A Natural L-Arginine Analog, L-Canavanine-Induced Apoptosis is Suppressedby Protein Tyrosine Kinase p56<SUP>lck</SUP> in Human Acute Leukemia Jurkat T Cells

Hae Sun Park(박해선),Do Youn Jun(전도연),Hyun Ju Woo(우현주),Seok Woo Rue(류석우),Sang Kook Kim(김경민),Kyung Min Kim(김상국),Wan Park(박완),Byung Jo Moon(문병조),Young Ho Kim(김영호) 한국생명과학회 2009 생명과학회지 Vol.19 No.11

L-arginine 구조유사체인 L-canavanine의 인체 급성백혈병 Jurkat T 세포에 대한 apoptosis 유도활성이 단백질 티로신키나아제 p56<SUP>lck</SUP>에 어떻게 조절되는지를 규명하기 위해 p56<SUP>lck</SUP>를 발현하는 Jurkat T 세포주 E6.1과 p56<SUP>lck</SUP>-결손 Jurkat T 세포주 JCaM1.6에 있어서 L-canavanine의 세포독성, L-canavanine에 의해 유도되는 apoptotic DNA fragmentation 및 apoptotic sub-G1 peak를 비교하여 본 바, p56<SUP>lck</SUP>-negative JCaM1.6 세포가 p56<SUP>lck</SUP>-positive E6.1 세포에 비해 L-canavanine의 apoptotis 유도활성에 훨씬 더 민감한 것으로 나타났다. 이러한 p56lck-negative JCaM1.6 세포의 민감성은 JCaM1.6 세포에 p56<SUP>lck</SUP> 유전자를 transfection시켜 발현시키면 현저히 감소되었다. L-Canavanine에 의해 유도되는 apoptosis관련 현상들을 p56<SUP>lck</SUP>-stable transfectant인 JCaM1.6/lck 세포와 empty vector-transfectant 인 p56<SUP>lck</SUP>-negaive JCaM1.6/vector 세포에서 Western blot analysis로 비교한 결과, L-canavanine에 의해 유도되는 mitochondrial membrane potential (Δψm)의 감소, caspase-9, -8, -7 및 -3의 활성화, 그리고 PARP 및 PLCγ-1의 분해가 JCaM1.6/vector 세포에 비해 JCaM1.6/lck 세포에서 더 약하게 나타났다. JCaM1.6/lck 세포를 2.5 mM L-canavanine으로 처리한 다음 세포 내 p56<SUP>lck</SUP> kinase 활성의 변화를 α-casein을 기질로 하여 시간 별로 측정한 결과, L-canavanine의 처리 후 15분만에 p56<SUP>lck</SUP> kinase의 활성이 약 1.6배 증가되었으며 이후 6시간 동안은 약 1.3~1.4 배정도 증가된 수준으로 kinase 활성이 유지되는 것으로 확인되었다. L-Canavanine에 의한 apoptosis의 개시에 Fas/FasL 상호작용이 관련되는지를 규명하기 위해 FADD-negative Jurkat T 세포주 I2.1, caspase-8-negative Jurkat T 세포주 I9.2 및 wild-type Jurkat T 세포주 A3에 대한 L-canavanine의 세포독성을 비교한 결과, A3와 I2.1 세포의 경우는 L-canavanine의 세포독성이 동일하게 나타났고, 특히 caspase-8가 결손된 I9.2 세포의 경우는 L-canavanine의 세포독성에 대한 민감성이 A3와 I2.1 세포에 비해 단지 미약하게만 완화되는 것으로 나타나, L-canavanine의한 apoptosis에는 Fas/FasL 상호작용이 관련되어 있지 않으며, 또한 caspase-8의 역할이 필수적이지 않음을 시사하였다. Jurkat T 세포에 있어서 L-canavanie에 의해 유도되는 sub-G1 peak 및 caspases 활성화에 미치는 pan-caspase inhibitor (z-VAD-fmk), caspase-9 inhibitor (z-LEHD-fmk), caspase-3 inhibitor (z-DEVD-fmk), caspase-4 inhibitor (z-LEVD-fmk) 및 caspase-12 inhibitor (z-ATAD-fmk)의 영향을 조사한 결과, L-canavanie에 의한 apoptosis는 Δψm의 감소, caspase-9 및 caspase -3의 활성화에 뒤따른 caspase-8 및 caspase-7의 활성화, 그리고 PARP의 분해의 순서로 유도되는 것으로 나타났으며, 아울러 caspase-9의 활성화와 함께 caspase-12의 활성화가 L-canavanine 처리에 따른 caspase-3의 활성화에 요구되는 것으로 확인되었다. 결론적으로, L-canavanine 처리에 의한 Jurkat T 세포의 apoptosis는 Δψm 감소, caspase-9, caspase-3 및 caspase-7의 활성화에 의해 유도되며, 이들 apoptosis 현상들은 p56<SUP>lck</SUP>에 의해 negative regulation되었다. To elucidate further the antitumor effects of a natural L-arginine analogue, L-canavanine, the mechanism underlying apoptogenic activity of L-canavanine and its modulation by protein tyrosine kinase p56<SUP>lck</SUP> was investigated in human Jurkat T cells. When the cells were treated with 1.25 to 2.5 mM L-canavanine for 36 h, several apoptotic events including mitochondrial membrane potential (Δψm) loss, activation of caspase-9, -3, -8, and -7, poly (ADP-ribose) polymerase (PARP) degradation, and DNA fragmentation were induced without alteration in the levels of Fas or FasL. These apoptotic changes were more significant in p56<SUP>lck</SUP>-deficient Jurkat clone JCaM1.6 than in p56<SUP>lck</SUP>-positive Jurkat clone E6.1. The L-canavanine-induced apoptosis observed in p56<SUP>lck</SUP>-deficient JCaM1.6 cells was significantly reduced by introducing p56<SUP>lck</SUP> gene into JCaM1.6 cells by stable transfection. Treatment of JCaM1.6/lck cells with L-canavanine caused a transient 1.6-fold increase in the kinase activity of p56<SUP>lck</SUP>. Both FADD-positive wild-type Jurkat T cell clone A3 and FADD-deficient Jurkat T cell clone I2.1 exhibited a similar susceptibility to the cytotoxicity of L-canavanine, excluding involvement of Fas/FasL system in triggering L-canavanine-induced apoptosis. The L-canavanine-induced apoptotic sub-G1 peak and activation of caspase-3, -8, and -7 were abrogated by pan-caspase inhibitor (z-VADfmk), whereas L-canavanine-induced activation of caspase-9 was not affected. These results demonstrated that L-canavanine caused apoptosis of Jurkat T cells via the loss of Δψm, and the activation of caspase-9, -3, -8, and -7, leading to PARP degradation, and that the p56<SUP>lck</SUP> kinase attenuated the Δψm loss and activation of caspases, and thus contributed as a negative regulator to L-canavanine-induced apoptosis.

