摘要
磷脂爬行酶1(phospholipid scramblase 1,PLSCR1)属于钙离子结合的、棕榈酰化II型膜蛋白,但最近的研究显示非棕榈酰化PLSCR1也能进入细胞核内并结合基因组DNA。目前,有关PLSCR1的生物学功能尚不完全清楚。在过去的工作中,本实验室报告蛋白激酶Cδ(PKCδ)通过序列活化JNK和STAT1信号分子调控PLSCR1的表达。在此基础上,我们发现PLSCR1在全反式维甲酸(ATRA)、佛波酯(PMA)诱导的白血病细胞分化过程中发挥作用。基于这些发现,本课题试图在利用四环素关闭的基因表达系统建立诱导表达PLSCR1的髓系白血病细胞系的基础上,研究PLSCR1对白血病细胞的生物学行为的影响,并初步探讨其分子机制。
本文主要取得如下结果:
(1)利用四环素-关闭基因表达系统建立了诱导表达PLSCR1的髓系白血病细胞系U937plscr1,并确定了诱导表达的PLSCR1主要分布在U937细胞的胞膜及胞浆中,少量分布于细胞核中;
(2) PLSCR1的诱导表达能有效抑制白血病细胞U937的增殖、伴随细胞周期阻滞在G1期,并促使其朝向粒系分化。与此同时,p27、p21、SKP2和c-Myc等多种细胞周期及分化相关蛋白的表达也发生改变;
(3)诱导表达的PLSCR1在下调抗凋亡蛋白bcl-2表达的同时,增强对化疗药etoposide引起的细胞凋亡的敏感性,表现为PKCδ、caspase-3的活化、线粒体跨膜电位的崩塌、凋亡核型的改变以及PARP剪切等显著加强.
综上所述,本文提供了PLSCR1在白血病细胞分化中发挥作用的直接证据,并提出PLSCR1可以发挥抗白血病作用的观点。这些工作拓展了人们对于PLSCR1生物学功能的认识,对于认识白血病细胞分化的分子机制,进而为识别诱导分化治疗白血病的药物靶标提供了重要理论基础。
Phospholipid scramblase 1 (PLSCR1) is a multiply palmitoylated, calcium-binding type II transmembrane protein, while unpalmitoylated PLSCR1 was also found to be localized into the nucleus where it binds to genomic DNA. However, the exact mechanisms of expression regulation of PLSCR1 gene and its biological functions remain largely unclear. More recently, our laboratory found that protein kinase Cδ(PKCδ) upregulates PLSCR1 expression via the sequential activation of c-Jun N-terminal kinase (JNK) and singnal transducer and activator of transcription 1 (STAT1). What’s more, PLSCR1 acts a role in the all-trans retinoic acid (ATRA) and phorbol 12-myristate 13-acetate (PMA) -induced leukemic cell differentiation. In the present work, we try to establish an inducible PLSCR1-expressing myeloid leukemic cell line to explore direct evidence of potential roles of PLSCR1 in the leukemic cell differentiation. Towards this end, the following original and interesting results were gotten:
(1) An inducible human PLSCR1-expressing leukemic cell line U937plscr1 was generated using tet-off gene inducible system and overexpressing PLSCR1 protein was found to be distributed largely in the plasma membrane and cytoplasm with a little in the nucleus.
(2) Upon PLSCR1 induction, the proliferation of U937plscr1 cells was arrested at G1 phase of the cell cycle. More importantly, PLSCR1 overexpression alone could also drive leukemic U937 cells to undergo differentiation towards granulocyte-like cells, as determined by morphology, differentiation markers and differentiation-related gene expressions. In aggrement with changes of these cellular behaviors, PLSCR1 expression also increased p27Kip1/p21Cip1 proteins and reduced c-Myc, SKP2 proteins, which induce growth arrest and cell differentiation.
(3) PLSCR1 induction increased cellular sensitivity to etoposide-induced apoptosis with decreased Bcl-2 protein and enhanced proteolytic cleavage of PKCδ, the ?Ψm collapse, caspase-3 activation, apoptotic nuclear changes, and PARP cleavage as well.
