Cyclin B1反义全长cDNA诱导细胞周期阻滞及抑制成瘤性的实验研究
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摘要
背景与目的:细胞周期蛋白B1(Cyclin B1)为有丝分裂期(M期)细胞周期蛋白,与细胞周期蛋白依赖性激酶(cyclin-dependent kinase,CDK1)结合形成成熟促进因子(muturior promoting factor,MPF),MPF的激活为真核细胞启动有丝分裂所必需,在肿瘤的发生发展中起重要作用;Cyclin B1在许多肿瘤细胞系均高表达,其高表达被视为肿瘤具有恶性潜能的标志之一。反义cDNA是反义技术中一种简便有效的下调靶基因的方法,但目前尚未见应用该方法靶向Cyclin B1基因抗肿瘤治疗的相关报道。
方法:构建含有小鼠Cyclin B1反义全长cDNA的重组质粒(pAS-mCLB1)并将pAS-mCLB1及空载体pcDNA3.1(+)分别转染小鼠Lewis肺癌(LL/2)和CT26结肠癌细胞系(CT26),建立了AS-mCLB1及pcDNA3.1(+)稳定转染子(Ac及Pc细胞);通过流式细胞仪检测Ac、Pc及未经转染(Nc)细胞的细胞周期分布及凋亡;分别用两步法半定量RT-PCR及Western blot比较上述三种细胞中mCyclin B1的mRNA及蛋白含量;MTT及细胞生长实验用于检测细胞增殖活性;在小鼠体内分别接种上述三种细胞,观察细胞在体內的成瘤性及荷瘤小鼠的生存时间;然后对Ac与Nc组的细胞及动物血清进行蛋白组学差异分析;免疫组化检测
Background and Objective: Cyclin B1, a cell cycle protein in mitosis phase, connecting with p34 kinase (CDK1) to form muturior promoting factor (MPF), is necessary for mitotic initiation in eukaryotic cells and it plays an important role in cancer development; cyclin B1 is over-expressed in a variety of tumors and is thought to be an indicator of the malignant potential of tumors. Antisense cDNA is a simple, easy and influential method to down-regulate the expression of target genes. It remains to be clarified whether down-regulation of cyclin Bl with full-length antisense cDNA of cyclin B1 (AS-CLB1) in tumor cells is an effective strategy for cancer therapy.Methods: A recombinant plasmid containing the full-length antisense cDNA of mouse cyclin Bl (pAS-mCLBl) and pcDNA3.1 empty vector were constructed and introduced into LL/2 and CT26 cells to establish AS-mCLB1 (Ac) and pcDNA3.1 (Pc) stable transfectants respectively. The cell cycles and apoptosis of Ac, Pc and untransfected (Nc) cells were determined using flow cytometry. mRNA and protein content of cyclin Bl were tested by two-step semi-quantitative RT-PCR and western blot assay respectively. The activity of cell proliferation was measured by MTT and cell growth assay.
Tumorigenicity and survival were observed in the mice which were implanted with Ac, Pc and Nc cells respectively. The proteomics of cells and animal serums were compared between the AC and NC groups. In the tumor tissues, the expression of mCyclin B1 was detected with immunohistochemistry and DNA Fragmentation Detection Kit was used to test DNA fragmentation associated with the apoptotic cells.Result: Prominent G1 arrest and apoptosis were shown and abnormal morphology with inhibition of cell growth appeared in the Ac cells in which persistent and evident down-regulation of mCyclin B1 expression was induced. Moreover, after implanting, tumors in the Ac group developed on day 9 versus day 5 in the controlled groups (Nc and Pc) in LL/2-bearing mice. In CT26-loaded mice, tumors in the Ac group developed on day 6 versus day 3 in the controlled after inoculation. Therefore, survival benefits might be achieved through the inhibition of tumorigenicity in the mice of Ac groups. Both in cells and animal serums, the differences of proteomics were apparent. The expression of mCyclin B1 protein was decreased and cell apoptotic rate was increased in the tumor tissues of Ac groups.Conclusion: To our understanding, this is the first report concerning the biological activities of cyclin Bl knockdown tumor cells using AS-mCLB1. These results suggested that the suppression of cyclin Bl by AS-CLBl could induce G1 arrest and apoptosis of tumor cells obviously, which involved in inhibiting the activity of tumor cells in vitro and in vivo efficiently. So AS-CLB1 might be a viable method for tumor biotherapy.
