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The effect of cyclooxygenase inhibitors on cell cycle regulation and proliferation of glioblastoma and lymphoma cell lines

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The effect of cyclooxygenase inhibitors on cell cycle regulation and proliferation of glioblastoma and lymphoma cell lines

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Content THE EFFECT OF CYCLOOXYGENASE INHIBITORS ON CELL CYCLE REGULATION AND PROLIFERATION OF GLIOBLASTOMA AND LYMPHOMA CELL LINES by Adel Kardosh A Thesis Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirement for the Degree MASTER OF SCIENCE (MOLECULAR MICROBIOLOGY AND IMMUNOLOGY) December 2004 Copyright 2004 Adel Kardosh Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. U M I N um ber: 1 4 2 4 2 4 6 INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. UMI UMI Microform 1424246 Copyright 2005 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGMENTS I would especially like to thank my research and thesis advisor, Dr. Axel H. Schonthal, whose encouragement and guidance has helped me become a meticulous and creative scientist. I appreciate his support, advice, and enthusiasm for my experience in research. In addition, I would like to extend my gratitude to Dr. Thomas C. Chen for his insightful scientific advice and his motivation for the advancement of science. I gratefully thank my thesis committee members, Dr. Florence Hofman and Dr. James Ou, for their time and insightful comments. I would also like to thank my lab mates for all their support. Finally, I am extremely thankful for the strong love of my family especially my parents, Issam and Ayda Kardosh, and habibi Patricia. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS A C K N O W LE D G M E N TS.............................................................................................................. ii LIST O F FIG U R ES A N D TABLES...........................................................................................v LIST O F A B B R EVIA TIO N S.......................................................................................................vii A B S T R A C T .....................................................................................................................................ix C H A PTER I: IN T R O D U C TIO N ................................................................................................ 1 1.1 Non-Steroidal Anti-Inflammatory Drugs.......................................................... 2 1.2 Glioblastoma............................................................................................................ 5 1.3 Lymphoma................................................................................................................ 6 1.4 The Cell Cycle Engine...........................................................................................9 CH A PTER II: MATERIALS & M E TH O D S ........................................................................... 13 M aterials.......................................................................................................................... 14 Tissue Culture................................................................................................................14 M TT Assay......................................................................................................................15 Cell Lysis and Protein Quantification......................................................................16 W estern Blot...................................................................................................................16 In vitro Kinase Assays.................................................................................................17 Cesium Chloride Gradient Plasmid Preparation................................................ 18 Transient Transfection by DNA/Calcium Phosphate Coprecipitation 19 Flow Cytometry Analysis........................................................................................... 20 Subcutaneous Tumor Growth in Nude M ice....................................................... 20 Animal Chow Preparation..........................................................................................21 Chapter III: Results..................................................................................................................... 22 The Anti-Proliferation Effect of Celecoxib..............................................................23 The Anti-Proliferation Effect of Celecoxib is Independent of C O X -2............ 28 Celecoxib Down-Regulate Cyclin Dependent Kinase Activity........................ 29 Celecoxib Down-Regulate Promoter Activity in Glioblastoma........................ 37 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. iv Inhibition of Tumor Growth by Celecoxib in Nude M ice................................................. 39 DMC, A Derivative of Celecoxib.............................................................................................41 Chapter IV: D IS C U S S IO N ........................................................................................................45 R E F E R E N C E S .............................................................................................................................52 A P P E N D IX A: LIST O F C O M P A N IE S ..................................................................................61 A P P E N D IX B: A N TIB O D IES AND PLA SM ID S..................................................................62 A P P E N D IX C: BUFFERS AND S O LU TIO N S .................................................................... 64 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. V LIST OF FIGURES AND TABLES Fig 1.1: Prostaglandin synthesis by COX enzymes. Fig 1.2: Cell cycle overview. Fig 3.1: COX expression levels. Fig 3.2: Cell proliferation in the presence of NSAIDs in glioblastoma. Fig 3.3: Cell proliferation in the presence of the various NSAIDs in lymphoma. Fig 3.4: Cell cytotoxicity induction in response to NSAIDs treatment. Fig 3.5: Cell cycle distribution in the presence of celecoxib. Fig 3.6: Levels of endogenous PGE2. Fig 3.7: The effect of exogenous PGE2 on cell proliferation. Fig 3.8: CDK activity in the presence of various NSAIDs. Fig 3.9: CDK activity in the presence of selective COX-2 inhibitors. Fig 3.10: CDK activity in the presence of celecoxib in glioblastoma. Fig 3.11: CDK activity in the presence of celecoxib in lymphoma. Fig 3.12: CDK activity in a timely dependent manner in the presence of celecoxib. Fig 3.13: Levels of cell cycle-regulatory proteins in the presence of celecoxib in glioblastoma. Fig 4.14: Levels of cell cycle-regulatory proteins in the presence of celecoxib in lymphoma. Fig 3.15: Promoter activity in the presence of celecoxib in glioblastoma. Fig 3.16: Tumor growth in mice treated with celecoxib. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig 3.17: Cell proliferation in the presence of the various selective COX-2 inhibitors plus DMC. Fig 3.18: CDK activity in the presence of celecoxib and DMC. Fig 3.19: Levels of cell cycle-regulatory proteins in the presence of DMC. Table 3.1 Comparison between COX-2 inhibitors and celecoxib’s derivative DMC. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF ABBREVIATIONS Amp: Ampicillin ATCC: American Type Culture Collection BSA: Bicinchoninic acid CDKs: Cyclin dependent kinases CDIs: Cdk inhibitors COX: cyclooxygenase enzyme COXIBs: Selective cyclooxygenase-2 inhibitors CNS: Central Nervous System DMSO: Dimethyl Sulfoxide FACS: Fluorescence-activated Cell Sorter Gi and G2 phases: Gap phases GBM: Malignant glioblastoma multiforme LDH: Lactate dehydrogenase M phase: Mitotic phase MTT: Tetrazolium salt NHL: Non-Hodgkin's lymphoma NSAIDs: Non-steroidal anti-inflammatory drugs PCNA: Proliferating cell nuclear antigen PCNSL: Primary CNS lymphoma Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. PGs: Prostaglandins PGE2: Prostaglandin E2 PLA2: Glycerophospholipids by phospholipase A2 PMSF: Phenyl methyl sulfonyl fluoride REAL system: American classification of Lymphoid Neoplasms system S phase: Synthetic phase TBST: Tris-buffered saline with 0.1% Tween 20 WBRT: Whole brain radiation therapy WHO: World Health Organization Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ix ABSTRACT The growth inhibitory effect of various non-steroidal anti-inflammatory drugs (NSAIDs) was studied in various Burkitt’s lymphoma and glioblastoma cell lines. We found that one of these drugs celecoxib, efficiently inhibited the proliferation of these cells in vitro — much more potently than any of the other cyclooxygenase (COX) inhibitors analyzed. Intriguingly, this effect appeared to be independent of the intracellular levels of COX-2. In order to study the molecular mechanisms underlying this growth-inhibitory effect, we determined the activity of cyclin-dependent kinases (CDKs), a group of enzymes that is essential for cell proliferation. Celecoxib was found to potently down-regulate CDK activity via a transcriptional block of the expression of the genes for cyclin A and cyclin B — two essential regulatory subunits of the CDK enzyme complex. Furthermore, we discovered that celecoxib suppresed tumor growth in experimental animals invivo. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 Chapter I INTRODUCTION Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2 Introduction 1.1 Non-Steroidal Anti-Inflammatory Drugs Non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to play a role in the treatment or prevention of various tumors. This postulation is strengthened by two recent large randomized, placebo-controlled clinical trials, demonstrating that aspirin might be effective in primary and secondary prevention of colorectal cancer by lowering the incident of adenomas 3 58. The mechanism by which these drugs exert their effect is believed to be due to the inhibition of cyclooxygenase (COX) enzymes, a family of enzymes that catalyze the conversion of arachidonic acid to prostaglandins 46. Two isoforms of COX enzyme exits: COX-1 and COX-2. COX-1 is a housekeeping gene and it is constitutively expressed in most tissues. In contrast, COX-2 is typically not expressed or is expressed at relatively low levels but can be induced by inflammatory stimuli, including cytokines, growth factors, and tumor promoters 63,74. In addition, many different types of human tumors have been shown to express elevated levels of COX-2, which include colon, pancreatic, prostate, gastric, and head and nick cancers; hence, it is thought that the increased expression of the enzyme might contribute to the carcinogenic process5 9'67'72. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3 Most NSAIDs, such as flurbiprofen, indomethacin and sulindac, are able to inhibit both COX-1 as well as COX-2, but treatment with these drugs is limited by normal tissue toxicity. New drugs have been designed for the CdMormambrar* POe2 P 0 & 2 TX A j Fig 1.1: Prostaglandin synthesis by COX enzymes. Arachadonic acid, which is freed from the membrane glycerophospholipids by phospholipase A2 (PLA2) family of enzymes, is catalyzed by COX enzymes to bicyclic endoperoxide intermediate PGG2, followed by reduction to PGH2, which is converted to one of several structurally related prostaglandins (PGs). Traditional non-steroidal anti-inflammatory drugs (NSAIDs) inhibit the activity of both COX-1 and COX-2, where as, the selective inhibitors (COXIBs) inhibit selectively COX-2. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4 treatment of adult arthritis 52, such as celecoxib and rofecoxib, which specifically inhibits the COX-2 enzyme, thereby, avoiding the severe side effects associated with the conventional NSAIDs 2 9 ,6 9 (Figure 1.1). The molecular processes underlying the anti-neoplastic properties of both NSAIDs and the selective COX-2 inhibitors are poorly understood 2 4 despite substantial evidence from clinical investigations 1 7 '6 6 ’70) and from animal studies 55’5 6 '76. Genetic experiments with mice 4 0 ,4 9 have demonstrated that COX-2 is an oncogene and its over-expression is sufficient to induce cellular transformation. However, the fact that NSAIDs and selective COX-2 inhibitors exert their anti-neoplastic effect through both COX-2-dependent and independent mechanisms, have complicated the notion that they pertain their effect through the inhibition of COX-2 68. For example, NSAIDs were shown to inhibit the proliferation of cancer cells that do not express COX proteins 5 25. In addition, transformed cell lines from knockout mice, where the genes for either COX-1 or COX-2 or both were deleted, demonstrate comparable sensitivity to NSAIDs-induced growth inhibition and cell death 84. Although the exact mechanism underlying these drug’s anti-proliferation effects has not been established and some evidence has emerged indicating a blockage in the Akt signaling pathway2 2,31. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5 1.2 Glioblastoma Glioblastomas or primary brain tumor develop mostly from astrocytes, oligodendriocytes, or Schwann cells and are among the most lethal malignancies in young children and adults, with a median survival of less than 2 years 41. Recent statistics on brain tumors incident estimate 18,400 patients would be newly diagnosed with primary malignant brain tumors and other nervous system tumors in 2004, and 12,640 deaths will occur in the U.S.2. The incidence rate for malignant brain tumors and other central nervous system tumors is 6.6 per 100,000 in general, 8.0 per 100,000 for males and 5.4 per 100,000 for females 57, with a mean age at diagnosis in the middle to late 50s 8. For individuals that are diagnosed with primary malignant brain tumors, survival declines rapidly after the first 2 years from diagnosis, with a 5-year relative survival rate of 20-30% 8’57. Based on histopathological features, primary brain tumors have been classified in four different grades by the World Health Organization (WHO). Grades I and II account for pilocytic astrocytomas and low grade astrocytomas, respectively, further transformation of these tumor result in anaplastic astocytomas, which is assigned Grade III, and finally Grade IV for malignant glioblastoma multiform (GBM) 60. Current treatment includes surgical resection, radiation therapy, and chemotherapy. Due to the high invasiveness of glioblastomas, they have Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 6 poor prognosis and thus, reductions in the efficiency of the treatment. The course of the development and prognosis of these tumors is not completely understood. However, certain abnormalities at the chromosomal and gene levels, such as the loss of tumor suppressor genes, gene mutations and abnormal expression of several genes, have been reported in glioblastomas that might be involved in tumor initiation, progression and angiogenesis 60. Recent studies have shown an upregulation in COX-2 levels in gliomas 32,54,61 1.3 Lymphoma Non-Hodgkin's lymphoma (NHL) involves malignant transformation of normal B-lymphocytes. The disease usually manifests with adenopathy involving the major lymph node chains throughout the body. In later stages, other organs can become involved due to haematogenous metastases, which arise from nodal sites or bone marrow involvement. Approximately 4% of cancers in the United States are NHL, and the lifetime risk of NHL is 2.08% 1 6 . It is estimated that 54,370 new cases of NHL will be diagnosed in the United States and 19,410 deaths will occur in 2004 2. The disease accounts for 5% of cancer-related deaths and is the leading cause of cancer- related death for people between 20 and 40 years of age. NHL is slightly more common in men with an incidence of 19.2 per 100,000 compared with 12.2 per 100,000 for women 1 6 . The median age at diagnosis is 65 years2 0 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 7 and the incidence of NHL increases with age and peaks in the 80-to-85-year age group. Lymphoid neoplasms are classified based on the Revised European- American classification of Lymphoid Neoplasms (REAL) system, which is a combination of immunophenotyping, morphologic and clinical characteristics introduced by the International Lymphoma Study Group 27. The low-grade lymphomas include the small cell lymphocytic lymphomas consistent with chronic lymphocytic leukemia, the follicular small cell cleaved lymphomas, and the follicular mixed lymphomas. The intermediate