Taken together, this work proposes that PLSCR1 exerts anti-leukemic effects through inducing growth arrest and differentiation as well as increasing sensitivity to apoptosis induction. These findings shed new insights for understanding the biological functions of PLSCR1 and provide the theoretic basis for further exploring the molecular mechanisms of leukemic cell differentiation and for recognizing new drug targets of differentiation therapy.
引文
1. 王振义,陈竺,主编。《肿瘤的诱导分化和凋亡疗法》。上海科学技术出版社,1998 年,第一版。
2. Sachs L. Control of normal cell differentiation and the phenotypic reversion of malignancy in myeloid leukemia. Nature 1978; 274:535-539.
3. Huang ME, Ye YC, Chen SR, Chai JR, Lu JX, Zhoa L, Gu LJ, Wang ZY. Use of all-trans retinoic acid in the treatment of acute promyelocytic leukemia. Blood 1988; 72: 567-572.
4. Leszczyniecka M, Roberts T, Dent P, Grant S, Fisher PB. Differentiation therapy of human cancer: basic science and clinical applications. Pharmacol Ther 2001; 90: 105-156.
5. Mistry AR, Pedersen EW, Solomon E, Grimwade D. The molecular pathogenesis of acute promyelocytic leukaemia: implications for the clinical management of the disease. Blood Rev 2003; 17: 71-97.
6. Daleke DL. Regulation of transbilayer plasma membrane phospholipids asymmetry. J Lipid Res 2003; 44:233-242.
7. Basse F, Stout JG, Sims PJ, Wiedmer T. Isolation of an erythrocyte membrane protein that mediates Ca2+-dependent transbilayer movement of phospholipid. J Biol Chem 1996; 271: 17205-17210.
8. Zhou Q, Zhao J, Stout JG, Luhm RA, Wiedmer T, Sims PJ. Molecular cloning of human plasma membrane phospholipid scramblase. A protein mediating transbilayer movement of plasma membrane phospholipids. J Biol Chem 1997; 272: 18240-18244.
9. Wiedmer T, Zhou Q, Kwoh DY, Sims PJ. Identification of three new members of the phospholipid scramblase gene family. Biochim Biophys Acta 2000; 1467:244-253.
10. Zhao J, Zhou Q, Wiedmer T, Sims PJ. Palmitoylation of phospholipid scramblase is required for normal function in promoting Ca2+-activated transbilayer movement of membrane phospholipids. Biochemistry 1998; 37: 6361-6366.
11. Verhoven B, Krahling S, Schlegel RA, Williamson P. Regulation of phosphatidylserine exposure and phagocytosis of apoptotic T lymphocytes.Cell Death Differ 1999; 6: 262-270.
12. Fadeel B, Gleiss B, Hogstrand K, Chandra J, Wiedmer T, Sims PJ, Henter JI, Orrenius S, Samali A. Phosphatidylserine exposure during apoptosis is a cell-type-specific event and does not correlate with plasma membrane phospholipid scramblase expression. Biochem Biophys Res Commun 1999; 266: 504-511.
13. Frasch SC, Henson PM, Kailey JM, Richter DA, Janes MS, Fadok VA, Bratton DL. Regulation of phospholipid scramblase activity during apoptosis and cell activation by protein kinase C delta. J Biol Chem 2000; 275: 23065-23073.
14. Zhou Q, Zhao J, Al-Zoghaibi F, Zhou A, Wiedmer T, Silverman RH, Sims PJ. Transcriptional control of the human plasma membrane phospholipid scramblase 1 gene is mediated by interferon-alpha. Blood 2000; 95: 2593-2599.
15. Sun J, Nanjundan M, Pike LJ, Wiedmer T, Sims PJ. Plasma membrane phospholipid scramblase 1 is enriched in lipid rafts and interacts with the epidermal growth factor receptor. Biochemistry 2002; 41: 6338-6345.
16. Janel N, Leroy C, Laude I, Toti F, Fressinaud E, Meyer D, Freyssinet JM, Kerbiriou-Nabias D. Assessment of the expression of candidate human plasma membrane phospholipid scramblase in Scott syndrome cells. Thromb Haemost 1999; 81: 322-323.
17. Zhou Q, Zhao J, Wiedmer T, Sims PJ. Normal hemostasis but defective hematopoietic response to growth factors in mice deficient in phospholipid scramblase 1. Blood 2002; 99: 4030-4038.