方法:构建含有小鼠Cyclin B1反义全长cDNA的重组质粒(pAS-mCLB1)并将pAS-mCLB1及空载体pcDNA3.1(+)分别转染小鼠Lewis肺癌(LL/2)和CT26结肠癌细胞系(CT26),建立了AS-mCLB1及pcDNA3.1(+)稳定转染子(Ac及Pc细胞);通过流式细胞仪检测Ac、Pc及未经转染(Nc)细胞的细胞周期分布及凋亡;分别用两步法半定量RT-PCR及Western blot比较上述三种细胞中mCyclin B1的mRNA及蛋白含量;MTT及细胞生长实验用于检测细胞增殖活性;在小鼠体内分别接种上述三种细胞,观察细胞在体內的成瘤性及荷瘤小鼠的生存时间;然后对Ac与Nc组的细胞及动物血清进行蛋白组学差异分析;免疫组化检测
Background and Objective: Cyclin B1, a cell cycle protein in mitosis phase, connecting with p34 kinase (CDK1) to form muturior promoting factor (MPF), is necessary for mitotic initiation in eukaryotic cells and it plays an important role in cancer development; cyclin B1 is over-expressed in a variety of tumors and is thought to be an indicator of the malignant potential of tumors. Antisense cDNA is a simple, easy and influential method to down-regulate the expression of target genes. It remains to be clarified whether down-regulation of cyclin Bl with full-length antisense cDNA of cyclin B1 (AS-CLB1) in tumor cells is an effective strategy for cancer therapy.Methods: A recombinant plasmid containing the full-length antisense cDNA of mouse cyclin Bl (pAS-mCLBl) and pcDNA3.1 empty vector were constructed and introduced into LL/2 and CT26 cells to establish AS-mCLB1 (Ac) and pcDNA3.1 (Pc) stable transfectants respectively. The cell cycles and apoptosis of Ac, Pc and untransfected (Nc) cells were determined using flow cytometry. mRNA and protein content of cyclin Bl were tested by two-step semi-quantitative RT-PCR and western blot assay respectively. The activity of cell proliferation was measured by MTT and cell growth assay.
Tumorigenicity and survival were observed in the mice which were implanted with Ac, Pc and Nc cells respectively. The proteomics of cells and animal serums were compared between the AC and NC groups. In the tumor tissues, the expression of mCyclin B1 was detected with immunohistochemistry and DNA Fragmentation Detection Kit was used to test DNA fragmentation associated with the apoptotic cells.Result: Prominent G1 arrest and apoptosis were shown and abnormal morphology with inhibition of cell growth appeared in the Ac cells in which persistent and evident down-regulation of mCyclin B1 expression was induced. Moreover, after implanting, tumors in the Ac group developed on day 9 versus day 5 in the controlled groups (Nc and Pc) in LL/2-bearing mice. In CT26-loaded mice, tumors in the Ac group developed on day 6 versus day 3 in the controlled after inoculation. Therefore, survival benefits might be achieved through the inhibition of tumorigenicity in the mice of Ac groups. Both in cells and animal serums, the differences of proteomics were apparent. The expression of mCyclin B1 protein was decreased and cell apoptotic rate was increased in the tumor tissues of Ac groups.Conclusion: To our understanding, this is the first report concerning the biological activities of cyclin Bl knockdown tumor cells using AS-mCLB1. These results suggested that the suppression of cyclin Bl by AS-CLBl could induce G1 arrest and apoptosis of tumor cells obviously, which involved in inhibiting the activity of tumor cells in vitro and in vivo efficiently. So AS-CLB1 might be a viable method for tumor biotherapy.
