[논문]인체 급성백혈병 Jurkat T 세포에 있어서 L-canavanine에 의해 유도되는 세포자살기전에 미치는 단백질 티로신 키나아제 p56lck의 저해 효과

박해선; 전도연; 우현주; 류석우; 김경민; 김상국; 박완; 문병조; 김영호

doi:10.5352/jls.2009.19.11.1529

인체 급성백혈병 Jurkat T 세포에 있어서 L-canavanine에 의해 유도되는 세포자살기전에 미치는 단백질 티로신 키나아제 p56lck의 저해 효과
A Natural L-Arginine Analog, L-Canavanine-Induced Apoptosis is Suppressed by Protein Tyrosine Kinase p56lck in Human Acute Leukemia Jurkat T Cells 원문보기

생명과학회지 = Journal of life science, v.19 no.11 = no.115, 2009년, pp.1529 - 1537

박해선 (경북대학교 생명과학부) , 전도연 (경북대학교 생명과학부) , 우현주 (경북대학교 생명과학부) , 류석우 (경북대학교 생명과학부) , 김경민 (대구경북대학교 상주캠퍼스, 생태자원응용학부) , 김상국 (경북대학교 생명과학부) , 박완 (경북대학교 생명과학부) , 문병조 (경북대학교 생명과학부) , 김영호 (경북대학교 생명과학부)

초록
AI-Helper

L-arginine 구조유사체인 L-canavanine의 인체 급성백혈병 Jurkat T 세포에 대한 apoptosis 유도활성이 단백질 티로신키나아제 $p56^{lck}$에 어떻게 조절되는지를 규명하기 위해 $p56^{lck}$를 발현하는 Jurkat T 세포주 E6.1과 $p56^{lck}$-결손 Jurkat T 세포주 JCaM1.6에 있어서 L-canavanine의 세포독성, L-canavanine에 의해 유도되는 apoptotic DNA fragmentation 및 apoptotic sub-$G_1$ peak를 비교하여 본 바, $p56^{lck}$-negative JCaM1.6 세포가 $p56^{lck}$-positive E6.1 세포에 비해 L-canavanine의 apoptotis 유도활성에 훨씬 더 민감한 것으로 나타났다. 이러한 $p56^{lck}$-negative JCaM1.6 세포의 민감성은 JCaM1.6 세포에 $p56^{lck}$ 유전자를 transfection시켜 발현시키면 현저히 감소되었다. L-Canavanine에 의해 유도되는 apoptosis관련 현상들을 $p56^{lck}$-stable transfectant인 JCaM1.6/lck 세포와 empty vector-transfectant 인 $p56^{lck}$-negaive JCaM1.6/vector 세포에서 Western blot analysis로 비교한 결과, L-canavanine에 의해 유도되는 mitochondrial membrane potential (${\Delta\Psi}m$)의 감소, caspase-9, -8, -7 및 -3의 활성화, 그리고 PARP 및 $PLC{\gamma}$-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 $p56^{lck}$ kinase 활성의 변화를 $\alpha$-casein을 기질로 하여 시간 별로 측정한 결과, L-canavanine의 처리 후 15분만에 $p56^{lck}$ 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-$G_1$ 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 ${\Delta\Psi}m$의 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 ${\Delta\Psi}m$ 감소, caspase-9, caspase-3 및 caspase-7의 활성화에 의해 유도되며, 이들 apoptosis 현상들은 $p56^{lck}$에 의해 negative regulation되었다.

Abstract ▼ AI-Helper

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 $p56^{lck}$ 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 (${\Delta\Psi}m$) 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 $p56^{lck}$-deficient Jurkat clone JCaM1.6 than in $p56^{lck}$-positive Jurkat clone E6.1. The L-canavanine-induced apoptosis observed in $p56^{lck}$-deficient JCaM1.6 cells was significantly reduced by introducing $p56^{lck}$ 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 $p56^{lck}$. 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-$G_1$ peak and activation of caspase-3, -8, and -7 were abrogated by pan-caspase inhibitor (z-VAD-fmk), 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 ${\Delta\Psi}m$, and the activation of caspase-9, -3, -8, and -7, leading to PARP degradation, and that the $p56^{lck}$ kinase attenuated the ${\Delta\Psi}m$ loss and activation of caspases, and thus contributed as a negative regulator to L-canavanine-induced apoptosis.