18. Sun J, Zhao J, Schwartz MA, Wang JY, Wiedmer T, Sims PJ. c-Abl tyrosine kinase binds and phosphorylates phospholipid scramblase 1. J Biol Chem 2001; 276: 28984-28990.
19. Nanjundan M, Sun J, Zhao J, Zhou Q, Sims PJ, Wiedmer T. Plasma membrane phospholipid scramblase 1 promotes EGF-dependent activation of c-Src through the epidermal growth factor receptor. J Biol Chem 2003; 278: 37413-37418.
20. Pastorelli C, Veiga J, Charles N, Voignier E, Moussu H, Monteiro RC, Benhamou M. IgE receptor type I-dependent tyrosine phosphorylation of phospholipid scramblase. J Biol Chem 2001; 276: 20407-20412.
21. Li Y, Rogulski K, Zhou Q, et al. The negative c-Myc target onzin affects proliferation and apoptosis via its obligate interaction with phospholipid scramblase 1. Mol Cell Biol 2006; 26:3401-3413.
22. Kametaka S, Shibata M, Moroe K, et al. Identification of phospholipids scramblase 1 as a novel interacting molecule with beta -secretase (beta -site amyloid precursor protein (APP) cleaving enzyme (BACE)). J Biol Chem 2003; 278:15239-15245.
23. Zhao KW, Li D, Zhao Q, et al. Interferon-alpha-induced expression of phospholipid scramblase 1 through STAT1 requires the sequential activation of protein kinase Cdelta and JNK. J Biol Chem 2005; 280:42707-42714.
24. Zhao KW, Li X, Zhao Q, Huang Y, Li D, Peng ZG, Shen WZ, Zhao J, Zhou Q, Chen Z, Sims PJ, Wiedmer T, Chen GQ. Protein kinase Cdelta mediates retinoic acid and phorbol myristate acetate-induced phospholipid scramblase 1 gene expression: its role in leukemic cell differentiation. Blood 2004; 104: 3731-3738.
25. Wiedmer T, Zhao J, Nanjundan M, Sims PJ. Palmitoylation of phospholipid scramblase 1 controls its distribution between nucleus and plasma membrane. Biochemistry 2003; 42: 1227-1233.
26. Ben-Efraim I, Zhou Q, Wiedmer T, Gerace L, Sims PJ. Phospholipid scramblase 1 is imported into the nucleus by a receptor-mediated pathway and interacts with DNA. Biochemistry 2004; 43: 3518-3526.
27. Zhou Q, Ben-Efraim I, Bigcas JL, et al. Phospholipid scramblase 1 binds to the promoter region of the inositol 1,4,5-triphosphate receptor type 1 gene to enhance its expression. J Biol Chem 2005; 280:35062-35068.
28. Kasukabe T, Okabe-Kado J, Honma Y. TRA1, a novel mRNA highly expressed in leukemogenic mouse monocytic sublines but not in nonleukemogenic sublines. Blood 1997; 89:2975-2985.
29. Yokoyama A, Yamashita T, Shiozawa E, Nagasawa A, Okabe-Kado J, Nakamaki T, Tomoyasu S, Kimura F, Motoyoshi K, Honma Y, Kasukabe T. MmTRA1b/phospholipid scramblase 1 gene expression is a new prognostic factor for acute myelogenous leukemia. Leuk Res 2004; 28: 149-157.
30. Dong B, Zhou Q, Zhao J, Zhou A, Harty RN, Bose S, Banerjee A, Slee R, Guenther J, Williams BR, Wiedmer T, Sims PJ, Silverman RH. Phospholipid scramblase 1 potentiates the antiviral activity of interferon. J Virol 2004; 78: 8983-8993.
31. Silverman RH, Halloum A, Zhou A, Dong B, Al-Zoghaibi F, Kushner D, Zhou Q, Zhao J, Wiedmer T, Sims PJ. Suppression of ovarian carcinoma cell growth in vivo by the interferon-inducible plasma membrane protein, phospholipid scramblase 1. Cancer Res 2002; 62: 397-402.
32. Boer J, Bonten-Surtel J, Grosveld G. Overexpression of the nucleoporin CAN/NUP214 induces growth arrest, nucleocytoplasmic transport defects, and apoptosis. Mol Cell Biol 1998; 18: 1236-1247.
