乳癌是全世界致死率極高的癌症。在過去的幾十年，臨床上常用tamoxifen這一藥物治療在乳癌病人；另外，也常利用TZD這一類的藥物進行臨床上的試驗和治療，例如:troglitazone、rosiglitazone等。不幸的，這些臨床上用藥常會造成嚴重的副作用。Cyclin D1 在細胞週期的調控上扮演重要的角色，使得G1時期進行到S時期，過去相關報導指出它會調控雌激素引導之上皮細胞的細胞生長能力。Cyclin D1在人類的乳癌當中有超過30%會大量表現，這影響到乳癌的治療和預後能力。因此，本研究中我們將利用一個新興合成的藥物cyclin D1抑制劑，合併臨床上的用藥治療乳腺癌。利用cyclin D1抑制劑降低cyclin D1在乳腺癌MCF-7 (存在雌激素受體)和MDA-MB 231 (不存在雌激素受體)細胞株中蛋白的表現量，並探討其抑制乳癌細胞生長的作用機轉。結果我們發現，cyclin D1抑制劑會降低cylin D1蛋白在MCF-7細胞株裡蛋白穩定度和表現量。而在MDA-MB 231細胞中，cyclin D1抑制劑不只會降低cyclin D1蛋白穩定度，亦會影響其轉錄層次。我們的實驗結果也顯示，cyclin D1抑制劑作用在兩株乳癌細胞株MCF-7和MDA-MB 231，會使得細胞週期停滯在G2/M期而導致細胞死亡。此外，我們也測試cyclin D1抑制劑和臨床上藥物tamoxifen以及TZDs合併使用，結果顯示並沒有明顯的加成性效果。

Breast cancer is the fifth most common cause of cancer death in the worldwide. In the past decades, tamoxifen has been used for clinical treatment for breast cancers. The derivatives of compound thiazolidinedione (TZDs) including troglitazone (rezulin) and rosiglitazone (avandia) are also in the stages of clinical trials. But in the earlier research, some studies reported that the use of these drugs was associated with some serious side effects. Cyclin D1 plays an important role in G1/S phase cell cycle progression and in growth factor- or estrogen-induced mammary epithelial cell proliferation. Cyclin D1 overexpression is also found in high percentage (over 30%) of human breast cancers, correlating with poor prognosis. In this study, we used a cyclin D1-ablative drug VGH No.47 to reduce the expression of cyclin D1 in human breast adenocarcinoma cell line MCF-7 (ER-positive) and MDA-MB-231 (ER-negative) and to study its effect on cell proliferation. Our results demonstrated that VGH No.47 decreased the protein stability of cyclin D1. Conversely, VGH No.47 reduced cyclin D1 at both transcriptional level and protein stability in ER-negative MDA-MB-231 cells. We found that VGH No.47 caused G2/M arrested in both breast cancer cell lines. In addition, we tested whether cyclin D1-ablative drug could sensitize breast cancer cells to tamoxifen and TZDs. We expect to lower the dose of tamoxifen, troglitazone or rosiglitazone to reduce the side effects, but the results do not meet our expectation and do not exhibit synergistic effect.

Contents-------------------------------------------------------------------------------------I
Acknowledgement-------------------------------------------------------------------------IV
Abbreviations ------------------------------------------------------------------------------VII
Abstract (in English) ---------------------------------------------------------------------VI
Abstract (in Chinese) ---------------------------------------------------------------------VII
Introduction -------------------------------------------------------------------------------- 1
Materials and Methods
1. Cell Lines ------------------------------------------------------------------------------ 4
2. Primers --------------------------------------------------------------------------------- 4
3. Antibodies ----------------------------------------------------------------------------- 5
4. Drugs ----------------------------------------------------------------------------------- 6
5. Cell culture
(a) Passage ----------------------------------------------------------------------------- 6
(b) Storage of frozen cell ------------------------------------------------------------- 6
(c) Defrost of frozen cell ------------------------------------------------------------- 7
6. MTT assay ----------------------------------------------------------------------------- 7
7. Transfection ---------------------------------------------------------------------------- 8
8. Promoter assay ------------------------------------------------------------------------- 8
9. Extraction of RNA purification ------------------------------------------------------ 8
10. Two-step RT-PCR (Reverse-transcription polymerase chain reaction) ------- 9
11. Western blot analysis ---------------------------------------------------------------- 9
12. Flow cytometry analysis ------------------------------------------------------------ 10
13. Reporter plasmid constructs -------------------------------------------------------- 10
14. Transformation ----------------------------------------------------------------------- 11
15. Site-directed mutagenesis
(a) Primer Design ---------------------------------------------------------------------- 12
(b) Mutant Strand Synthesis Reaction (Thermal Cycling) ----------------------- 12
16. Plasmid purification
(a) Plasmid mini-purification -------------------------------------------------------- 13
(b) Plasmid midi-purification -------------------------------------------------------- 13