grade lymphomas include the follicular large cell lymphomas, the diffuse small cell lymphomas, and the diffuse mixed small cell and large cell lymphomas. The high-grade lymphomas include: the immunoblastic lymphomas, the lymphoblastic lymphomas, and the small noncleaved cell lymphomas. In some occasions, lymphomas arise in a non-lymphoid organ. For example,it may develop in the skin or the gastrointestinal tract, where lymphocytes are an important component of the normal structure of these organs, or in the CNS, which contains no lymph nodes or lymphatics. The origin of B cell primary CNS lymphoma (PCNSL) is not fully understood but several hypotheses exist1 3. B cells may transform outside the CNS and then "home" into the CNS. This mechanism would suggest that these tumors have unique surface markers which would induce migration into the CNS, however, primary CNS lymphoma cells have the same cell surface receptors Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 8 as standard systemic non-Hodgkin's lymphoma cells 50. Alternatively, a B cell passing through the CNS, even intravascularly, may become transformed during this passage and then remain in the brain. The incidence of primary central nervous system lymphoma (PCNSL) in the United States increased more than tenfold between 1973 and 1992 1 0 . This increase is in part due to the AIDS epidemic 1 0 , although the incidence of CNS lymphoma has increased in non-AIDS populations as w e ll1 2 . The current method used in the treatment of systemic lymphoma is not effective in PCNSL. Therefore, currently the approach for treating PCNSL is a methotrexate-based chemotherapy in combination with cranial radiotherapy, i.e., whole brain radiation therapy (WBRT). Even though the treatment with WBRT has prolonged survival, it usually causes irreversible and progressive damage to the CNS. Such damage may include vascular injury, causing ischemia of surrounding tissue, demyelination of white matter, and necrosis 1 1 resulting in a progressive neurological syndrome that is characterized by dementia, gait ataxia, and urinary dysfunction 1. Thus, the patient’s quality of life is considerably reduced. It seems that the prevalence of these neurotoxic side effects correlate with the increase of radiation dose and with the age of the patient—occurring in as many as 90% of patients who are older than 60 years. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 9 1.4 The Cell Cycle Engine The cell cycle consists of four phases, which all proliferating cells need to complete; the S (synthetic) phase, where DNA mass is duplicated, the M (mitotic) phase in which homologes chromatides are separated and distributed evenly to the daughter cells, and the two other phases Gi and G2 (gap) which separate the S phase and the M phase. In the gap phases, the cell is allowed to grow and prepare for the next phase. Proliferating cells constantly progress through the cell cycle until the cell receives an anti mitotic signal or terminal differentiation is initiated. To ensure accurate cell division and maintenance of genomic information, the process of cell division is tightly regulated by holoenzymes that consist of cyclins, which are the regulatory subunits and cyclin dependent kinases (cdk’s) as the catalytic subunit21. The expression levels of the different cyclins are altered throughout the cell cycle whereas cdk levels are relatively maintained constant. When arrested cells exit Go and enter the cell cycle in G^ cyclin D is the first cyclin to become up regulated 43, cyclin D complex with cdk4 or cdk6 in G 1, and induces cyclin E/cdk2 association in late G 1 34. Unlike all other cyclins, cyclin D levels depend on the level of growth factors. In late G 1 early S phases cyclin A levels increase, forming a complex with cdk2, the complex activity peaks during S phase where it acts on the replication machinery as a down stream target6. Cyclin A also complex with cdkl (cdc2) in late G2 , but Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 10 the major regulator of the G2/M transition is cyclin B/cdk1 complex. Finally, a reduction in cyclin B levels is required for the cell to exit the mitotic phase. Given the importance of the integrity of the genome and, thereby, cell cycle, Cyclin dependent kinase (CDK) complexes need to be tightly regulated. Therefore, many aspects are involved to insure this regulation: First, for the cdks to become active they need to bind with their respective cyclin, further more the subcellular location of the cyclin is significant, for example, cyclin B is transported in to the nucleus just before the mitotic entry 30. Second, for the complex to become fully active it requires specific phosphorelation of the threonine-160/161 by the cdk activating kinase (cak) 64, and dephosphorelation at two inhibitory sites, threonine-14 and tyrosine- 15, by two dual specific phosplatase cdc25 45. The reverse phosphorelation of these two sites by wee-1 like kineases inactivates CDK. Third, Cdk inhibitors (CDIs), which are divided into two families, the ink4 family which includes p15 and p16 that target cdk4 and cdk6 26, and cip/kip family including p21 and p2 745, a more broader inhibitors that target cdks as well as cyclins. Cdk inhibitors play a major role in the cell cycle check points, which are stations in the cell cycle were the cell arrests due to damage or stress that may affect the accurate transmission of the genomic information. The two main checkpoints in the cell cycle occur at the G 1/S phase and G2/M phase boundary (Figure 1.2). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 11 The universal inhibitor p21 can interfere at both Gi and G2 checkpoints by forming a quaternary complex with cyclin/cdk as well as the proliferating cell nuclear antigen (PCNA) that is involved in replication 4. Furthermore, ink4 proteins inhibit the assembly and activity of cyclin D cdk4/6 complex by inhibiting the activities of the cyclin/cdk complex in G 1 phase. Cdc2-cyc!m B Cdc2-cycim A n / ' : Go W T G |i; | „ C dk4-cyclin D ' C dk6-cyclin D / / I f Restriction / / J-if •/ Cdk2—cydtn a * Cdk2-cyd<n E Fig 1.2: Cell cycle overview.The cell cycle is divided into four phases G1 (gap 1), S (DNA synthesis), G2 (gap 2), and M (mitosis) phase. The point after wich cellular divition must occur is the restriction point and is located at late G 1. Progression in the cell cycle is promoted by the formation of cdk/cyclin complexes. The relative activity of each complex in the cell cycle is represented by the thickness of the arcs. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 12 The transcriptional repressor Rb in its unphosphorelated state, is found bound to the transcription factor E2F. Partial phosphorelation of Rb by cyclin D/cdk4 complex promotes the dissociation of some Rb/E2F complexes. The released E2F initiates the transcription of cyclin E, which complex with cdk2 and regulated its own synthesis in a positive feedback loop by fully phosphorelating Rb causing it to release E2F. At this point, the so-called restriction point is passed and the entry into the cell cycle becomes irreversible 21. The G2/S transition is thought to follow the same mechanism involving the Rb related proteins p107 or p130 that are bound to E2F4 and inactivated by the cyclinB/cdkl complex4. The G 1 checkpoint is primarily regulated by a tumor suppression protein p53 1 8. This protein is upregulated due to DNA damage and it induces the expression of p21, thereby, arresting the cells to initiate DNA repair before the cell enters replication. The interaction of p21 with PCNA may even stimulate excision repair by itself44. The G2 checkpoint is mostly regulated by a p53 independent pathway(s) that has not been elucidated yet. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 13 Chapter II MATERIALS & METHODS Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 14 Materials & Methods Materials Celecoxib was obtained at 200 mg Celebrex® capsules (Pfizer, New York, NY). They were opened manually and the powdery content was dissolved in DMSO at 100 mM. Rofecoxib (Vioxx®, Merck, Whitehouse Station, NJ) and valdecoxib (Bextra®, Pfizer) caplets were suspended in H2O to disintegrate the excipient, and the active ingredient was dissolved in DMSO at 25 mM. NS-398 was acquired from Calbiochem (San Diego, CA) in soluble form. All traditional NSAIDs were purchased from Sigma (St. Louis, MO) in powder form and dissolved in DMSO at 100 mM. Tissue Culture Four different glioblastoma cell lines were used: LN229, LN18, U87 and A172. A172 and U87 cells were obtained from the American Type Culture Collection (ATCC). LN229 and LN18 were obtained from Frank B. Furnari and Webster K. Cavenee (University of California in San Diego, La Jolla, CA). Cells were routinely cultured on 100mm tissue culture dishes (Becton-Dickinson) in DMEM (Cellgro). In addition, three different Burkitt’s lymphoma cell lines were used: Raji and Ramos cells were obtained from the ATCC. The A6876 cell line, a gift from Parkash Gill (University of Southern California, Los Angeles, CA), Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 15 was established from a patient with non-Hodgkin’s lymphoma. Cells were regularly cultured in 100mm dishes (VWR International) in RPM11640 (Cellgro). All media were supplemented with 10% fetal bovine serum (qualified Australian sourced; GIBCO BRL), 100 U/ml Penicillin, and 0.1 mg/ml streptomycin (p/s; GIBCO). Cells were kept in a humidified atmosphere with 5% CO2 at 37°C. Glioblastomas were subcultured by trypsinization with 0.05% trypsin in 530 pM EDTA (GIBCO) twice per week. Lymphomas were collected, centrifuged at 1200 rpm for 3.5 min and resuspended with fresh media every 2-3 days. MTT Assay Lymphoma cells were seeded at 1.5x105 cells/ml, and glioblastoma cells were seeded at 3x104 cells/ml in 96 well plates. The next day, the various drugs were supplemented to yield the required concentrations. 48 hours thereafter, MTT dye was added, and the cells were incubated for another 4 hours before the reaction was terminated with solubilization solution. Cell proliferation was determined by measuring the optic density at 490 nm in an ELISA plate reader. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 16 Cell Lysis and Protein Quantification Cell were washed twice with phosphate buffered saline (PBS; Cellgro), scratched from the culture plate with a cell lifter, collected in 1 ml ice cold PBS, and centrifuged for 15 sec at 10k rpm. Pellets were lysed in 100- 500 pi RIPA detergent lysis buffer (Appendix C) containing 1 mM PMSF (Sigma) and one tablet of Complete Proteinase Inhibitor Cocktail (Roche) per 8 ml of lysis buffer for 10 min on ice and centrifuged for 15 min at 14k rpm at 4°C; supernatants were stored at -80°C. Protein concentration was determined by using bicinchoninic acid (BSA) assay kit (Pierce, Rockford, IL). Western Blot Equal amount of total protein (25-50 pl/sample) from cell lysates and a 1X final concentration of SDS sample buffer, were denatured for 5 min at 95°C and run on a 7-15% acrylamide mini gel with Laemmli electrophoresis buffer (Appendix C) for 1.5-2 hours at 100 V. Gels and nitrocellulose membrane (Amersham Pharmacia Biotech) were incubated in Bjerrum and Schajer-Nielsen Transfer buffer (Appendix C) for 15 min prior to a semi-dry electrophoretic transfer on a BioRad transfer apparatus for 45-75 min at 15 V. Gels were stained with Coomassie Blue and destained to confirm efficient transfer and equal loading. Membrane was blocked with Tris-buffered saline with 0.1% Tween 20 (TBST) 5% non-fat dry milk (Ralphs) for 2-3 hours at Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 17 room temperature. Membranes were incubated with the primary antibody (Appendix B), (1:500 or 1:1,000 in 5% TBST milk) over night at 4°C, washed three times with TBST, then incubated with the secondary antibody coupled to horseradish peroxidase (1:5,000 in 5% TBST milk) for 45 min at 4°C. After multiple washes, secondary antibodies were detected by chemiluminescence using a SuperSignal West pico/femto peroxide (Pierce, Rockford, IL) for 1 min and immediately exposed to CL Xposure films (Pierce) for 1, 5, and 10 min. A western blot was repeated at least once to confirm the results. In vitro Kinase Assays Cells were lysed in RIPA buffer (Appendix C), and protein concentrations were determined using the bicinchoninic acid (BSA) protein assay reagent (Pierce, Rockford, IL) .100 pg of each lysate was used for immunoprecipitation with 1 pg of anti-cyclin B or 200 pg for immunoprecipitation with anti-cdk2 antibodies (Appendix B). The antigen- antibody complexes were collected with proteinA-agarose, washed twice with RIPA and three times with kinase buffer (Appendix C). 25 pi kinase buffer contained 2 pg histone H1 protein, 50 pM ATP and 5 pCi [y-32P]ATP (3000 Ci mmol'1 ) was added, the mixture was incubated on a rocking platform at room temperature for 25 min. The reaction was stooped by adding 40 pi of 2x Laemmli sample buffer (Appendex C) then boiled for 8 min. the products Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 18 were separated on an 11% acrylamide gel and exposed to CL Xposure films (Pierce). The gel was stained with Coomassie blue and dried to confirm equal loading. All reactions were repeated at least once. Cesium Chloride Gradient Plasmid Preparation DH5 -a bacteria containing cycA-luciferase (luc), cycB-luc, 3200cdc2- luc, PP2A-luc, and CMV-luc (Appendix B) ampicillin resistant plasmids were grown in 1 ml LB Amp+ (50 ng/ml) medium for 4 h at 37°C. Midi cultures (200ml LB Amp+ ) were inoculated with 100 pi of the mini cultures and incubated at 37°C overnight. Bacteria culture was centrifuged for 10 min at 5K rpm and 4°C. Pallets were lysed in 20 ml lysozyme solution (2 mg/ml; Appendix C) for 1 hour on ice followed by 20 ml of 0.2 M NaAc/1 % SDS for 5 min on ice, and finally 1 hour on ice with the addition of 15 ml 3 M NaAc pH 4.8, lysates were centrifuged for 20 min at 10K rpm and 4°C. Supernatants were precipitated with 100 ml EtOH for 1 h at -20°C, centrifuged for 20 min at 10K rpm and 4°C; pellets were resuspended in 10 ml 0.1 M NaAc/ 50 mM Tris pH 8.0 and Precipitated once more with 50 ml EtOH at -20°C. Pallets were allowed to dry after centrifugation for 10 min at 10K rpm and 4°C, suspended in 9 ml TE (Appendex C) with Ethidium Bromide (0.2 mg/ml) and CsCI (1 mg/ml), centrifuged in sealed ultra-centrifuged tube (16x67 mm; Beckman) for 16 h at 55K rpm and RT using a Vti65 rotor in a Beckman Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 19 ultracentrifuge. Plasmeds were visualized under UV light, transferred to new centrifuge tube with a 20q1 needle and centrifuged in TE/EtBr/CsCI for 6 hours at 65K rpm and RT. The ethidium bromide was extracted from the isolated DNA by mixing repeatedly with 2x volume of isoamyl alcohol, centrifuged for 3 min at 1500 rpm and RT, the organic phase was discarded. The CsCI was removed by dialysis against 3x2 | TE for 48 h at 4°C. The DNA yield was determined by measuring the O.D. at 260 nm. The DNA (0.1- 0.5pg) was run on a 0.8% agarose gel with 0.5pg/ml EtBr. Transient Transfection By DNA/Calcium Phosphate Coprecipitation Cells (5 x 105 ) were seeded in a 60 mm cell culture dishes (Corning) a day prior to the experiment. 2 pg DNA Plasmid per plate was precipitated with 125 mM calcium phosphate in HEPES buffered saline (heBS; Appendex C) for 20 min under sterile conditions. For MOCK transfection 0 pg plasmid was added. Each transfection was preformed in triplicates and split to each treatment condition by adding 500 pi to 4.5 ml DMEM (USC core facilities; supplemented with 10% FBS and 1% P/S) there by every triplet treatment was comparable to the other treatments. The cells were then incubated for 16h at 37°C before the medium was changes back to the standard culture medium for 5-8h. After recovery the cells were treated with 70 pM celecoxib for 30h and then harvested, centrifuged for 15 sec at 10K rpm and lysed in Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 20 50 pi 5x reporter lysis buffer (RLB; Promega) in a single freeze-thaw cycle at -80°C. Supernatants were kept and tested for protein concentration by BSA kit (Pierce), protein amounts were equalized to 10-30 pg on 40 pi RLB and supplemented with 100 pi luciferase reagent (Pierce) immediately before measuring the luciferase activity in a luminometer on a 10sec interval following a 2 sec delay. Background activity was determent by MOCK transfection activity. Flow Cytometry Analysis 1x106 cells per sample