33. Lanotte M, Martin-Thouvenin V, Najman S, Balerini P, Valensi F, Berger R. NB4, a maturation inducible cell line with t(15;17) marker isolated from a human acute promyelocytic leukemia (M3). Blood 1991; 77: 1080-1086.
34. Chen GQ, Shi XG, Tang W, Xiong SM, Zhu J, Cai X, Han ZG, Ni JH, Shi GY, Jia PM, Liu MM, He KL, Niu C, Ma J, Zhang P, Zhang TD, Paul P, Naoe T, Kitamura K, Miller W, Waxman S, Wang ZY, de The H, Chen SJ, Chen Z. Use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL): I. As2O3 exerts dose-dependent dual effects on APL cells. Blood 1997; 89: 3345-3353.
35. Zhu J, Shi XG, Chu HY, Tong JH, Wang ZY, Naoe T , Waxman S, Chen SJ, Chen Z. Effect of retinoic acid isomers on proliferation, differentiation and PML relocalization in the APL cell line NB4. Leukemia 1995; 9: 302-309.
36. Nakajima H, Kizaki M, Ueno H, Muto A, Takayama N, Matsushita H, Sonoda A, Ikeda. All-trans and 9-cis retinoic acid enhance 1,25-dihydroxyvitamin D3-induced monocytic differentiation of U937 cells. Leuk Res 1996; 20:665-676.
37. Stegmaier K, Ross KN, Colavito SA, O’Malley S, Stockwell BR, Golub TR. Gene expression-based high-throughput screening(GE-HTS) and application to leukemia differentiation. Nature Genetics 2004; 36:257-263.
38. Coleman ML, Marshall CJ, Olson MF. Ras promotes p21(Waf1/Cip1) protein stability via a cyclin D1-imposed block in proteasome-mediated degradation.EMBO J 2003; 22: 2036-3046
39. Friedman AD. Runx1, c-Myb, and C/EBPalpha couple differentiation to proliferation or growth arrest during hematopoiesis. J Cell Biochem 2002; 86:624-629.
40. Mitani K. Molecular mechanisms of leukemogenesis by AML1/EVI-1. Oncogene 2004; 23:4263-4269.
41. Koschmieder S, Rosenbauer F, Steidl U, Owens BM, Tenen DG. Role of transcription factors C/EBPalpha and PU.1 in normal hematopoiesis and leukemia. Int J Hematol 2005; 81:368-377.
42. Yamanaka R, Lekstrom-Himes J, Barlow C, Wynshaw-Boris A, Xanthopoulos KG. CCAAT/enhancer binding proteins are critical components of the transcriptional regulation of hematopoiesis (Review). Int J Mol Med 1998; 1:213-221.
43. Sherr CJ and Roberts JM. CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev 1999; 13:1501–1512.
44. Steinman RA. Cell cycle regulators and hematopoiesis. Oncogene 2002; 21: 3403-3413.
45. Rots NY, Iavarone A, Bromleigh V, Freedman LP. Induced differentiation of U937 cells by 1, 25-dihydroxyvitamin D3 involves cell cycle arrest in G1 that is preceded by a transient proliferative burst and an increase in cyclin expression. Blood 1999; 93: 2721-2729.
46. Kikuchi J, Furukawa Y, Iwase S, Terui Y, Nakamura M, Kitagawa S Kitagawa M, Komatsu N, Miura Y. Polyploidization and functional maturation are two distinct processes during megakaryocytic differentiation: involvement of cyclin-dependent kinase inhibitor p21 in polyploidization. Blood 1997; 89: 3980-3990.
47. Wang Z, Su ZZ, Fisher PB, Wang S, VanTuyle G., Grant S. Evidence of a functional role for the cyclin-dependent kinase inhibitor p21(WAF1/CIP1/MDA6) in the reciprocal regulation of PKC activator-induced apoptosis and differentation in human myelomonocytic leukemia cells. Exp Cell Res 1998; 244: 105-116.
48. Munoz-Alonso MJ, Acosta JC, Richard C, Delgado MD, Sedivy J, Leon J. p21Cip1 and p27Kip1 induce distinct cell cycle effects and differentiation programs in myeloid leukemia cells. J Biol Chem 2005; 280:18120-18129.