引文
1. Porter LA, Singh G, Lee JM. Abundance of cyclin B1 regulates gamma-radiation-induced apoptosis. Blood, 2000; 95(8):2645-2650.
2. O'Connor PM, Ferris DK, White GA, et al. Relationships between cdc2 kinase, DNA cross-linking, and cell cycle perturbations induced by nitrogen mustard. Cell Growth Differ, 1992; 3(1):43-52.
3. Sofia JC, Jang SJ, Khuri FR, et al. Overexpression of cyclin B1 in early-stage nonsmall cell lung cancer and its clinical implication. Cancer Res, 2000; 60(15):4000-4004.
4. Allan K, Jordan RC, Ang LC, et al. Overexpression of cyclin A and cyclin B1 proteins in astrocytomas. Arch Pathol Lab Med, 2000; 124(2):216-220.
5. Kallakury, BV, Sheehan CE, Rhee S J, et al. The prognostic significance of proliferation-associated nucleolar protein p120 expression in prostate adenocarcinoma: a comparison with cyclins A and B1, Ki-67, proliferating cell nuclear antigen, and p34cdc2. Cancer (Phila), 1999; 85(7):1569-1576.
6. Porter LA, Cukier IH, Lee JM. Nuclear localization ofcyclin B1 regulates DNA damage-induced apoptosis. Blood, 2003; 101 (5): 1928-1933.
7. Wang A, Yoshimi N, Ino N, et al. Overexpression of cyclin B1 in human colorectal cancers. J Cancer Res Clin Oncol, 1997; 123(2): 124-127.
8. Dutta A, Chandra R, Leiter LM, et al. Cyclins as markers of tumor proliferation: immunocytochemical studies in breast cancer. Proc Natl Acad Sci USA, 1995; 92(12):5386-5390.
9. Flanagan WM, Su LL, Wagner RW. Elucidation of gene function using C-5 propyne antisense oligonucleotides. Nat Biotechnol, 1996; 14(9):1139-1145.
10. Friedenstein A J, Petrakova KV, Kurolesova AI, et al. Heterotopic of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues. Transplantation, 1968; 6(2):230-247.
11. Owen M. Marrow stromal stem cells. J Cell Sci Suppl, 1988; 10:63-76.
12. Kopen GC, Prockop DJ, Phinney DG. Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. Proc Natl Acad Sci USA, 1999;96(19):10711-10716.
13. Weiss B, Davidkova G, Zhou LW. Antisense RNA gene therapy for studying and modulating biological processes. Cell Mol Life Sci, 1999;55(3):334-358.
14. Hnatowich DJ. Antisense and nuclear medicine. J Nucl Med, 1999;40(4):693-703.
15. Agrawal S, Zhao Q. Antisense therapeutics. Curr Opin Chem Biol, 1998;2(4):519-528.
16. Kashihara N, Maeshima Y, Makino H. Antisense oligonucleotides. Exp Nephrol, 1998; 6(1):84-88.
17. Cao GD, Wang SW, Wu SS, et al. Retrovirus-mediated antisense RNA to bcl-2 alter the biological behavior of stomach carcinoma MGC-803 cell lines. World J Gastroenterol, 1998; 4(Suppl 2):45-48.
18. Morris MC, Chaloin L, Choob M, et al. Combination of a new generation of PNAs with a peptide-based carrier enables efficient targeting of cell cycle progression. Gene Ther, 2004; 11 (9):757-764.
19. Yuan J, Yan R, Kramer A, et al. Cyclin B1 depletion inhibits proliferation and induces apoptosis in human tumor cells. Oncogene, 2004;23(34):5843-5852.
20. Rishi AK, Zhang L, Boyanapalli M, et al. Identification and characterization of a cell cycle and apoptosis regulatory protein-1 as a novel mediator of apoptosis signaling by retinoid CD437. J Biol Chem,2003; 278(35):33422-33435.
