title: Octa-arginine mediated delivery of wild-type Lnk protein inhibits TPO-induced M-MOK megakaryoblastic leukemic cell growth by promoting apoptosis.
abstract: BACKGROUND
  Lnk plays a non-redundant role by negatively regulating cytokine signaling of TPO, SCF or EPO. Retroviral expression of Lnk has been shown to suppress hematopoietic leukemic cell proliferation indicating its therapeutic value in cancer therapy. However, retroviral gene delivery carries risks of insertional mutagenesis. To circumvent this undesired consequence, we fused a cell permeable peptide octa-arginine to Lnk and evaluated the efficacy of inhibition of leukemic cell proliferation in vitro.
  
  
  RESULTS
  In this study, proliferation assays, flow cytometry, Western Blot analyses were performed on wild-type (WT), mutant Lnk R8 or BSA treated M-MOK cells. We found that delivered WT, but not mutant Lnk R8 blocked TPO-induced M-MOK megakaryoblastic leukemic cell proliferation. In contrast, WT Lnk R8 showed no growth inhibitive effect on non-hematopoietic HELA or COS-7 cell. Moreover, we demonstrated that TPO-induced M-MOK cell growth inhibition by WT Lnk R8 was dose-dependent. Penetrated WT Lnk R8 induced cell cycle arrest and apoptosis. Immunoprecipitation and Western blots data indicated WT Lnk R8 interacted with endogeneous Jak2 and downregulated Jak-Stat and MAPK phosphorylation level in M-MOK cells after TPO stimulation. Treatment with specific inhibitors (TG101348 and PD98059) indicated Jak-Stat and MAPK pathways were crucial for TPO-induced proliferation of M-MOK cells. Further analyses using TF-1 and HEL leukemic cell-lines showed that WT Lnk R8 inhibited Jak2-dependent cell proliferation. Using cord blood-derived CD34+ stem cells, we found that delivered WT Lnk R8 blocked TPO-induced megakaryopoiesis in vitro.
  
  
  CONCLUSIONS
  Intracellular delivery of WT Lnk R8 fusion protein efficiently inhibited TPO-induced M-MOK leukemic cell growth by promoting apoptosis. WT Lnk R8 protein delivery may provide a safer and more practical approach to inhibit leukemic cell growth worthy of further development.
authors.affiliation: Department of Pediatrics, Tohoku University School of Medicine, Sendai, Japan.
authors.forename: Chung Yeng
authors.initials: CY
authors.lastname: Looi
authors.forename: Miki
authors.initials: M
authors.lastname: Imanishi
authors.forename: Satoshi
authors.initials: S
authors.lastname: Takaki
authors.forename: Miki
authors.initials: M
authors.lastname: Sato
authors.forename: Natsuko
authors.initials: N
authors.lastname: Chiba
authors.forename: Yoji
authors.initials: Y
authors.lastname: Sasahara
authors.forename: Shiroh
authors.initials: S
authors.lastname: Futaki
authors.forename: Shigeru
authors.initials: S
authors.lastname: Tsuchiya
authors.forename: Satoru
authors.initials: S
authors.lastname: Kumaki
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D019556:COS Cells; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002522:Chlorocebus aethiops; D004789:Enzyme Activation / Q000187:drug effects; D005419:Flavonoids / Q000494:pharmacology; D018014:Gene Transfer Techniques; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D053614:Janus Kinase 2 / Q000378:metabolism; D007947:Leukemia, Megakaryoblastic, Acute / Q000201:enzymology / Q000473:pathology*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D050505:Mutant Proteins / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D011506:Proteins / Q000378:metabolism*; D011759:Pyrrolidines / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D050791:STAT Transcription Factors / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology; D013926:Thrombopoietin / Q000494:pharmacology*; D000096926:Benzenesulfonamides
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D005419:Flavonoids; D047908:Intracellular Signaling Peptides and Proteins; D053614:Janus Kinase 2; D020928:Mitogen-Activated Protein Kinases; D050505:Mutant Proteins; D009842:Oligopeptides; D011506:Proteins; D011759:Pyrrolidines; D011993:Recombinant Fusion Proteins; D050791:STAT Transcription Factors; D013449:Sulfonamides; D013926:Thrombopoietin; C490778:SH2B3 protein, human; C448619:octaarginine; C528327:fedratinib; D000096926:Benzenesulfonamides; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one