where harvested by trypsinization, centrifugated for 5 min at 1200 rpm, washed in PBS and resuspended in 5 ml 70% cold ethanol for fixation, cells were stored in -20°C over night. The next day, cells were washed again and resuspended in 2 ml PBS, 40 pg/ ml DNAse- free RNAse A and 20 pg/ml proidium iodine were added, cells were incubated 30 min in 37°C and the proidium iodine incubation was on a FACS-scan analyzer. Subcutaneous Tumor Growth in Nude Mice Four to six-week old male athymic nude mice 20-30 grams (Harlan, Indianapolis, IN) were maintained in a pathogen-free environment. In the experiment with glioblastoma, U87 cells were grown in 100 mm tissue culture Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 21 dishes, the cells were trypsinized and resuspended in sterile PBS, centrifuged at 1200 rpm 5 min. the supernatant was aspirated, and the cells were resuspended in PBS and counted using a hemocytometer, 3 x 106per 100 p J cells were innoculated subcutaneously in the right frank of the animals, in the case of lympohoma cells, Animals were first irradiated with 300 centigray (cGY) with a MARK 11 3 7 Cs irradiator, for the depletion of mice natural killer cells, 4 days after radiation, 3 x 106 Raji lymphoma cell line that were grown in 100 mm tissue culture dishes, and collected by centrifugation at 1200 rpm 5 min, and then washed and resuspended in PBS. The cells were innoculated subcutaneously in the right frank. Upon tumor formation, animals were randomly divided into two groups: treated (1000 ppm in animal chow) and Control (normal animal chow). When tumor reached to 20 mm in diameter all animals were sacrificed. Animal Chow Preparation Powder form of the animal chow was obtained and mixed properly by with the drug, by continually adding small volumes to a mixing camber, at a concentration of 1000 ppm. Sterile water was added to the mix, creating dough like mixture, which was made into small cubes and dried in a vacuum oven. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 22 Chapter III RESULTS Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 23 Results The anti-proliferation effect of celecoxib Even though the majority of studies investigating the anti-proliferative effect of the various traditional NSAIDs and the selective COX-2 inhibitors have focused on colorectal cancer, more recent investigation have shown that COX inhibitors may exert this inhibitory effect in other cancers as well 2 4 ,29,66 Therefore, we set out to verify if these drugs could be considered in the treatment of other cancers,such as glioblastoma and lymphoma. We investigated four different glioblastoma cell lines: LN229, LN18, U87 and A172 and two Burkitt’s lymphoma cell lines Raji and Ramos and another cell line A6876 that was established from a patient with non-Hodgkin’s lymphoma. All glioblastoma cell lines tested had comparable COX-1 levels, but varying COX-2 levels (Figure 3.1A). U87 exhibited the highest levels of COX-2, while LN229 and LN18 displayed moderate levels, and A172 showed undetectable levels. Likewise, lymphoma cells had no detectable levels of COX-2 enzyme (Figure 3.1 B). In order to test the growth inhibitory effect of the various traditional NSAIDs and the selective COX-2 inhibitors, we incubated the cells with increasing concentrations of various drugs and determined cell proliferation with an MTT assay. We chose giloblastoma cell lines LN229 and A172 because they demonstrated different COX-2 levels. Both cell lines were Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 24 incubated with increasing concentration of celecoxib (a selective COX-2 inhibitor), or sulindac, flurobiprofen and indomethacin (traditional COX-1 and COX-2 inhibitors). As shown in figure 3.2 (top panels), celecoxib is clearly Fig 3.1: COX expression COX-1 levels. (A) Cell lysates from logarithmically growing COX-2 LN229, LN18, A172, and U87 cells were prepared PP2A and analyzed by Western blot with either COX-1 or COX-2 specific antibodies (as indicated). As a control for equal loading in each cox -2 lane, the membranes were also probed with an antibody ig G against the protein phosphatase type 2A (PP2A). (B) COX-2 protein was immunoprecipitated using a specific COX-2 antibody and analyzed by western blot, Ramos and Raji lymphoma cells. As a control various glioblastoma cells were analyzed as well. a much more potent inhibitor of cell proliferation than the rest of the traditional COX-2 inhibitors. Furthermore, celecoxib’s anti-proliferation effect was compared to other selective COX-2 inhibitors, valdecoxib, rofecoxib and NS-398, which proved much less effective than celecoxib (Figure 3.2 bottom panels). While celecoxib completely inhibited cell growth at 100 pM, the other drugs exhibited minimal effects at concentration up to 500pM. When lymphoma cells were incubated with increasing concentrations of various U8” LN 229 A 172 Raji Ramos LN229 LN18 A172 U87 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 25 drugs a similar picture emerged, celecoxib was clearly the most potent inhibitor (Figure 3.3). As shown in Figure 4.2B, the traditional m E I ” & 26 60 75 100 250 S O O 0 26 60 75 100 K » m E I 8 C**W^ 0 26 60 75 100 2SO 500 0 26 S O 76 100 250 600 Concentration of Drug [|iM] Fig 3.2: Ceil proliferation in the presence of NSAIDs in glioblastoma. Cell proliferation was analyzed with a commercially available MTT assay as recommended by the manufacturer. Cells were seeded at 3x104 cells/ml in 96 well plates. The various NSAIDs were supplemented to yield the respective final concentrations as indicated for 48 hours. Values shown are the mean (± SD) of at least six measurements. NSAIDs, sulindac, flurbiprofen, and indomethacin, were not very effective in inhibiting Raji cells. In addition, when we compared celecoxib to the other selective COX-2 inhibitors rofecoxib and valdecoxib, interestingly, they were less effective as well. Celecoxib completely inhibited the cells at a Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 26 concentration of 100 pM, whereas both refocoxib and valdicoxib had no effect at the same concentration. While at a significantly higher concentration of 500 pM both rofecoxib and valdecoxib inhibited the cells by only 50%, in comparison, celecoxib achieved the same effect at a concentration of 50 pM, i.e. approximately a 10-fold difference. Similar results were seen in Ramos and Raji cell line as well (Figure not shown). « « •0 2 L o 0 0 $ 0 Concentration of Colocoxlb Conc«n!ration of Various Drugs ||iM ] Fig 3.3: Cell proliferation in the presence of the various NSAIDs in lymphoma. Cell proliferation was analyzed by an MTT assay. Cells were seeded at 1.5x10s cells/ml in 96 well plates. A) The various lymphoma cells were treated with in increasing concentration of celecoxib. B) Raji cells treated with the various drugs were supplemented to yield the respective final concentrations as indicated. All treatments were for 48 hours. (± SD) of at least six measurements. These results were further confirmed by measuring the cytotoxicity which the various drugs induced. As shown in Figure 3.4A, celecoxib induces the highest level of cytotoxicity compared to the other NSAIDs (Figure 3.4B), where no major effect was seen, and the selective COX-2 inhibitors showed a 10 fold less inhibition than celecoxib. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 27 ■ R<rf*coxib ■ Vntdscoxlb a Indanothacin* □ Suilndsc 1200 - - 0 Celecoxib W H I* less# Concentration [fiM] Concentration jjtM] Fig 3.4: Cell cytotoxicity induction in response to NSAIDs treatment. Raji cells were cultured in the presence of increasing concentration of (A) celecoxib or (B) the various NSAIDs for 48 hours. Cell cytotoxicity was measured using a commercially available LDH ELISA kit. The inhibitory effects of celecoxib were further established by analyzing the cell cycle distribution. LN229 cells were treated with either 35 or 70 pM celecoxib. after treatment, the percentage of cells in the G1 phase of the cell cycle increased from 69% to 82% and 87%, respectively (Figure 3.5). tool < 5 * |is| |10 | n |j 0.0 35 70 Concentration of Celecoxib [#/M] Fig 3.5: Cell cycle distribution in the presence of celecoxib. Cell cycle distribution of LN229 cells was analyzed by fluorescence-activated cell sorting (FACS). The cells were treated with various concentrations of celecoxib as indicated for 36 hours. The cells were fixed and incubated with RNAse A and propidium iodine (PI). Cell cycle distribution was determined with a FACScan analyzer. The numbers shown are the percent of cells in the respective cell cycle phase. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 28 However, the percentage of cells in the S phase decreased from 13% to 8% and 4%, respectively, and G2/M phase cells decreased from 18% to 10% and 9%, respectively. In addition, Raji cells treated with 70 pM celecoxib, exhibited an increase in the percentage of cells in the Gi phase of the cell cycle increasing from 68% to 84.5% (Figure not shown). Conversely, the percentage of cells in the S phase decreased from 19.6% to 5.6%, and the G2/M phase decreased from 12.3% to 9.8%. The anti-proliferation effect of celecoxib is independent of COX-2 The surprising observation was that the selective COX-2 inhibitors exerted a different cell proliferation effect which suggests that they might undertake different inhibitory effects on the COX-2 enzyme and, thereby, on PGE2 production levels. To determine this, we measured the PGE2 levels in LN229 and A172 cells after treatment with the different drugs. As shown in figure 3.6, no significant differences were found between the various drugs. Treatment of LN229 with 75 pM of celecoxib, rofecoxib, or valdecoxib resulted in similar reduction of PGE2 levels 85% to 92%. In A172 cells, which harbor undetectable levels of COX-2, the PGE2 levels were low before any treatment. These results demonstrate an absence of correlation between the anti-proliferative effects of celecoxib and its inhibition of COX-2, since the rest of the inhibitors inhibited COX-2 similarly, but had no anti-proliferative effect. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 29 ■ u s b i ■Am Control C«lec<sx*b Rofc coxib Valdccoxib b jr t o Fig 3.6: Levels of endogenous PGE2. LN229 and A172 cells were cultured in the presence or absence of 75 pM of the indicated selective COX-2 inhibitors, for 24 hours. The concentration of PGE2 in the growth medium was determined by commercially available ELISA kit. Shown is the average PGE2 concentration (pg/ml) from four measurements from two independent experiments (± SD). Moreover, when LN229 and A172 (Figure 3.7A) and Raji (Figure 3.7B) cells were supplemented with various concentration of exogenous PGE2 , the anti-proliferative effects were unaffected Thus, these data suggest that the inhibitory effects of celecoxib might be independent of COX-2 inhibition, but rather mediated by other pathway(s). Celecoxib down-regulates cyclin dependent kinase activity. To gain an insight to the possible pathway that might underlie the anti proliferative effects of celecoxib, we investigated the activity of cyclin dependent kinases (CDKs), which represent the crucial regulator of cell proliferation control. First, we determined the enzymatic activity of cyclinB/cdkl complex, the key determinant of progression of cells through Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 30 G2/M phase of the cell cycle. Cells were treated with increasing concentration of the various NSAIDs or the selective COX-2 inhibitors, and the CDK activity was determined by an in-vitro kinase assay. As shown in figure 3.8, celecoxib clearly was the most potent inhibitor of CDK activity. § [|« M ] IM 3 E } [ m M J C iK w ak Tr**tm«nt 0.1 1.0 [jtM ] PG Ej )iM ] Celecoxib TmtwH Fig 3.7: The effect of exogenous PGE2 on cell proliferation. (A) LN229 and (B) Raji Cells were grown in a 96-well plate, treated with 0, 75, or 100 pM celecoxib in the presence or absence of 0.1 or 1.0 pM PGE2. Cell proliferation was determined by MTT assay Shown is the mean (±SD) from three different measurements. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 31 In LN229 cells, 70 pM celecoxib completely abolished CDK activity, while in A172 cells the activity was reduced nearly 80%. In comparison, all the other NSAIDs required higher concentrations. In LN229 cells, indomethacin inhibited CDK activity at a concentration of 300 pM and in A172 cells the LN229 A172 0 70 300 500 0 70 300 500 IliM ] Celecoxib « » •x#-3 * P-H1 100 7 <5 «5 100 21 <5 <5 % c.p.m Indomethacin * * * * * ■ 1 ■ H P-H1 100 84 23 6 100 104 98 97 % cp.m Flurbiprofen m m m m . ■ & > ^ *-3?P-H1 100 107 103 20 100 103 96 S O % c.p.m Sulindac - ♦ “ P -H t 100 168 134 51 100 117 115 92 % c.p.m Piroxicam — — - * . 3 *P-H 1 100 101 127 S3 100 106 100 97 % c.p.m Ketoprofen ■ — ■ * » 3 V -H 1 100 117 105 107 100 137 100 109 % c.p.m Fig 3.8: CDK activity in the presence of various NSAIDs. LN229 and A172 cells were cultured in the presence or absence of the indicated concentrations of the various NSAIDs specified on the left side of the panel. After 36 hours, cell lysates were prepared and analyzed for the enzymatic activity of cyclinB/cdkl complexes in vitro. The amount of radioactivity incorporated into the substrate histone H1 protein is reflected in each panel and is indicated by the arrow labeled 3 2 P-H1. Incorporated radioactivity was determined as counts per minute (c.p.m.). The relative amounts of kinase activity are shown below each panel as % c.p.m. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 32 same concentration reduced CDK activity by 77% while at 500 pM the CDK activity was reduced by 94%. Other NSAIDs requires even higher concentration. Flurobipofen reduced CDK activity by 80% in LN229 cells at a concentration of 500 pM but had no major effect in A172 cells (Figure 3.8). Furthermore, when kinase activity was compared between cells treated with the various selective COX-2 inhibitors, celecoxib again wiped out CDK activity at a concentration of 100 pM (Figure 3.9). Fig 3.9: CDK activity in the 3i presence of selective COX-2 + inhibitors. LN229 and A172 cells were cultured in the presence or absence of the selective COX-2 « < I3 2 p.hi inhibitors celecoxib, rofecoxib, or valdecoxib, at 100 pM each for 36 hours. As a control one set of cells *9 q received the same volume of DMSO. The arrow labeled 3 2 P-H1 indicates the amount of radioactivity H 1 incorporated into the substrate histone H1 protein. As a control, each gel was stained with Coomassie blue to verify that equal amounts of antibody (IgG) and substrate (H1) were recovered and loaded onto the gel. One such stained gel is shown at the bottom. Next, we investigated the effect of celecoxib on other CDKs, cyclinE/cdk2 and cycliA/cdk2 complex, the major determinant of late G1/S phase of the cell cycle. We used a specific antibody for cdk2 to immunopurify both complexes and to compare the results to the activity of the G2/M regulatory complex CyclinB/cdk1, we used an antibody for cyclinB to pull LN229 A172 < » — Coom, Blue Stain * Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 33 down the complex. We tested the four glioblastoma cell lines LN229, LN18, U87 and A172 and two lymphoma cell lines Raji and A6876. As shown in figure 3.10, in glioblastoma cells 70 pM celecoxib inhibited the activity of the CDK complexes. Celecoxib was somewhat more effective in LN229 and U87 a - c d k 2 a - c y c l m B l 0 7 2 0 7 0 C o 0 7 2 0 7 0 C O [p M ] C elecoxib L N 229 m m m m *k*.3 2 p - h i 100 89 77 7 99 100 112 99 13 125 % c pm L N 18 m m m m *<#-3 I P-H 1 100 112 127 1« 141 100 85 115 19 131 % c p m U -8 7 mmm m 1 0 0 m m » 83 100 102 07 8 97 % c p m A -172 m m ■ ' ■ too 1 0 1 8 8 28 1 1 2 100 97 » 24 102 % cpm . ^ ^ ^ • m 1 9 G Cooro, •i "Wii"- jnqpp" Blue Stein m h i Fig 3.10: CDK activity in the presence of celecoxib in glioblastoma. LN229, LN18, U87, and A172 cells were cultured in the presence or absence of the indicated concentrations of celecoxib for 36 hours. Cell lysates were prepared and immunoprecipitated with antibodies against either cdk2 (a-cdk2) or cyclin B (a-cyclin B1). The purified immunocomplexes were analyzed for associated kinase activity in vitro. As a control, each gel was stained with Coomassie blue to verify that equal amounts of antibody (IgG) and substrate (H1) were recovered and loaded onto the gel. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cells (approximately 90%) than in LN18 (roughly 82%) and in A172 (around 75%). Nonetheless, celecoxib was able to down-regulate the activity of the major G1/S and G2/M cell cycle regulatory kinases. Similarly, in lymphoma cells, celecoxib inhibited CDK activity in a concentration dependent manner, leading to a complete inhibition of the activity of CDK complexes at a concentration of 70 pM (Figure 3.11). 26 60 rs C h m h * tl>M c«leeoxlb) C < K 2 cyctn 6 Oximas** igS 8u« * Hi A6876 0 17 35 50 70 Co (MM] C«leeoxib ,JAH1 »p.Hl ig O a § = H I Fig 3.11 CDK activity in the presence of celecoxib in lymphoma. Raji and A6876 cells were cultured in the presence or absence of the indicated concentrations of the celecoxib, for 36 hours. Cell lysates were prepared and analyzed for the enzymatic activity of cyclinB/cdkl, cyclinA/cdk2, and cyclinE/cdk2 complexes in vitro. The amount of radioactivity incorporated into the substrate histone H1 protein is reflected in each panel and is indicated by the arrow labeled 3 2 P-H1. In addition, we investigated the inhibitory effect of celecoxib in lymphoma cells in a timely kinetic fashion in which they were incubated for different time points consecutively up to 60 hours at a concentration of 50 pM celecoxib. As shown in figure 3.12, CDK activity is down-regulated 36 hours after treatment in both Raji and A6876 lymphoma cells. We further investigated the mechanism underlying celecoxib’s inhibition of CDK activity by analyzing Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 35 the expression levels of various proteins that are involved in the execution and regulation of CDK activity. We examined cyclin A and cyclin B, the regulatory subunits of CDK s, cdkland cdk4, the catalytic subunits of CDK, and P21C ip 1 and P27K ip 1 , which are inhibitory proteins of CDK activity at a posttranslational level. C*c8 Coem #*** 8 H j « A6876 0 50 0 60 3 8 16 24 36 60 cyclin 8 Raji 0 s o 0 6 0 8 > 6 2 4 3 8 4 8 8 0 [pM} Celecoxib m «m~ 32P-H1 (l)M[ C*l»«omb (li| li«atn<«fli — — I9 © mi Fig 3.12: CDK activity in a timely dependent manner in the presence of celecoxib. Raji and A6876 cells were treated with 50 pM celecoxib, in a timely kinetic manner. Cell lysates were prepared and analyzed Cell lysates were prepared and analyzed for the enzymatic activity of cyclinB/cdkl complex in-vitro. The amount of radioactivity incorporated into the substrate histone H1 protein is reflected in each panel and is indicated by the arrow labeled 3 2 P-H1. As a control, each gel was stained with Coomassie blue to verify that equal amounts of antibody (IgG) and substrate (H1) were recovered and loaded onto the gel. One such stained gel is shown at the bottom. As shown in figure 3.13, celecoxib, at a concentration of 70 pM down- regulated cyclin A in both LN229 and A172. Cyclin B and cdk2 were down- Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 36 regulated in LN229, but less pronounced in A172 and with regards to CDK inhibitory proteins, there was no major change in the levels of either one. With respect to A6876 lymphoma cells, as shown in figure 3.14, celecoxib down-regulated both cyclin A and cyclin B in a concentration dependant [pM] Celecoxib cycA cycB cdki p27 p21 Erk1 Fig 3.13: Levels of cell cycle-regulatory proteins in the presence of celecoxib. LN229 and A172 cells were cultured in the presence of the indicated concentrations of celecoxib for 36 hours. In parallel, some of the cell cultures received only DMSO (indicated as Co). Equal amounts of cell lysates were analyzed by Western blot analysis with antibodies against the various proteins specified. As a loading control, some of the blots were probed with an antibody against extracellular signal-regulated kinase 1 (Erk1). manner whereas no major change occurred on the levels of cdk4 up to a concentration of 70 pM celecoxib . With regards to both p21C ip 1 and p27K ip1, there was no major change in the expression of p21C ip1, whereas for p27K ip 1 , there was a slight increase in its levels at a concentration of 17 pM that did not change with the increase in celecoxib’s concentration. In Raji cells, celecoxib inhibited cyclin A and cyclin B more potently. There was a major LN229 Celts A-172 Cells 0 7 20 70 Co 0 7 20 70 Co 1 t 1 1 . — m m M il — > • ..w in — ^RHRP PUMP m mH P - mm mm mm* 4^||||p PM tM P Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 37 drop in the levels of both proteins at a concentration of 50 pM. On the other hand, p27K ip 1 was significantly up-regulated with the increasing concentration, and peaking at 50 pM celecoxib, no detectable levels of p21C ip 1 were observed. A6876 « 0 K > * 5 0 ® - 0 25 50 75 100 Co [m M ] cycA C ycA cycB C ycB edk4 p27 m p21 m R a ji 0 2 $ s o 7 S B U S O — *—*' — — - — 1 1 1 ’ pM C « !»M X lb Fig 4.14: Levels of cell cycle-regulatory proteins in the presence of celecoxib. A6876 and Raji cells were cultured in the presence of the indicated concentrations of celecoxib for 36 hours. In parallel, some of the cell cultures received only DMSO (indicated as Co). Equal amounts of cell lysates were analyzed by Western blot analysis with antibodies against the various proteins specified. Celecoxib down-regulates promoter activity in glioblastoma. The reduction in protein levels of the various CDK subunits due to the treatment of cells with celecoxib might be executed by several different mechanisms. Since no stimulation in protein levels was observed for the CDK inhibitors P21C ip 1 and P27K ip1, this excludes posttranslational inhibition of CDK activity, but rather indicate that the regulation might be at the level of expression of the receptive CDK subunits. In order to determine this, we analyzed the activity of the respective gene-regulatory elements by Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 38 transiently transfecting luciferase reporter constructs under the control of either the cyclin A promoter, the cyclin B promoter, or the cdkl promoter and determined the activity in the presence and absence of celecoxib. As shown in figure 3.15, the activities of all the three promoters were significantly down- regulated in response to celecoxib. To ensure that celecoxib’s effect was 200 cdkl cyclinA cyclinB PP2A CMV Fig 3.15: Promoter activity in the presence of celecoxib. LN229 cells were transiently transfected with various promoter-luciferase constructs and cultured further in the presence or absence of 70 pM celecoxib for 30 hours. The cells were harvested and equal amounts of total cellular protein were analyzed for luciferase activity. Shown is the mean (± SD) from at least five individual transfections. The luciferase activity in the absence of celecoxib was set to 100%. One-way ANOVA between control and celecoxibtreated cells: one asterisk (*) p=0.000; two asterisks (**) p=0.002. NS, not significant. specific we used 2 controls which are: PP2A-luc, which contain the promoter taken from the gene encoding the catalytic subunit of protein phosphatase type 2A, and CMV-luc, a strong viral promoter taken from cytomegalovirus (CMV). Neither of the two promoters was inhibited by celecoxib (Figure 3.15), but rather celecoxib stimulated PP2A promoter. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Thus, our data demonstrate the inhibitor effect of celecoxib was not due to general toxic effect of the drug, but rather to a targeted effect on a specific signal transduction pathways(s) resulting in the down-regulation of cyclin A and cyclin B and, thereby, inhibiting cellular proliferation. Inhibition of tumor growth by celecoxib in nude mice. In order to investigate the inhibitory effects of the selective COX-2 inhibitor invivo, we implanted tumor cells subcutaneously in the right paws of male nude mice. The mice were randomly distributed into 2 groups. One group received celecoxib supplemented in the diet at a concentration of 1000 ppm, the other group received a normal diet. In the glioblastoma group, U87 cells were implanted and one week later, the mice received the special diet. As shown in figure 3.16A the growth of U87 cells in animals that received celecoxib in their diet was significantly reduced when compared with animals on the control diet. The experiment was terminated at day 50 after implantation. There was an approximately two-fold difference between the treated and untreated group. In the lymphoma group, two experiments were conducted: in the first experiment, the animals received the treated diet immediately after implantation (Figure 3.16B), whereas, in the second experiment, treatment of the mice began after three weeks of implantation, i.e., after the appearance of palpable tumors (Figure 3.16C). A statistically significant difference (p<0.05) between the control and treated group became Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Tumor vofum# i n m m * 40 H 0 c *I# tO X < fe £ £ E .3 o > o B £ A t SO 0 30 32 40 *13 45 Day* *f*«r Tow tu * S 3 t u it « U y % <n»i tm yUM M ton n it 3 3 *3 3 5 3 ? 