21. Choi JA, Kim JY, Lee JY, et al. Induction of cell cycle arrest and apoptosis in human breast cancer cells by quercetin. Int J Oncol, 2001;19(4):837-844.
22. Shaw MM, Gurr WK, Watts PA, et al. Ganciclovir and penciclovir, but not acyclovir, induce apoptosis in herpes simplex virus thymidine kinase-transfonned baby hamster kidney cells. Antivir Chem Chemother, 2001; 12(3):175-186.
23. Maier TJ, Schilling K, Schmidt R, et al. Cyclooxygenase-2 (COX-2)-dependent and -independent anticarcinogenic effects of celecoxib in human colon carcinoma cells. Biochem Pharmacol, 2004;67(8): 1469-1478.
24. Seth P, Katayose D, Li Z, et al. A recombinant adenovirus expressing wild type p53 induces apoptosis in drug-resistant human breast cancer cells: a gene therapy approach for drug-resistant cancers. Cancer Gene Ther, 1997;4(6):383-390.
25. Weinstein IB, Begemann M, Zhou P, et al. Disorders in cell circuitry associated with multistage carcinogenesis: exploitable targets for cancer prevention and therapy. Clin Cancer Res, 1997; 3(12 Pt 2):2696-2702.
26. Masato T, Naohiko S, Toshinori O, et al. Identification of the p33INGl-regulated genes that include cyclin B1 and proto-oncogene DEK by using cDNA microarray in a mouse mammary epithelial cell line NmuMG. Cancer Res, 2002; 62(8):2203-2209.
27. Lim KM, Yeo WS, Chow VT. Antisense abrogation of DENN expression induces apoptosis of leukemia cells in vitro, causes tumor regression in vivo and alters the transcription of genes involved in apoptosis and the cell cycle. Int J Cancer, 2004; 109(1):24-37.
28. Craig C, Wersto R, Kim M, et al. A recombinant adenovirus expressing p27Kipl induces cell cycle arrest and loss ofcyclin-Cdk activity in human breast cancer cells. Oncogene, 1997; 14(19):2283-2289.
29. Dubrez L, Coll JL, Hurbin A, et al. Cell cycle arrest is sufficient for p53-mediated tumor regression. Gene Ther, 2001; 8(22):1705-1712.
30. Zhan Q, Antinore MJ, Wang XW, et al. Association with Cdc2 and inhibition of Cdc2/Cyclin B1 kinase activity by the p53-regulated protein Gadd45. Oncogene, 1999; 18(18):2892-2900.
31. Azzam EI, Toledo SM de, Waker AJ, et al. High and low fluences of (?) -particles induce a G1 checkpoint in human diploid fibroblasts. Cancer Res, 2000; 60(10):2623-2631.
32. Kornmarm M, Arber N, Korc M. Inhibition of basal and mitogen-stimulated pancreatic cancer cell growth by cyclin D1 antisense is associated with loss of tumorigenicity and potentiation of cytotoxicity to cisplatinum. J Clin Invest, 1998; 101 (2):344-352.
33. Meadus WJ. A semi-quantitative RT-PCR method to measure the in vivo effect of dietary conjugated linoleic acid on porcine muscle PPAR gene expression. Biol Proced Online, 2003; 5:20-28.
34. D(?)ndas N. A semi-quantitative method for the estimation of adenosine A1 receptor mRNA levels in rat kidney. Indian J Pharmacol, 2004;36(3):167-170.
35. Ladanyi A, Gallai M, Paku S, et al. Expression of a Decorin-Like Molecule in Human Melanoma. Pathol Oncol Res, 2001; 7(4):260-266.
36. Christine S, Michael K O'Connor, Elizabeth RB, et al. Treatment of prostate cancer by radioiodine therapy after tissue-specific expression of the sodium iodide symporter. Cancer Res, 2000; 60(22):6526-6530.