주제어

AI 본문요약
AI-Helper

* AI 자동 식별 결과로 적합하지 않은 문장이 있을 수 있으니, 이용에 유의하시기 바랍니다.

데이터처리

Values represent the mean ± standard deviation (SD) of these experiments. The statistical significance was calculated with Student's f-test. P values less than 0.

성능/효과

However, current results suggested that although L-canavaine was previously reported to induce unfolded protein response (UPR) by accumulating structurally aberrant proteins in the ERZ and thus over- expression of ubiquitin was able to confer tolerance to the cytoxicity of L-canavanine [2Z27]Z ER stress-mediated activa- tion of caspase42 rather than caspase-4 appeared to be in- volved in L-canavanine-induced apoptotic signaling in order to support the proper activation of caspase-3 mediated by caspase-9. Consequently, current results indicated that the L-canavanine-induced apoptotic signaling pathway was mediated by activation of caspase-9 and -3, where ER stress-mediated caspase42 activation might be required for its proper progression, leading to the activation of caspase-7 and caspase-8.
6/lck. The results demonstrate that L-canavanine induces apoptotic cell death of Jurkat cells through provoking mitochondrial membrane potential (Aem) reduction, caspase-9 activation, and subsequent activation of caspase-3, and -7, leading to degradation of poly (ADP-ribose) polymerase (PARP), which can be significantly suppressed by p56lck.
6/vector cellsz could express the p56lck protein. These results demonstrated that although the p56lck was not a pre- requisite for L~canavanine~induced apoptosisz it could con- tribute to suppress the L~canavanine4nduced apoptosis by reducing the loss of Aym and the activation of caspase-9, -8, -7, , -and -3.
IB and IC). These results indicated that the higher susceptibility of p56lck-deficient JCaM1.6 cells’ as compared with p56lck-pos- itive E6.1 cells’ toward the cytotoxicity of L-canavanine was attributable to induced apoptosis that could be augmented by the lack of p56lck.

참고문헌 (32)

Bae, M. A., D. Y. Jun, K. M. Kim, S. K. Kim, J. S. Chun, D. Taub, W. Park B. J. Moon, and Y. H. Kim. 2005. Protein tyrosine kinases, $p56^{lck}$ and $p59^{fyn}$ , and MAP kinase JNK1 provide an early signal required for upregulation of Fas ligand expression in aburatubolactam C-induced apoptosis of human Jurkat T cells. Journal of Microbiology and Biotechnology 15, 756-766
Chen, Y. and P. W. Piper. 1995. Consequences of the overexpression of ubiquitin in yeast: elevated tolerances of osmostress, ethanol and canavanine, yet reduced tolerances of cadmium, arsenite and paromomycin. Biochimica et Biaphysica Acta 1268, 59-64

상세보기
Ding, Y, Y. Matsukawa, N. OhtaniFujita, D. Kato, S. Dao, T. Fujii, Y. Naito, T. Yoshikawa, T. Sakai, and G. A. Rosenthal. 1999. Growth inhibition of A549 human lung adenocarcinoma cells by L-canavanine is associated with p21/WAF1 induction. Japanese Journal of Cancer Research 90, 69-74

상세보기
Friesen, C., I. Herr, P. H. Krammer, and K. M. Debatin. 1996. Involvement of the CD95 (APO-1/FAS) receptor/ligand system in drug-induced apoptosis in leukemia cells. Nature Medicine 2, 574-578

상세보기
Green, M. H. and F. J. Ward. 1983. Enhancement of human tumor cell killing by L-canavanine in combination with gamma radiation. Cancer Research 43, 4180-4182

상세보기
Green, M. H., T. L. Brooks, J. Mendelsohn, and S. B. Howell. 1980. Antitumor activity of L-canavanine against L1210 murine leukemia. Cancer Research 40, 535-537

상세보기
Hannun, Y. A. 1997. Apoptosis and dilemma of cancer chemotheraphy. Blood 89, 1845-1853
Jang, M. H., D. Y. Jun, S. W. Rue, K. H. Han, W. Park, and Y. H. Kim. 2002. Arginine antimetabolite L-canavanine induces apoptotic cell death in human Jukat T cells via caspase-3 activation regulated by Bcl-2 or Bcl-xL. Biochemical and Biophysical Research Communications 295, 283-288