49. Carrano AC, Eytan E, Hershko A, Pagano M. SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27. Nat Cell Biol 1999;1:193–199.
50. Krucher NA, Rubin E, Tedesco VC, Roberts MH, Sherry TC, De Leon G. Dephosphorylation of Rb (Thr-821) in response to cell stress. Exp Cell Res.2006 May 10; [Epub ahead of print]
51. Gonda TJ and Metcalf D. Expression of myb, myc and fos proto-oncogenes during the differentiation of a murine myeloid leukaemia. Nature 1984; 310:249-251.
52. Larsson LG, Pettersson M, Oberg F, Nilsson K, Luscher B. Expression of mad, mxi1, max and c-myc during induced differentiation of hematopoietic cells: opposite regulation of mad and c-myc. Oncogene 1994; 9:1247-1252.
53. Colby WW, Chen EY, Smith DH, Levinson AD. Identification and nucleotide sequence of a human locus homologous to the v-myc oncogene of avian myelocytomatosis virus MC29. Nature 1983; 301:722-725.
54. Coppola JA and Cole MD. Constitutive c-myc oncogene expression blocks mouse erythroleukaemia cell differentiation but not commitment. Nature 1986; 320:760-763.
55. Hoffman-Liebermann B and Liebermann DA. Suppression of c-myc and c-myb is tightly linked to terminal differentiation induced by IL6 or LIF and not growth inhibition in myeloid leukemia cells. Oncogene 1991; 6:903-909.
56. Holt JT, Redner RL and Nienhuis AW. An oligomer complementary to c-myc mRNA inhibits proliferation of HL-60 promyelocytic cells and induces differentiation. Mol Cell Biol 1998; 8:963-973.
57. Johansen LM, Iwama A, Lodie TA, Sasaki K, Felsher DW, Golub TR. c-Myc is a critical target for c/EBPalpha in granulopoiesis. Mol Cell Biol 2001; 21:3789-3806.
58. Reyland ME, Anderson SM, Matassa AA, Barzen KA, Quissell DO. Protein kinase C delta is essential for etoposide-induced apoptosis in salivary gland acinar cells. J Biol Chem 1999; 274:19115-19123.
59. Matassa AA, Carpenter L, Biden TJ, Humphries MJ, Reyland ME. PKCdelta is required for mitochondrial-dependent apoptosis in salivary epithelial cells. J Biol Chem 2001; 27: 29719-29728.
60. Li L, Lorenzo PS, Bogi K, Blumberg PM, Yuspa SH. Protein kinase Cdelta targets mitochondria, alters mitochondrial membrane potential, and induces apoptosis in normal and neoplastic keratinocytes when overexpressed by an adenoviral vector. Mol Cell Biol 1999; 19: 8547-8558.
61. Song MG, Gao SM, Du KM, Xu M, Yu Y, Zhou YH , Wang Q, Chen Z, Zhu YS, Chen GQ. Nanomolar concentration of NSC606985, a camptothecinanalog, induces leukemic-cell apoptosis through protein kinase Cdelta-dependent mechanisms. Blood 2005; 105: 3714-3721.
62. Yu A, McMaster CR, Byers DM, Ridgway ND, Cook HW. Stimulation of phosphatidylserine biosynthesis and facilitation of UV-induced apoptosis in Chinese hamster ovary cells overexpressing phospholipid scramblase 1. J Biol Chem 2003; 278: 9706-9714.
63. Bailey K, Cook HW, McMaster CR. The phospholipid scramblase PLSCR1 increases UV induced apoptosis primarily through the augmentation of the intrinsic apoptotic pathway and independent of direct phosphorylation by protein kinase C delta. Biochim Biophys Acta 2005; 1733:199-209
64. Kasukabe T, Kobayashi H, Kaneko Y, Okabe-Kado J, Honma Y. Identity of human normal counterpart (MmTRA1b) of mouse leukemogenesis-associated gene (MmTRA1a) product as plasma membrane phospholipid scramblase and chromosome mapping of the human MmTRA1b/phospholipid scramblase gene. Biochem. Biophys. Res. Commun 1998; 249: 449-455.