Results
1. Cyclin D1-ablative drug reduced the viability of breast cancer in dose-
dependent manner--------------------------------------------------------------------- 15
2. Cyclin D1-ablative drug reduced cyclin D1 expression in breast cancer in time-dependent manner --------------------------------------------------------------- 15
3. The cyclin D1-ablative drug induced breast cancer MCF-7 and MDA-MB 231 G2/M phase arrest ---------------------------------------------------------------------- 16
4. The effects of cyclin D1-ablative drug on the gene expression in breast cancer cells --------------------------------------------------------------------------------------- 17
5. The effects of cyclin D1-ablative drug on MCF-7 and MDA-MB 231 cells --- 17
6. The cyclin D1-ablative drug results in the proteosome-mediated degradation of cyclin D1 in MCF-7 breast cancer cells --------------------------------------------- 18
7. The cyclin D1-ablative drug induced phosphorylation of p38 to degrade
cyclin D1 --------------------------------------------------------------------------------- 19
8. The cyclin D1-ablative drug results in the proteosome-mediated degradation of cyclin D1 in MDA-MB 231 breast cancer cells ------------------------------------ 19
9. Identification of the cyclin D1 promoter response element ----------------------- 20
Discussion---------------------------------------------------------------------------------------22
Figure Legends
Table 1. Cyclin D1-ablative drug induced MCF-7 cells G2/M phase arrest in flow
cytometry assay -------------------------------------------------------------------26
Table 2. Cyclin D1-ablative drug induced MDA-MB 231 cells G2/M phase
arrest in flow cytometry assay --------------------------------------------------27
Table 3. KinasePhos predict phosphorylation sites of cyclin D1 ---------------------28
Table 4. NetPhosK predict phosphorylation sites of cyclin D1 -----------------------29
Fig.1 Cyclin D1-ablative drug reduced the cell viability of breast cancer in dose-dependent manner -------------------------------------------------------------30
Fig.2 Cyclin D1-ablative drug reduced the expression of cyclin D1 in breast
cancer in time-dependent manner -------------------------------------------------31
Fig.3 The effects of cyclin D1-ablative drug on the gene expression in breast
cancer cells ---------------------------------------------------------------------------32
Fig.4 The effects of cyclin D1-ablative drug on cell cycle protein expression in MCF-7 cells --------------------------------------------------------------------------33
Fig.5 The effects of cyclin D1-ablative drug on cell cycle protein expression in MDA-MB 231 cells -----------------------------------------------------------------35
Fig.6 The cyclin D1-ablative drug affected the proteosome-mediated
degradation of cyclin D1 in MCF-7 cells ----------------------------------------37
Fig.7 The cyclin D1-ablative drug induced phosphorylation of p38 to degrade cyclin D1, but not major ------------------------------------------------------------38
Fig.8 The cyclin D1-ablative drug affects the proteosome-mediated degradation
of cyclin D1 in MDA-MB-231 cells ----------------------------------------------40
Fig.9 Identification of the cyclin D1 promoter response element --------------------41
Fig.10 Identification of the cyclin D1 promoter response element -------------------42
Fig.11 The affect of VGH No.47 on the potency of NF-κB responsive element within cyclin D1 -------------------------------------------------------------------44
Fig.12 Point mutations identified the Sp1 and LEF-1 responsive region within cyclin D1 promoter ----------------------------------------------------------------45
Fig.13 Overview of the mechanism of cyclin D1-ablative drug on
breast cancer cells------------------------------------------------------------------47
References--------------------------------------------------------------------------------------49
Appendix----------------------------------------------------------------------------------------58

Aaltonen K, Amini RM, Landberg G, Eerola H, Aittomaki K, Heikkila P, Nevanlinna H, Blomqvist C. Cyclin D1 expression is associated with poor prognostic features in estrogen receptor positive breast cancer. Breast Cancer Res Treat 2008;60(11):2825-30.
Aaltonen K, Blomqvist C, Amini RM, Eerola H, Aittomaki K, Heikkila P, Nevanlinna H. Familial breast cancers without mutations in BRCA1 or BRCA2 have low cyclin E and high cyclin D1 in contrast to cancers in BRCA mutation carriers. Clin Cancer Res 2008;14(7):1976-83.