37. Anderson IC, Shipp MA, Docherty AP, et al. Combination therapy including a gelatinase inhibitor and cytotoxic agent reduces local invasion and metastasis of murine Lewis lung carcinoma. Cancer Res, 1996;56(4):715-718.
38. Ermert L, Hocke AC, Duncker H-R, et al. Comparison of different detection methods in quantitative microdensitometry. Am J Pathol, 2001;158(2):407-417.
39. Andreoletti O, Berthon P, Levavasseur E, et al. Phenotyping of protein-prion (PrPsc)-accumulating cells in lymphoid and neural tissues of naturally scrapie-affected sheep by double-labeling immunohistochemistry. J Histochem Cytochem, 2002; 50(10): 1357-1370.
40. Gharbi S, Gaffney P, Yang A, et al. Evaluation of two-dimensional differential gel electrophoresis for proteomic expression analysis of a model breast cancer cell system. Mol Cell Proteomics, 2002; 1 (2):91-98.
41. Dunbar B, Wilson SB. A buffer exchange procedure giving enhanced resolution to polyacrylamide gels prerun for protein sequencing. Anal Biochem, 1994; 216(1):227-228.
42. G(?)rg A, Obermaier C, Boguth G, et al. The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis, 2000; 21 (6): 1037-1053.
43. Kubota K, Wakabayashi K, Matsuoka T. Proteome analysis of secreted proteins during osteoclast differentiation using two different methods: two-dimensional electrophoresis and isotope-coded affinity tags analysis with two-dimensional chromatography. Proteomics, 2003; 3(5):616-626.
44. Arber N, Doki Y, Han EKH, et al. Antisense to cyclin D1 inhibits the growth and tumorigenicity of human colon cancer cells. Cancer Res, 1997; 57(8):1569-1574.
45. Zhou P, Jiang W, Zhang YJ, et al. Antisense to cyclin D1 inhibits growth and reverses the transformed phenotype of human esophageal cancer cells. Oncogene, 1995; 11(3):571-580.
46. Weiss B, Davidkova G, Zhang SP. Antisense strategies in neurobiology. Neurochem Int, 1997; 31(3):321-348.
47. Kawasaki H, Machida M, Komatsu M, et al. Specific regulation of gene expression by antisense nucleic acids: a summary of methodologies and associated problems. Artificial Organs, 1996; 20(8):836-848.
48. Tu SP, Jiang XH, Lin MC, et al. Suppression of survivin expression inhibits in vivo tumorigenicity and angiogenesis in gastric cancer. Cancer Res, 2003; 63(22):7724-7732.
49. Darzynkiewicz Z, Gong J, Juan G, et al. Cytometry of cyclin proteins. Cytometry, 1996; 25(1):1-13.
50. Barrett KL, Demiranda D, Katula KS. Cyclin bl promoter activity and functional cdkl complex formation in G1 phase of human breast cancer cells. Cell Biol Int, 2002; 26(1):19-28.
51. Shen M, Feng Y, Gao C, et al. Detection of cyclin b l expression in g(1)-phase cancer cell lines and cancer tissues by postsorting Western blot analysis. Cancer Res, 2004; 64(5): 1607-1610.
52. Addinall SG, Mayr PS, Doyle S, et al. Phosphorylation by cdc2-CyclinB1 kinase releases cytoplasmic dynein from membranes. J Biol Chem, 2001; 276(19): 15939-15944.
53. Dell KR, Turck CW, Vale RD. Mitotic phosphorylation of the dynein light intermediate chain is mediated by cdc2 kinase. Biochem Biophys Res Commun, 2003; 307(2):401-407.
54. Song SY, Meszoely IM, Coffey RJ, et al. K-Ras-independent effects of the farnesyl transferase inhibitor L-744,832 on cyclin B1/Cdc2 kinase activity, G2/M cell cycle progression and apoptosis in human pancreatic ductal adenocarcinoma cells. Neoplasia, 2000; 2(3):261-272.
