摘要
CDK4分子是一种细胞周期依赖性激酶,是细胞周期G1-S期的调控中心。CDK4蛋白在Gl中后期呈表达高峰并与细胞周期蛋白D1(CyclinDl)结合成复合体而被激活,介导Rb基因产物磷酸化,从而促进细胞G1-S期的转化和细胞增殖。国内外研究证明该基因的扩增和过度表达广泛存在于人类的多种肿瘤组织及其细胞中, CDK4基因的异常与多种肿瘤的发生密切相关。为了以CDK4为突破点寻找肿瘤的诊断、鉴别诊断及抗肿瘤治疗的新途径,该分子的研究已成为一个新的研究热点。
本研究从肝癌组织中获得总RNA,逆转录PCR反应获得人CDK4基因,将目的基因插入原核表达载体pET28a(+)的启动子下游,构建了原核表达质粒pET28a(+)-CDK4。将重组质粒pET28a(+)-CDK4用氯化钙法转化入宿主菌BL21(DE3)中,筛选出含重组质粒的基因工程菌。工程菌经IPTG诱导表达和SDS-PAGE电泳分析及Western blotting鉴定表明含有人pET28a(+)-CDK4重组质粒的工程菌BL21(DE3)可以表达出34kd左右的CDK4蛋白质。本研究的成果可以进一步研究CDK4的生物活性,筛选CDK4的配体和阻断剂以及制备单克隆抗体。
CDK4 is an important member of CDK family, which plays a key role inregulating cell cycle. It combines with cyclin D1 and propels the cell cyclethrough the G1 checkpoint, which a critical phase of cell proliferation. CDK4can modulate cell cycle progression from G1 to S phase, facilitate cellproliferation, accelerate cell division and lead to tumorigenesis and progress.The amplification and overexpression of CDK4 gene in many tumor tissueand tumor cells imply this gene is closely related to many tumors. In order tofind new path of tumor diagnosis, differential diagnosis and antineoplastictherapy, research about CDK4 has been a new point.
Human CDK4 gene locates in the 12th chromosome, has 912 base pairsin cDNA and products a polypeptide chain including 303 amino acids with34Kd. CDK4 protein is a member of the Ser/Thr protein kinase family and itsactivation involves both association with cyclinD1 and phosphorylation of aconserved threonine residue excited by Cdk-activating kinase (CAK). Theternary complex of cyclinD1/CDK4/P21 in mid-G1 phase, under effect ofCAK, stimulate the activity of CDK4, which is responsible for thephosphorylation of retinoblastoma gene product (Rb) and the release of E2Ffactor, and then propels the cell cycle alteration of G1-S phase and cellproliferation. In the cell progression, fortification of provocative element(CylinD1) and attenuation of inhibitor (Cyclin-dependent kinase Inhibitors,CKIs) may lead to cell proliferation and division, ultimately result incanceration.
Many researches have confirmed amplification and overexpression ofCDK4 gene in hepatocelluar carcinoma, malignant glial tumor, malignantmelanoma, breast cancer, skin cancer, epithelial ovarian tumor, and so on. Atsame time, the inhibition of gene knockout, siRNA targeting CDK4 andCDK4 inhibitor on many tumor tissues or tumor cells is obvious. So CDK4gene is an important target of antineoplaston, and it is essential to establish amodel of selecting antineoplaston aiming directly at CDK4. Althoughfrequencies of cells in division are more than in normal cells, the content islittle. So it is difficult to abstract many CDK4 active complexes for screeningantineoplastic medicine. In this study, we cloned human CDK4 gene fromhepatoma, constructed prokaryote expression plasmid and induced CDK4protein expression. This protein can be used to further study bioactivity ofCDK4, screen suppressor and prepare monoclonal antibody of CDK4.Although DNA recombination technique can get amount of target geneproducts, the high yields of foreign gene expression are correlated to not onlythe relationship between host strain, vector and target gene, but also promoter,self-character of gene, growing temperature, concentration of inducer (IPTG)and base sequence around initiation codon.In this experiment, we designed a pair of specific primer according toCDK4 cDNA sequence and obtained human CDK4 gene from hepatoma tissuewith RT-PCR method. Sequencing analysis following TA clone indicated thegene is identical with that in Genebank (NM00075) and the CDK4 gene clonewas successful. Subsequently, we insert CDK4 gene into the downstream ofT7 promotor of prokaryotic expression vector pET28a(+) and successfulconstruct prokaryotic expression recombinant plasmid pET28a(+)-CDK4