Alao JP. The regulation of cyclin D1 degradation: roles in cancer development and the potential for therapeutic invention. Molecular Cancer 2007; 24(6):6-24.
Albanese C, Wu K, D'Amico M, Jarrett C, Joyce D, Hughes J, Hulit J, Sakamaki T, Fu M, Ben-Ze'ev A, Bromberg JF, Lamberti C, Verma U, Gaynor RB, Byers SW, Pestell RG. IKKalpha regulates mitogenic signaling through transcriptional induction of cyclin D1 via Tcf. Mol Biol Cell 2003;14(2):585-99.
Bonofiglio D, Aquila S, Catalano S, Gabriele S, Belmonte M, Middea E, Qi H, Morelli C, Gentile M, Maggiolini M, Ando S. Peroxisome proliferator-activated receptor-gamma activates p53 gene promoter binding to the nuclear factor-kappaB sequence in human MCF7 breast cancer cells. Mol Endocrinol 2006;20(12):3083-92.
Bonofiglio D, Gabriele S, Aquila S, Catalano S, Gentile M, Middea E, Giordano F, Ando S. Estrogen receptor alpha binds to peroxisome proliferator-activated receptor response element and negatively interferes with peroxisome proliferator-activated receptor gamma signaling in breast cancer cells. Clin Cancer Res 2005;11(17):6139-47.
D'Amico M, Hulit J, Amanatullah DF, Zafonte BT, Albanese C, Bouzahzah B, Fu M, Augenlicht LH, Donehower LA, Takemaru K, Moon RT, Davis R, Lisanti MP, Shtutman M, Zhurinsky J, Ben-Ze'ev A, Troussard AA, Dedhar S, Pestell RG. The integrin-linked kinase regulates the cyclin D1 gene through glycogen synthase kinase 3beta and cAMP-responsive element-binding protein-dependent pathways. J Biol Chem 2000;275(42):32649-57.
Diehl JA, Cheng M, Roussel MF, Sherr CJ. Glycogen synthase kinase-3beta regulates cyclin D1 proteolysis and subcellular localization. Genes Dev 1998;12(22):3499-511.
Dulic V, Stein GH, Far DF, Reed SI. Nuclear accumulation of p21Cip1 at the onset of mitosis: a role at the G2/M-phase transition. Mol Cell Biol 1998;18(1):546-57.
Germain D, Russell A, Thompson A, Hendley J. Ubiquitination of free cyclin D1 is independent of phosphorylation on threonine 286. J Biol Chem 2000;275(16):12074-9.
Garrison JB, Kyprianou N. Novel targeting of apoptosis pathways for prostate cancer therapy. Curr Cancer Drug Targets 2004;4(1):85-95.
Garrison JB, Kyprianou N. Doxazosin induces apoptosis of benign and malignant prostate cells via a death receptor-mediated pathway. Cancer Res 2006;66(1):464-72.
Garrison JB, Shaw YJ, Chen CS, Kyprianou N. Novel quinazoline-based compounds impair prostate tumorigenesis by targeting tumor vascularity. Cancer Res 2007;67(23):11344-52.
Grinstein E, Jundt F, Weinert I, Wernet P, Royer HD. Sp1 as G1 cell cycle phase specific transcription factor in epithelial cells. Oncogene 2002;21(10):1485-92.
Guastamacchia E, Resta F, Triggiani V, Liso A, Licchelli B, Ghiyasaldin S, Sabba C, Tafaro E. Evidence for a putative relationship between type 2 diabetes and neoplasia with particular reference to breast cancer: role of hormones, growth factors and specific receptors. Curr Drug Targets Immune Endocr Metabol Disord 2004;4(1):59-66.
Hiscox S, Jiang WG, Obermeier K, Taylor K, Morgan L, Burmi R, Barrow D, Nicholson RI. Tamoxifen resistance in MCF7 cells promotes EMT-like behaviour and involves modulation of beta-catenin phosphorylation. Int J Cancer 2006;118(2):290-301.
Huang JW, Shiau CW, Yang J, Wang DS, Chiu HC, Chen CY, Chen CS. Development of small-molecule cyclin D1-ablative agents. J Med Chem 2006;49(15):4684-9.
Huang JW, Shiau CW, Yang YT, Kulp SK, Chen KF, Brueggemeier RW, Shapiro CL, Chen CS. Peroxisome proliferator-activated receptor gamma-independent ablation of cyclin D1 by thiazolidinediones and their derivatives in breast cancer cells. Mol Pharmacol 2005;67(4):1342-8.
Ishii Y, Waxman S, Germain D. Tamoxifen stimulates the growth of cyclin D1-overexpressing breast cancer cells by promoting the activation of signal transducer and activator of transcription 3. Cancer Res 2008;68(3):852-60.