55. Shimizu T, O'Connor PM, Kohn KW, et al. Unscheduled activation of cyclin B1/Cdc2 kinase in human promyelocytic leukemia cell line HL60 cells undergoing apoptosis induced by DNA damage. Cancer Res, 1995;55(2):228-231.
56. Ling YH, Tornos C, Perez-Soler R. Phosphorylation of Bcl-2 is a marker of M phase events and not a determinant of apoptosis. J Biol Chem, 1998;273(30): 18984-18991.
57. Scaife RM. G2 cell cycle arrest, down-regulation of cyclin B, and induction of mitotic catastrophe by the flavoprotein inhibitor diphenyleneiodonium. Mol Cancer Ther, 2004; 3(10): 1229-1237.
58. Wersto RP, Rosenthal ER, Seth PK, et al. Recombinant, replication-defective adenovirus gene transfer vectors induce cell cycle dysregulation and inappropriate expression of cyclin proteins. J Virol, 1998; 72(12):9491-9502.
2. O'Connor PM, Ferris DK, White GA, et al. Relationships between cdc2 kinase, DNA cross-linking, and cell cycle perturbations induced by nitrogen mustard. Cell Growth Differ, 1992; 3(1):43-52.
3. Sofia JC, Jang SJ, Khuri FR, et al. Overexpression of cyclin B1 in early-stage nonsmall cell lung cancer and its clinical implication. Cancer Res, 2000; 60(15):4000-4004.
4. Allan K, Jordan RC, Ang LC, et al. Overexpression of cyclin A and cyclin B1 proteins in astrocytomas. Arch Pathol Lab Med, 2000; 124(2):216-220.
5. Kallakury, BV, Sheehan CE, Rhee S J, et al. The prognostic significance of proliferation-associated nucleolar protein p120 expression in prostate adenocarcinoma: a comparison with cyclins A and B1, Ki-67, proliferating cell nuclear antigen, and p34cdc2. Cancer (Phila), 1999; 85(7):1569-1576.
6. Porter LA, Cukier IH, Lee JM. Nuclear localization ofcyclin B1 regulates DNA damage-induced apoptosis. Blood, 2003; 101 (5): 1928-1933.
7. Wang A, Yoshimi N, Ino N, et al. Overexpression of cyclin B1 in human colorectal cancers. J Cancer Res Clin Oncol, 1997; 123(2): 124-127.
8. Dutta A, Chandra R, Leiter LM, et al. Cyclins as markers of tumor proliferation: immunocytochemical studies in breast cancer. Proc Natl Acad Sci USA, 1995; 92(12):5386-5390.
9. Flanagan WM, Su LL, Wagner RW. Elucidation of gene function using C-5 propyne antisense oligonucleotides. Nat Biotechnol, 1996; 14(9):1139-1145.
10. Friedenstein A J, Petrakova KV, Kurolesova AI, et al. Heterotopic of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues. Transplantation, 1968; 6(2):230-247.
11. Owen M. Marrow stromal stem cells. J Cell Sci Suppl, 1988; 10:63-76.
12. Kopen GC, Prockop DJ, Phinney DG. Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. Proc Natl Acad Sci USA, 1999;96(19):10711-10716.
13. Weiss B, Davidkova G, Zhou LW. Antisense RNA gene therapy for studying and modulating biological processes. Cell Mol Life Sci, 1999;55(3):334-358.
14. Hnatowich DJ. Antisense and nuclear medicine. J Nucl Med, 1999;40(4):693-703.
15. Agrawal S, Zhao Q. Antisense therapeutics. Curr Opin Chem Biol, 1998;2(4):519-528.
16. Kashihara N, Maeshima Y, Makino H. Antisense oligonucleotides. Exp Nephrol, 1998; 6(1):84-88.
17. Cao GD, Wang SW, Wu SS, et al. Retrovirus-mediated antisense RNA to bcl-2 alter the biological behavior of stomach carcinoma MGC-803 cell lines. World J Gastroenterol, 1998; 4(Suppl 2):45-48.