Son of Seveless Bins to the SH3 Domain of Src-Type Tyrosine Kinase

Park, Changwon,Choi, youngbong,Yun, Yungdae Korean Society of Molecular Biology 1998 Molecules and cells Vol.8 No.5

P56 LCK Inhibitor Identification by Pharmacophore Modelling and Molecular Docking

Nagakumar Bharatham,Kavitha Bharatham,이근우 대한화학회 2007 Bulletin of the Korean Chemical Society Vol.28 No.2

Pharmacophore models for lymphocyte-specific protein tyrosine kinase (P56 LCK) were developed using CATALYST HypoGen with a training set comprising of 25 different P56 LCK inhibitors. The best quantitative pharmacophore hypothesis comprises of one hydrogen bond acceptor, one hydrogen bond donor, one hydrophobic aliphatic and one ring aromatic features with correlation coefficient of 0.941, root mean square deviation (RMSD) of 0.933 and cost difference (null cost-total cost) of 66.23. The pharmacophore model was validated by two methods and the validated model was further used to search databases for new compounds with good estimated LCK inhibitory activity. These compounds were evaluated for their binding properties at the active site by molecular docking studies using GOLD software. The compounds with good estimated activity and docking scores were evaluated for physiological properties based on Lipinskis rules. Finally 68 compounds satisfied all the properties required to be a successful inhibitor candidate.

P56 LCK Inhibitor Identification by Pharmacophore Modelling and Molecular Docking

Bharatham, Nagakumar,Bharatham, Kavitha,Lee, Keun-Woo Korean Chemical Society 2007 Bulletin of the Korean Chemical Society Vol.28 No.2

Pharmacophore models for lymphocyte-specific protein tyrosine kinase (P56 LCK) were developed using CATALYST HypoGen with a training set comprising of 25 different P56 LCK inhibitors. The best quantitative pharmacophore hypothesis comprises of one hydrogen bond acceptor, one hydrogen bond donor, one hydrophobic aliphatic and one ring aromatic features with correlation coefficient of 0.941, root mean square deviation (RMSD) of 0.933 and cost difference (null cost-total cost) of 66.23. The pharmacophore model was validated by two methods and the validated model was further used to search databases for new compounds with good estimated LCK inhibitory activity. These compounds were evaluated for their binding properties at the active site by molecular docking studies using GOLD software. The compounds with good estimated activity and docking scores were evaluated for physiological properties based on Lipinski's rules. Finally 68 compounds satisfied all the properties required to be a successful inhibitor candidate.