Jeruss JS, Mittendorf EA, Tucker SL, Gonzalez-Angulo AM, Buchholz TA, Sahin AA, Cormier JN, Buzdar AU, Hortobagyi GN, Hunt KK. Combined use of clinical and pathologic staging variables to define outcomes for breast cancer patients treated with neoadjuvant therapy. J Clin Oncol 2008;26(2):246-52.
Joyce D, Albanese C, Steer J, Fu M, Bouzahzah B, Pestell RG. NF-kappaB and cell-cycle regulation: the cyclin connection. Cytokine Growth Factor Rev 2001;12(1):73-90.
Joyce D, Bouzahzah B, Fu M, Albanese C, D'Amico M, Steer J, Klein JU, Lee RJ, Segall JE, Westwick JK, Der CJ, Pestell RG. Integration of Rac-dependent regulation of cyclin D1 transcription through a nuclear factor-kappaB-dependent pathway. J Biol Chem 1999;274(36):25245-9.
Kaushal N, Bansal MP. Inhibition of CDC2/Cyclin B1 in response to selenium-induced oxidative stress during spermatogenesis: potential role of Cdc25c and p21. Mol Cell Biochem 2007;298(1-2):139-50.
Kavurma MM, Khachigian LM. Sp1 inhibits proliferation and induces apoptosis in vascular smooth muscle cells by repressing p21WAF1/Cip1 transcription and cyclin D1-Cdk4-p21WAF1/Cip1 complex formation. J Biol Chem 2003;278(35):32537-43.
Khoshnoud MR, Fornander T, Johansson H, Rutqvist LE. Long-term pattern of disease recurrence among patients with early-stage breast cancer according to estrogen receptor status and use of adjuvant tamoxifen. Breast Cancer Res Treat 2008;107(1):71-8.
Kumar AP, Bhaskaran S, Ganapathy M, Crosby K, Davis MD, Kochunov P, Schoolfield J, Yeh IT, Troyer DA, Ghosh R. Akt/cAMP-responsive element binding protein/cyclin D1 network: a novel target for prostate cancer inhibition in transgenic adenocarcinoma of mouse prostate model mediated by Nexrutine, a Phellodendron amurense bark extract. Clin Cancer Res 2007;13(9):2784-94.
Lin SY, Xia W, Wang JC, Kwong KY, Spohn B, Wen Y, Pestell RG, Hung MC. Beta-catenin, a novel prognostic marker for breast cancer: its roles in cyclin D1 expression and cancer progression. Proc Natl Acad Sci U S A 2000;97(8):4262-6.
Love RR, Van DN, Quy TT, Linh ND, Tung ND, Shen TZ, Hade EM, Young GS, Jarjoura D. Survival after adjuvant oophorectomy and tamoxifen in operable breast cancer in premenopausal women. J Clin Oncol 2008;26(2):253-7.
Marchal JA, Nunez MC, Suarez I, az-Gavilan M, Gomez-Vidal JA, Boulaiz H, Rodriguez-Serrano F, Gallo MA, Espinosa A, Aranega A, Campos JM. A synthetic uracil derivative with antitumor activity through decreasing cyclin D1 and Cdk1, and increasing p21 and p27 in MCF-7 cells. Breast Cancer Res Treat 2007;105(3):237-46.
Masiuk M, Urasinska E, Domagala W. Intranuclear nucleolin distribution during cell cycle progression in human invasive ductal breast carcinomas in relation to estrogen receptor status. Anticancer Res 2007;27(6B):3957-62.
Moriuchi A, Ido A, Nagata Y, Nagata K, Uto H, Hasuike S, Hori T, Hirono S, Hayashi K, Tsubouchi H. A CRE and the region occupied by a protein induced by growth factors contribute to up-regulation of cyclin D1 expression in hepatocytes. Biochem Biophys Res Commun 2003;300(2):415-21.
Mukherji A, Janbandhu VC, Kumar V. GSK-3beta-dependent destabilization of cyclin D1 mediates replicational stress-induced arrest of cell cycle. FEBS Lett 2008;582(7):1111-6.
Nagata D, Suzuki E, Nishimatsu H, Satonaka H, Goto A, Omata M, Hirata Y. Transcriptional activation of the cyclin D1 gene is mediated by multiple cis-elements, including SP1 sites and a cAMP-responsive element in vascular endothelial cells. J Biol Chem 2001;276(1):662-9.
Opitz OG, Rustgi AK. Interaction between Sp1 and cell cycle regulatory proteins is important in transactivation of a differentiation-related gene. Cancer Res 2000;60(11):2825-30.