18. Morris MC, Chaloin L, Choob M, et al. Combination of a new generation of PNAs with a peptide-based carrier enables efficient targeting of cell cycle progression. Gene Ther, 2004; 11 (9):757-764.
19. Yuan J, Yan R, Kramer A, et al. Cyclin B1 depletion inhibits proliferation and induces apoptosis in human tumor cells. Oncogene, 2004;23(34):5843-5852.
20. Rishi AK, Zhang L, Boyanapalli M, et al. Identification and characterization of a cell cycle and apoptosis regulatory protein-1 as a novel mediator of apoptosis signaling by retinoid CD437. J Biol Chem,2003; 278(35):33422-33435.
21. Choi JA, Kim JY, Lee JY, et al. Induction of cell cycle arrest and apoptosis in human breast cancer cells by quercetin. Int J Oncol, 2001;19(4):837-844.
22. Shaw MM, Gurr WK, Watts PA, et al. Ganciclovir and penciclovir, but not acyclovir, induce apoptosis in herpes simplex virus thymidine kinase-transfonned baby hamster kidney cells. Antivir Chem Chemother, 2001; 12(3):175-186.
23. Maier TJ, Schilling K, Schmidt R, et al. Cyclooxygenase-2 (COX-2)-dependent and -independent anticarcinogenic effects of celecoxib in human colon carcinoma cells. Biochem Pharmacol, 2004;67(8): 1469-1478.
24. Seth P, Katayose D, Li Z, et al. A recombinant adenovirus expressing wild type p53 induces apoptosis in drug-resistant human breast cancer cells: a gene therapy approach for drug-resistant cancers. Cancer Gene Ther, 1997;4(6):383-390.
25. Weinstein IB, Begemann M, Zhou P, et al. Disorders in cell circuitry associated with multistage carcinogenesis: exploitable targets for cancer prevention and therapy. Clin Cancer Res, 1997; 3(12 Pt 2):2696-2702.
26. Masato T, Naohiko S, Toshinori O, et al. Identification of the p33INGl-regulated genes that include cyclin B1 and proto-oncogene DEK by using cDNA microarray in a mouse mammary epithelial cell line NmuMG. Cancer Res, 2002; 62(8):2203-2209.
27. Lim KM, Yeo WS, Chow VT. Antisense abrogation of DENN expression induces apoptosis of leukemia cells in vitro, causes tumor regression in vivo and alters the transcription of genes involved in apoptosis and the cell cycle. Int J Cancer, 2004; 109(1):24-37.
28. Craig C, Wersto R, Kim M, et al. A recombinant adenovirus expressing p27Kipl induces cell cycle arrest and loss ofcyclin-Cdk activity in human breast cancer cells. Oncogene, 1997; 14(19):2283-2289.
29. Dubrez L, Coll JL, Hurbin A, et al. Cell cycle arrest is sufficient for p53-mediated tumor regression. Gene Ther, 2001; 8(22):1705-1712.
30. Zhan Q, Antinore MJ, Wang XW, et al. Association with Cdc2 and inhibition of Cdc2/Cyclin B1 kinase activity by the p53-regulated protein Gadd45. Oncogene, 1999; 18(18):2892-2900.
31. Azzam EI, Toledo SM de, Waker AJ, et al. High and low fluences of (?) -particles induce a G1 checkpoint in human diploid fibroblasts. Cancer Res, 2000; 60(10):2623-2631.
32. Kornmarm M, Arber N, Korc M. Inhibition of basal and mitogen-stimulated pancreatic cancer cell growth by cyclin D1 antisense is associated with loss of tumorigenicity and potentiation of cytotoxicity to cisplatinum. J Clin Invest, 1998; 101 (2):344-352.