judged by double restriction enzymes digestion.The use of E.coli for protein expression is well documented for itsadvantages of low cost, easy transformation and high protein yields, whileeukaryotic expression system is strict with cell culture condition and itsexpression level is low, cost is also high. So in this experiment, we usedprokaryotic expression system pET28a (+) to express CDK4 protein. VectorpET28a (+) is one of prokaryotic expression series in Novagen company withmany characters: 1) A kana antibiotic resistance selectable marker. 2) Thenucleotide sequences in translation initiation and the distance between SDsequence and AUG are mostly optimized. 3) It has powerful T7 promoter,terminator and multiple cloning sites. 4) The downstream and upstream ofmultiple cloning sites respectively is His-tag sequence which can fuse totarget gene and express fusion protein include 6 histidines. Because 6continual histidines can reversibly combine with Ni with high specificity, sowe can purify the protein with affinity chromatograph.Obtaining target protein with genetic engineering required not onlyexcellent expression system but also corresponding host bacteria. We selectedEscherichia coli BL21(DE3), the widely used host bacteria according topET28-a(+) in this experiment. In this bacterium, there are mutation of ompTgene coding tunica adventitia protease which can degrade outer membraneprotein and Lon gene coding Lon protease which can degrade theuncomplicated unfolding protein and foreign protein. Thus, expression inBL21 (DE3) is benefit for higher yields of intact recombinant protein.In this experiment, we transformed the recombinant plasmidpET28a(+)-CDK4 into Escherichia coli BL21 (DE3) with CaCl2 method
followed by selecting and identifying clonal bacteria, inducing expression byIPTG, identifying protein expression with SDS-PAGE electrophoresis andWestern blotting. Result display that we successfully obtain CDK4 protein.This protein can be used to further study bioactivity of CDK4, select itsligands and series inhibitors, prepare antibody and subsequently studyantineoplastic effect. The target of medicine selected by this method is definite,and it can shorten obviously study process of medicine. The result of ourstudy makes it is possible to built specific and sensitive model of selectingmedicine and provides new path for tumor gene diagnosis and differentialdiagnosis and antineoplastic therapy.
引文
1. Kamb A, Gruis NA, Weaver FJ, et al. A cell cycle regulator potentially involved in genesis of many tumor types. Science. 1994 Apr 15;264 (5157): 436-440
2. Hartwel LH,Kastau MB. Cell Cycle Control and Cancer. Science. 1994 Dec 16;266 (5192): 1821-1828
3. Sherr CJ. Cancer Cell Cycles. Science. 1996 Dec 6;274 (5293): 1672-1677
4. Nurse P. Regulation of the eukaryotic cell cycle. Eur J Cancer. 1997 Jun;33 (7): 1002-1004
5. Li A, Blow JJ. The origin of CDK regulation. Nat Cell Biol. 2001 Aug;3(8): 182-184.
6. Matsushime H, Quelle DE, Shurtleff SA, et al. D-type cyclin-dependent kinase activity in mammalian cells. Mol Cell Biol. 1994 Mar;14(3): 2066-2076
7. Hunter T, Pines J. Cyclins and cancer II: cyclinD and CDK inhibitors come of age. Cell. 1994 Nov 18;79 (4): 573-580
8. De Bondt HL, Rosenblat J, Jancarik J, et al. Crystal structure of cyclin-dependent kinase 2. Nature. 1993 Jun 17;363 (6430): 595-602.
9. He J, Olson JJ, James CD. Lack of p16INK4 or retinoblastome protein (PRb) or amplification-associated over expression of CDK4 is observed in distinct sub-sets of malignant glial tumors and cell lines. Cancer Res. 1995 Nov 1;55(21):4833-4836.
10. Houillier C, Lejeune J, Benouaich-Amiel A,et al. Prognostic impact of molecular markers in a series of 220 primary glioblastomas. Cancer. 2006 Mar, 27 (Epub ahead of print)
11. Muthusamy V, Hobbs C, Nogueira C, et al. Amplification of CDK4 and MDM2 in malignant melanoma. Genes Chromosomes Cancer. 2006 May;45(5): 447-454.
12. Yu Q, Sicinska E, Geng Y, et al. Requirement for CDK4 kinase function in breast cancer. Cancer Cell. 2006 Jan;9(1): 23-32.
13. Edmunds SC, Kelsell DP. Double jeopardy: Ras and CDK4 co-expre--ssion in skin cancer. Trends in Molecular Medicine. 2002 Dec;8 (12): 548
14. Kita Y, Masaki T, Funakoshi F. Expression of G1 phase-related cell cycle molecules in naturally developing hepatocellular carcinoma of Long-Evans Cinnamon rats. Int J Oncol. 2004 May;24(5):1205-11
15. Shionzawa T, Nikaido T, Shimizu M,et al. Immunohistochemical analysis of the expression of CDK4 and p16 in human endometrioid--type endometrial carcinoma. Caner. 1997;80(12): 2250-2256
16. Khatib ZA, Matsushime H, Valentine M, et al. Coamplification of the CDK4 gene with MDM2 and GL1 in human sarcomas. Cancer Res. 1993 Nov 15;53(22): 5535-5541
17. Yan KX, Liu BC, Shi XL, et al. Role of cyclinD1 and CDK4 in the carcino genesis induced by silica. Biomed Environ Sci. 2005, Oct;18(5): 286-296.