Prasad CP, Gupta SD, Rath G, Ralhan R. Wnt signaling pathway in invasive ductal carcinoma of the breast: relationship between beta-catenin, dishevelled and cyclin D1 expression. Oncology 2007;73(1-2):112-7.
Prasad S, Kaur J, Roy P, Kalra N, Shukla Y. Theaflavins induce G2/M arrest by modulating expression of p21waf1/cip1, cdc25C and cyclin B in human prostate carcinoma PC-3 cells. Life Sci 2007;81(17-18):1323-31.
Qin C, Burghardt R, Smith R, Wormke M, Stewart J, Safe S. Peroxisome proliferator-activated receptor gamma agonists induce proteasome-dependent degradation of cyclin D1 and estrogen receptor alpha in MCF-7 breast cancer cells. Cancer Res 2003;63(5):958-64.
Qin C, Singh P, Safe S. Transcriptional activation of insulin-like growth factor-binding protein-4 by 17beta-estradiol in MCF-7 cells: role of estrogen receptor-Sp1 complexes. Endocrinology 1999;140(6):2501-8.
Rowlands TM, Pechenkina IV, Hatsell S, Cowin P. Beta-catenin and cyclin D1: connecting development to breast cancer. Cell Cycle 2004;3(2):145-8.
Sharma C, Pradeep A, Pestell RG, Rana B. Peroxisome proliferator-activated receptor gamma activation modulates cyclin D1 transcription via beta-catenin-independent and cAMP-response element-binding protein-dependent pathways in mouse hepatocytes. J Biol Chem 2004;279(17):16927-38.
Shtutman M, Zhurinsky J, Simcha I, Albanese C, D'Amico M, Pestell R, Ben-Ze'ev A. The cyclin D1 gene is a target of the beta-catenin/LEF-1 pathway. Proc Natl Acad Sci U S A 1999;96(10):5522-7.
Smalley MJ, Dale TC. Wnt signalling in mammalian development and cancer. Cancer Metastasis Rev;18(2):215-30.
Smalley MJ, Dale TC. Wnt signaling and mammary tumorigenesis. J Mammary Gland Biol Neoplasia 2001;6(1):37-52.
Takahashi-Yanaga F, Sasaguri T. GSK-3beta regulates cyclin D1 expression: a new target for chemotherapy. Cell Signal 2008;20(4):581-9.
Vogel VG. Recent results from clinical trials using SERMs to reduce the risk of breast cancer. Ann N Y Acad Sci 2006;1089:127-42.
Wang C, Pattabiraman N, Zhou JN, Fu M, Sakamaki T, Albanese C, Li Z, Wu K, Hulit J, Neumeister P, Novikoff PM, Brownlee M, Scherer PE, Jones JG, Whitney KD, Donehower LA, Harris EL, Rohan T, Johns DC, Pestell RG. Cyclin D1 repression of peroxisome proliferator-activated receptor gamma expression and transactivation. Mol Cell Biol 2003;23(17):6159-73.
Wang PS, Chou FS, Porchia L, Saji M, Pinzone JJ. Troglitazone inhibits cell migration, adhesion, and spreading by modulating cytoskeletal rearrangement in human breast cancer cells. Mol Carcinog 2008.
Watanabe G, Albanese C, Lee RJ, Reutens A, Vairo G, Henglein B, Pestell RG. Inhibition of cyclin D1 kinase activity is associated with E2F-mediated inhibition of cyclin D1 promoter activity through E2F and Sp1. Mol Cell Biol 1998;18(6):3212-22.
Wheler J, Johnson M, Seidman A. Adjuvant therapy with aromatase inhibitors for postmenopausal women with early breast cancer: evidence and ongoing controversy. Semin Oncol 2006;33(6):672-80.
Weng JR, Chen CY, Pinzone JJ, Ringel MD, Chen CS. Beyond peroxisome proliferator-activated receptor gamma signaling: the multi-facets of the antitumor effect of thiazolidinediones. Endocr Relat Cancer 2006;13(2):401-13.
Yan GZ, Ziff EB. Nerve growth factor induces transcription of the p21 WAF1/CIP1 and cyclin D1 genes in PC12 cells by activating the Sp1 transcription factor. J Neurosci 1997;17(16):6122-32.
Yang CC, Wang YC, Wei S, Lin LF, Chen CS, Lee CC, Lin CC, Chen CS. Peroxisome proliferator-activated receptor gamma-independent suppression of androgen receptor expression by troglitazone mechanism and pharmacologic exploitation. Cancer Res 2007;67(7):3229-38.