33. Meadus WJ. A semi-quantitative RT-PCR method to measure the in vivo effect of dietary conjugated linoleic acid on porcine muscle PPAR gene expression. Biol Proced Online, 2003; 5:20-28.
34. D(?)ndas N. A semi-quantitative method for the estimation of adenosine A1 receptor mRNA levels in rat kidney. Indian J Pharmacol, 2004;36(3):167-170.
35. Ladanyi A, Gallai M, Paku S, et al. Expression of a Decorin-Like Molecule in Human Melanoma. Pathol Oncol Res, 2001; 7(4):260-266.
36. Christine S, Michael K O'Connor, Elizabeth RB, et al. Treatment of prostate cancer by radioiodine therapy after tissue-specific expression of the sodium iodide symporter. Cancer Res, 2000; 60(22):6526-6530.
37. Anderson IC, Shipp MA, Docherty AP, et al. Combination therapy including a gelatinase inhibitor and cytotoxic agent reduces local invasion and metastasis of murine Lewis lung carcinoma. Cancer Res, 1996;56(4):715-718.
38. Ermert L, Hocke AC, Duncker H-R, et al. Comparison of different detection methods in quantitative microdensitometry. Am J Pathol, 2001;158(2):407-417.
39. Andreoletti O, Berthon P, Levavasseur E, et al. Phenotyping of protein-prion (PrPsc)-accumulating cells in lymphoid and neural tissues of naturally scrapie-affected sheep by double-labeling immunohistochemistry. J Histochem Cytochem, 2002; 50(10): 1357-1370.
40. Gharbi S, Gaffney P, Yang A, et al. Evaluation of two-dimensional differential gel electrophoresis for proteomic expression analysis of a model breast cancer cell system. Mol Cell Proteomics, 2002; 1 (2):91-98.
41. Dunbar B, Wilson SB. A buffer exchange procedure giving enhanced resolution to polyacrylamide gels prerun for protein sequencing. Anal Biochem, 1994; 216(1):227-228.
42. G(?)rg A, Obermaier C, Boguth G, et al. The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis, 2000; 21 (6): 1037-1053.
43. Kubota K, Wakabayashi K, Matsuoka T. Proteome analysis of secreted proteins during osteoclast differentiation using two different methods: two-dimensional electrophoresis and isotope-coded affinity tags analysis with two-dimensional chromatography. Proteomics, 2003; 3(5):616-626.
44. Arber N, Doki Y, Han EKH, et al. Antisense to cyclin D1 inhibits the growth and tumorigenicity of human colon cancer cells. Cancer Res, 1997; 57(8):1569-1574.
45. Zhou P, Jiang W, Zhang YJ, et al. Antisense to cyclin D1 inhibits growth and reverses the transformed phenotype of human esophageal cancer cells. Oncogene, 1995; 11(3):571-580.
46. Weiss B, Davidkova G, Zhang SP. Antisense strategies in neurobiology. Neurochem Int, 1997; 31(3):321-348.
47. Kawasaki H, Machida M, Komatsu M, et al. Specific regulation of gene expression by antisense nucleic acids: a summary of methodologies and associated problems. Artificial Organs, 1996; 20(8):836-848.
48. Tu SP, Jiang XH, Lin MC, et al. Suppression of survivin expression inhibits in vivo tumorigenicity and angiogenesis in gastric cancer. Cancer Res, 2003; 63(22):7724-7732.
49. Darzynkiewicz Z, Gong J, Juan G, et al. Cytometry of cyclin proteins. Cytometry, 1996; 25(1):1-13.
50. Barrett KL, Demiranda D, Katula KS. Cyclin bl promoter activity and functional cdkl complex formation in G1 phase of human breast cancer cells. Cell Biol Int, 2002; 26(1):19-28.
51. Shen M, Feng Y, Gao C, et al. Detection of cyclin b l expression in g(1)-phase cancer cell lines and cancer tissues by postsorting Western blot analysis. Cancer Res, 2004; 64(5): 1607-1610.
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