Quantitative Structure-Activity Relationships and Molecular Docking Studies of P56 LCK Inhibitors

Bharatham, Nagakumar,Bharatham, Kavitha,Lee, Keun-Woo Korean Chemical Society 2006 Bulletin of the Korean Chemical Society Vol.27 No.2

Three-dimensional quantitative structure-activity relationship (3D-QSAR) models were developed for 67 molecules of 2-amino-benzothiazole-6-anilide derivatives against lymphocyte-specific protein tyrosine kinase (P56 LCK). The molecular field analysis (MFA) and receptor surface analysis (RSA) were employed for QSAR studies and the predictive ability of the model was validated by 15 test set molecules. Structure-based investigations using molecular docking simulation were performed with the crystal structure of P56 LCK. Good correlation between predicted fitness scores versus observed activities was demonstrated. The results suggested that the nature of substitutions at the 2-amino and 6-anilide positions were crucial in enhancing the activity, thereby providing new guidelines for the design of novel P56 LCK inhibitors.

Quantitative Structure-Activity Relationships and Molecular Docking Studies of P56 LCK Inhibitors

Nagakumar Bharatham,Kavitha Bharatham,이근우 대한화학회 2006 Bulletin of the Korean Chemical Society Vol.27 No.2

Cytotoxicity of Anti-CD4 Antibody Activated CD4+ T-Lymphocytes against Herpesvirus-Infected Target Cells is Dependent on p56lck and p59fyn Protein Tyrosine Kinase Activity

Choi,Sang Hoon,Jang,Yong-Suk,Oh,Chan-Ho The Korea Science and Technology Center 1998 BMB Reports Vol.31 No.4

MHC unrestricted, antigen nonspecific killing by CD4+ T-cells against virally-infected target cells was induced following cross-linking of DC4 molecules. The cytotoxicity of antibody-activated CD4+ T-cells was abolished by genistein (4',5,7-trihydroxyisoflavone), a protein tyrosine kinase (PTK) inhibitor, but not by H-7, a protein kinase C(PKC) inhibitor. Genisteintreated human or bovine peripheral blood CD4+ T-cells lacked PTK activity and failed to kill virally-infected target cells even after cross-linking of CD4 molecules. The cross-linking of CD4 molecules did not induce effector cell proliferation or the transcription of TNFβ. TNFβ synthesis was up-regulated by incubating antibody activated effector cells with bovine herpesvirus type 1 (BHV-1) infected D17 target cells. Anti-TNFβ antibody partially abrogated direct effector cell-mediated antiviral cytotoxicity. On the other hand, this antibody effectively neutralized antiviral activity of effector and target cell culture supernatants against BHV-1 infected D17 cells. The inhibition level of the antiviral activity by the antibody was dependent on effector and target cell ratio. These findings have importance to define the mechanisms of how CD4 cytotoxic cells control viral infection.

Cytotoxicity of Anti-CD4 Antibody Activated $CD4^+$ T-Lymphocytes against Herpesvirus-Infected Target Cells is Dependent on $p56^{lck}$ and $p59^{fyn}$ Protein Tyrosine Kinase Activity

Choi, Sang-Hoon,Jang, Yong-Suk,Oh, Chan-Ho Korean Society for Biochemistry and Molecular Biol 1998 Journal of biochemistry and molecular biology Vol.31 No.4

MHC unrestricted, antigen nonspecific killing by $CD4^+$ T-cells against virally-infected target cells was induced following cross-linking of CD4 molecules. The cytotoxicity of antibody-activated $CD4^+$ T-cells was abolished by genistein (4',5,7-trihydroxyisoflavone), a protein tyrosine kinase (PTK) inhibitor, but not by H-7, a protein kinase C (PKC) inhibitor. Genisteintreated human or bovine peripheral blood $CD4^+$ T-cells lacked PTK activity and failed to kill virally-infected target cells even after cross-linking of CD4 molecules. The cross-linking of CD4 molecules did not induce effector cell proliferation or the transcription of TNF ${\beta}$. TNF ${\beta}$ synthesis was up-regulated by incubating antibody activated effector cells with bovine herpesvirus type 1 (BHV-1) infected D17 target cells. Anti-TNF ${\beta}$ antibody partially abrogated direct effector cell-mediated antiviral cytotoxicity. On the other hand, this antibody effectively neutralized antiviral activity of effector and target cell culture supernatants against BHV-1 infected D17 cells. The inhibition level of the antiviral activity by the antibody was dependent on effector and target cell ratio. These findings have importance to define the mechanisms of how CD4 cytotoxic cells control viral infection.