18. Grillo M, Bott MJ, Khandke N, et al. Validation of cyclin D1/CDK4 as an anticancer drug target in MCF-7 breast cancer cells: Effect of regulated overexpression of cyclin D1 and siRNA-mediated inhibition of endogenous cyclin D1 and CDK4 expression. Breast Cancer Res Treat. 2006, Jan;95 (2):185-194
19. Malumbres M, Barbacid M, et al. Is Cyclin D1-CDK4 kinase a bona fide cancer target? Cancer Cell. 2006, Jan;9 (1): 24-34
20. Thomas MP, Mcinnes C, et al. Structure-based discovery and optimizati--on of potential cancer therapeutics targeting the cell cycle. IDrugs. 2006 Apr;9(4): 273-278.
21. Masciullo V, Scambia G, Marone M, et al. Altered expression of cyclinD1 and CDK4 genes in ovarian carcinomas. Int J Cancer. 1997 Aug 22;74(4): 390-395
22. Takahashi, Hiroyuki, Menjo, et al. CDK4 activation is dependent on the subunit rearrangement in the complexes. Biochem and Biophys Res Commun. 2000 Jan 7;267 (1): 388-393
23. Ezhevsky SA, Nagahara H, Vocero-Akbani AM, et al. Hypo-Phosphorylaion of the retinoblastoma protein (pRb) by cyclin D: Cdk4/6 complexes results in active pRb. Proc Natl Acad Sci USA. 1997 Sep 30;94(20): 10699-10704
24. Ewen ME. The cell cycle and retinblastoma protein family. Cancer Metastasis Rev. 1994 Mar;13(1): 45-56
25. Weinberg RT. The retinoblastoma protein and cell cycle control. Cell, 1995, 81: 323-330
26. Nurse P. A long twentieth century of the cell cycle and beyond. Cell. 2000 Jan, 100(1): 71 -78
27. Serrano M, Hannon GJ, Beach D. A new regulatory motif in cell-cycle control causing specfic inhibiton of cyclin D/ CDK4. Nature. 1993 Dec 16;366 (6454): 704-707
28. Hiyama H, Iavarone A, Reeves SA, et al. Regulation of the CDK inhibitor p21 gene during cell-cycle progression is under the control of transcription factor E2F. Oncogene. 1998 Mar 26;16(12): 1513-1523.
29. Gillett C, Fantl V, Smith R, et al. Amplification and overexpression of cyclinD1 in breast cancer detected by immunohistochemical staining. Cancer Res. 1994 Apr 1;54(7): 1812-1817
30. Lukas J, Parry D, Aaggaard L, et al. Retinoblastoma-protein dependent cell-cycle inhibition by tumor suppressor p16. Nature. 1995 Jun 8;375 (6531): 503-506 .
31. Harper JW, Elledge SJ. CDK inhibitors in development and Cancer. Curr Opin Genet Dev. 1996 Feb;6(1): 56-64.
32. Tagliati F,Chiara Zatelli M, Bottoni A,et al. Role of complex Cyclin D1/Cdk4 in somatostatin subtype 2 receptor-mediated inhibition of cell proliferation of a medullary thyroid carcinoma cell line in vitro. Endocrinology. 2006, Apr 6 [Epub ahead of print]
33. Ewen ME, Sluss HK, Whitehouse LL, et al. TGF beta inhibition of Cdk4 synthesis is linked to cell cycle arrest. Cell. 1993, Sep 24;74(6): 1009-1020.
34. 胡和平,满文波,王建等。实时荧光定量 PCR 检测原发性肝癌中CDK4 基因的表达。第二军医大学学报。2003,24(7):773 -775
35. 陈学敏,李厚祥。TGF-β1, TGF-βRII 和 CDK4 在原发性肝细胞肝癌中的表达及其意义。2005,Feb 14(2)146-147
36. He J, Olson JJ, James CD, et al. Lack of p16INK4 or retinoblastoma protein (pRb) or amplification-associated overexpression of cdk4 is observed in distinct subsets of malignant glial tumors and cell lines. Cancer Res. 1995, Nov 1;55(21): 4833-4836
37. Youyi Dong, Li Sui, Katsuyoshi, et al. CyclinD1-CDK4 complex: A possible critical factor for cell proliferation and prognosis in laryngeal squamous cell carcinomas. Int J Cancer. 2001;95: 209-215
38. Peter M, Herskowitz I. Joining the complex: cyclin-dependent kinase inhibitory proteins and the cell cycle. Cell. 1994 Oct, 21;79(2): 181-184.
39. Pines J. Cyclins and cyclin-development kinases: a biochemical view. Biochem J. 1995 Jun;308: 697-711.
40. Morgan DO. Principls of CDK regulation. Nature. 1995 Mar, 374 (6518): 131-134.
41. Srudier FW, Rosenberg AH, Dunn JJ, et al. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185: 60-89
42. Gottesman S,Maurizi MR. Regulation by proteolysis: energy-dependent proteases and their targets. Microbiol Rev. 1992 Dec;56: 592-621