MM1.R cells. Expression array profiling identified differential kinase profiles between MM1.R and MM1.S cells. Among these kinases, protein kinase Cβ (PKCβ) was found by immunoprecipitation and in vitro kinase studies to be a candidate kinase responsible for apoptin phosphorylation. Indeed, shRNA knockdown or drug-mediated inhibition of PKCβ significantly reduced apoptin phosphorylation. Furthermore, apoptin-mediated cell death proceeded through the upregulation of PKCβ, activation of caspase-9/3, cleavage of the PKCδ catalytic domain, and downregulation of the MERTK and AKT kinases. Collectively, these results elucidate a novel pathway for apoptin activation involving PKCβ and PKCδ. Further, they highlight the potential of apoptin and its cellular regulators to purge bone marrow used in autologous transplantation for multiple myeloma.
Jiang
Jie
J
Department of Haematological and Molecular Medicine, The Rayne Institute, King's College London, United Kingdom.
Cole
Daryl
D
Westwood
Nigel
N
Macpherson
Lee
L
Farzaneh
Farzin
F
Mufti
Ghulam
G
Tavassoli
Mahvash
M
Gäken
Joop
J
D036022:Capsid Proteins / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D045325:HCT116 Cells; D018922:HL-60 Cells; D006801:Humans; D020014:K562 Cells; D016086:Lentivirus / Q000235:genetics; D007938:Leukemia / Q000201:enzymology / Q000235:genetics / Q000628:therapy*; D009101:Multiple Myeloma / Q000201:enzymology / Q000235:genetics / Q000628:therapy*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D064546:Protein Kinase C beta; D034741:RNA, Small Interfering / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D020298:U937 Cells
D036022:Capsid Proteins; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; C091717:VP3 protein, Chicken anemia virus; D049452:Green Fluorescent Proteins; D011493:Protein Kinase C; D064546:Protein Kinase C beta