상세보기
Jun, D. Y., J. S. Kim, H. S. Park, C. R. Han, Z. Fang, M. H. Woo, I. K. Rhee, and Y. H. Kim. 2007. Apoptogenic activity of Zanthoxylum schinifolium toward human acute leukemia Jurkat T cells is associated with ER stress-mediated caspase-8 activation that stimulates mitochondria-dependent or -independent caspase cascade. Carcinogenesis 28, 1303-1313

상세보기
Jun, D. Y., H. S. Park, J. S. Kim, J. S. Kim, W. Park, B. H. Song, H. S. Kim, D. Taub, and Y. H. Kim. 2008. 17 $\alpha$ -estradiol arrests cell cycle progression at $G_{2}$ /M and induces apoptotic cell death in human acute leukemia Jurkat T cells. Toxicology and Applied Pharmacology 231, 401-412

상세보기
Juo, P., M. S. Woo, C. J. Kuo, P. Signorelli, H. P. Biemann, Y. A. Hannun, and J. Blenis. 1999. F ADD is required for multiple signaling events downstream of the receptor Fas. Cell Growth and Differentiation 10, 797-804
Kim, Y. H., M. J. Buchholz, and A. A. Nordin. 1993. Murine T-lymphocyte proliferation induced by interleukin 2 correlates with a transient increase in $p56^{lck}$ kinase activity and the tyrosine phosphorylation of a 97-kDa protein. Proceedings of National Academy of Sciences USA 90, 3187-3191

상세보기
Mengoli, M., N. Bangi, G. Luccarini, V. N. Ronchi, and D. Serafini-Fracassini. 1989. Daucus carota cell cultures: polyamines and effect of polyamine biosynthesis inhibitors in the preembryogenic phase and different embryo stages. Journal of Plant Physiology 134, 389-394

상세보기
Momoi, T. 2004. Caspases involved in ER stress-mediated cell death. Journal of Chemical Neuroanatomy 28, 101-105

상세보기
Muller, M., S. Strand, H. Hug, E. M. Heinemann, H. Walczak, W. J. Hofmann, W. Stremmel P. H. Krammer, and P. R. Galle. 1997. Drug-induced apoptotsis in hepatoma cells is mediated by the CD95 (APO-1/Fas) receptor/ligand system and involves activation of wild-type p53. Journal of Clinical Investigation 99, 403-413

상세보기
Nagarkatti, N., and B. A. Davis. 2003. Tamoxifen induces apoptosis in $Fas^{+}$ tumor cells by upregulating the expression of Fas ligand. Cancer Chemotherapy and Pharmacology 51, 284-290

상세보기
Robertson, A. T., R. C. Bates, and E. R. Stout. 1984. Reversible inhibition of bovine parvovirus DNA replication by aphidicolin and L-canavanine. Journal of General Virology 65, 1497-1505

상세보기
Rosenthal, G. 1998. The protective action of a higher plant toxic product. Bioscience 38, 104-108
Rosenthal, G. A. 1977. The biological effects and mode of action of L-canavanine, a structural analog of L-canavanine. Quarterly Review of Biology 52, 155-178

상세보기
RosenthaL G. A., J. -M. Reichart, and J. A. Hoffman. 1989. L-canavanine incorporation into vitellogenin and macromolecular conformation. Journal of Biological Chemistry 264, 13693-13696
Schwartz, M., A. Altman, Y. Cohen, and T. Arzee. 1997. Inhibition of polyamine biosynthesis by L-canavanine and its effect on meristematic activity, growth, and development of Zea mays roots. Israel Journal of Plant Sciences 45, 23-30

상세보기
Seu, J. H., B. H. Song, and C. B. Yu. 1973. Studies on the inhibitory substance of yeast growth: effect on the nitrogen uptake. Korean Journal of Applied Microbiology and Bioengineering 1, 3-11
Slee, E. A., H. Zhu, S. C. Chow, M. MacFarlane, D. W. Nicholson, and G. M. Cohen. 1996. Benzyloxycarbonyl-ValAla-Asp (OMe) fluoromethylketone (z-VAD-fmk) inhibits apoptosis by blocking the processing CPP32. Biochemical Journal 315, 21-24
Swaffar, D. S. and C. Y. Ang. 1999. Growth inhibitory effect of L-canavanine against MIA PaCa-2 pancreatic cancer cells is not due to conversion to its toxic metabolite canaline. Anticancer Drugs 10, 113-118

상세보기
Swaffar, D. S., C. Y. Ang, P. B. Desai, G. A. Rosenthal, D. A. Thomas, P. A. Crooks, and W. J. John. 1995. Combination therapy with 5-fluoruracil and L-canavanine: in vitro and in vivo studies. Anticancer Drugs 6, 586-593

상세보기
Thomas, D. A., G. A. Rosenthal, D. V. Gold, and K. M. Dickey. 1986. Growth inhibition of a rat colon tumor by L-canavanine. Cancer Research 46, 2898-2903

상세보기
Tsirigotis, M., M. Zhang, R. K. Chiu, B. G. Wouters, and D. A. Gray. 2001. Sensitivity of mammalian cells expressing mutant ubiquitin to protein-damaging agents. Journal of Biological Chemistry 276, 46073-46078

상세보기
Twiddy, D. and K. Cain. 2007. Caspase-9 cleavage, do you need it? Biochemical Journal 405, e1-e2

상세보기
Veillette, A., I. D. Horak, E. M. Horak, M. A. Bookman, and J. B. Bolen. 1988. Alterations of the lymphocyte-specific protein tyrosine kinase ( $p56^{lck}$ ) during T-cell activation. Molecular and Cellular Biology 8, 4353-4361

상세보기
Worthen, D. R., L. Chien, C. P. Tsuboi, X. Y. Mu, M. M. Bartik, and P. A. Crooks. 1998. L-Canavanine modulates cellular growth, chemosensitivity and P-glycoprotein substrate accumulation in cultured human tumor cell lines. Cancer Letter 132, 229-239

상세보기
Zamzami, N., P. Marchetti, M. Caste do, C. Zanin, J. L. Vayssiere, P. X. Petit, and G. Kroemer. 1995. Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. Journal of Experimental Medicine 181, 1661-1672

상세보기
Zou, H., R. Yang, J. Hao, J. Wang, C. Sun, S. W. Fesik, J. C. Wu, K. J. Tomaselli, and R. C. Armstrong. 2003. Regulation of the Apaf-1/ caspase-9 apoptosome by caspase-3 and XIAP. Journal of Biological Chemistry 278, 8091-8098

상세보기

저자의 다른 논문 :

LOADING...

표제어: PCR

동의어: Packet Collision Rate

용어 설명 출처 목록 (6)

용어 설명: PCR은 세균 특이성이 있는 primer를 이용하여 적은 수의 세균이 있을지라도 쉽게 검출할 수 있는 유용한 방법이며, 이를 이용하여 구강 내 치면세균막이나 타액에서 직접 세균을 검출할 수 있게 되었다[8].

내보내기 구분	파일저장 인쇄 메일전송
구성항목	기본정보 상세정보 관리번호, 논문명, 저널/프로시딩명, 저자 , 발행년, 권, 호, 시작페이지, 끝페이지, 발행기관 관리번호, 논문명, 대등논문명, 저자 , 저널/프로시딩명, 발행기관, 발행년, 발행언어, 권, 호, 시작페이지, 끝페이지, ISBN, ISSN, 주제분야, 키워드, 초록(한글), 초록(영문), 저자(소속기관)
저장형식	Text(ASCII format) Excel format RefWorks Direct Export RIS format (for Reference Manager, ProCite, EndNote), Scholar's Aids, Mendeley
메일정보	받는사람 (필수) @ 보내는사람 (선택) @ 제목 내용 KISTI 검색결과 이메일 서비스
안내	총 건의 자료가 검색되었습니다. 다운받으실 자료의 인덱스를 입력하세요. (1-10,000) 검색결과의 순서대로 최대 10,000건 까지 다운로드가 가능합니다. 데이타가 많을 경우 속도가 느려질 수 있습니다.(최대 2~3분 소요) 다운로드 파일은 UTF-8 형태로 저장됩니다. 파일의 내용이 제대로 보이지 않을실 때는 웹브라우저 상단의 보기 -> 인코딩 -> 자동선택 여부를 확인하십시오. ~ Text(ASCII format) Excel format