4 1 4 3 4 * y v «n»« lu n w t in t|itin t* U « rt Fig 3.16: Tumor growth in mice treated with celecoxib. (A) 10s U87 cells were implanted subcutaneously in to 10 nude male mice. The mice were randomly distributed into two groups. A group of five animals received celecoxib in their diet (at a concentration of 1000 ppm), the other group received normal diet. Beginning on day 30, the tumor size in each animal was measured with a caliper every two to three days. Shown is the average of each group. The difference between the two groups became statistically significant starting day 43 (p<0.05). (B), (C) 2.5x10s Raji cells were implanted subcutaneously in to 12 nude mail mice per experiment. The mice were randomly distributed into two groups. One group received celecoxib in their diet (at a concentration of 1000 ppm), the other group received normal diet. The tumor size in each animal was measured with a caliper every two to three days. Shown is the average of each group. (B) Treatment of the animals started immediately after implantation, a statistically significant difference (p<0.05) between the two groups became apparent on day 24 and after. (C) Treatment of the animals began after three weeks if implantation, the difference between the two groups became statistically significant starting day 34 (p<0.05). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 41 apparent in the first experiment after day 24,though it took till day 34 in the second experiment. Nevertheless, it is clear that celecoxib reduced the growth of the primary tumor. DMC, a derivative of celecoxib DMC (4-[-5-(2,5-dimethylphenyl)-3(trifluoromethyl)-1 H-pyrazol-1 -yl]benzene- sulfonamide) is a new drug which has been developed on the platform of celecoxib. It maintains celecoxib’s anti-proliferative effect, but does not Chemical Structure Drug COX-2 Inhibition proliferation Inhibition Q SO.**. Celecoxib + sofmt, DMC + Refecoxib - Table 3.1: Comparison between COX-2 inhibitors and celecoxib’s derivative DMC. Shown, is the different structure and activity of refecoxib and celecoxib the selective COX-2 inhibitor, and DMC, a derivative of celecoxib that have maintained its anti proliferative effect, but does not inhibit COX-2 enzyme. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 42 inhibit COX-2 enzyme 52. DMC contains one methyl group more than celecoxib on one of its rings (Table 3.1). To further investigate the contribution of COX-2 inhibition to the antiproliferative effect of celecoxib we compared DMC to celecoxib. Two lymphoma cell lines were incubated with increasing concentration of celecoxib, DMC, and two of the selective COX-2 inhibitors, valdcoxib and rofecoxib. Cell proliferation was measured using an MTT assay. As shown in figure 3.17, both celecoxib and DMC o < n o o OMC .... 0MC 0 25 SO 75 100 250 500 0 25 50 75 100 250 500 Drug Treatm ent [|iM] Drug Treatm ent [|iM] Fig 3.17: Cell Proliferation in the presence of the Various selective Cox-2 inhibitors plus DMC. Cell proliferation was analyzed by an M TT assay. Raji and Ramos Cells were seeded at 1.5x105 cells/ml in 96 well plates, treated with in increasing concentration of the various drugs as indicated. All treatments were for 48 hours. (± SD) of at least six measurements. exhibited similar antiproliferative effect, at a concentration of 100 pM the cells were completely inhibited. In comparison, the other COX-2 inhibitors did not Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 43 demonstrate any inhibitory effect at an equal concentration. A significantly higher concentration was required of rofecoxib and valdecoxib to inhibit the cell growth. Next, we analyzed the cyclin dependent kinase activity of celecoxib and DMC, Raji cells were treated in a concentration dependent manner with both drugs, and kinase activity was determined after 48 hours. As shown in figure 3.18, both drugs essentially yield comparable results, although it appears that DMC is slightly more potent than celecoxib. At a concentration of 50 pM, CDK activity was significantly down-regulated in both drugs and at a concentration of 75 pM DMC, no kinase activity was detected in DMC treated cells whereas when compared to celecoxib treated cells, were a minor kinase activity was seen. Raj I m o x so n c m so |pMj C»*»c<w* e x » n m & K d i J d * * " f d ^ C *8 m ....... m m i m m m m m m m 10 0 ____ w. « *- m Cow** ...... ... ............................... .... SttA • ’“i t m w r m - HI * — w i p m - J#s H! Fig 3.18: CDK activity in the presence of celecoxib and DMC. Raji cells were cultured in the presence or absence of the indicated concentrations of the celecoxib and DMC, for 36 hours. Cell lysates were prepared and analyzed for the enzymatic activity of cyclinB/cdkl, in vitro. The amount of radioactivity incorporated into the substrate histone H1 protein is reflected in each panel and is indicated by the arrow labeled 3 2 P- H1. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 44 These results were further demonstrated by analyzing the protein levels of various regulators and inhibitors of CDK activity. As shown in figure 3.19, cycin A and cyclin B expression levels were down-regulated in an increasing manner as the concentration of DMC increased. However, no change was observed in the levels of cdk2, the catalytic subunit of the CDK. With regards to p21C ip 1 and p27K ip1, the inhibitors of CDK, a significant increase was observed in the levels of p27K ip1, whereas, for p21C ip 1 very low levels were detected. This data compares to the results seen with Raji cells when treated with celecoxib (Figure 3.14). Thus, all responses that were investigated were comparable in response to either drugand we suspect that both drugs impinge on the same signal transduction pathway(s) to exert their anti-proliferative effect, independent of COX-2. Raj) Fig 3.19: Levels of cell cycle- tuM] DMC regulatory proteins in the 0 25 50 dm so CVCB - P27 CycA mm — presence of DMC. Raji cells were cultured in the presence of the indicated concentrations of DMC for 36 hours. In parallel, some of the cell cultures received only DMSO. Equal amounts of cell lysates were analyzed by Western blot analysis with antibodies against the various proteins specified. P21 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 45 Chapter IV DISCUSSION Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 46 Discussion Some of the traditional non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin, ibuprofen, etc., are known to reduce the incidence of colon cancer. They exert their effect by inhibiting both cyclooxygenase-1 (COX-1) and COX-2. However, due to their inhibition of COX-1 enzyme, which is a housekeeping protein, these drugs cause significant side effects in patients, such as erosion and ulceration of the gastric mucosa. Therefore, new drugs were developed which selectively target COX-2 enzyme. These new drugs, such as celecoxib, rofecoxib, or valdecoxib appear to offer the therapeutic benefit of traditional NSAIDs with less of the associated side effects 1 5 . Nonetheless, very little is known about the possible usefulness of these drugs in the therapy of other cancers. In this study, we investigated whether celecoxib and other COX inhibitors would be effective in the treatment and management of glioblastoma, an advanced stage brain tumor with poor prognosis for long-term survival. COX-2 and its reaction products, prostaglandins, are commonly found in elevated levels in gliom as8'3 2 '35 '62. Further more, in clinically more aggressive tumors such as glioblastoma multiforme, COX-2 is highly expressed and is an indicator of poor survival 61. In addition, the efficiency of these drugs was tested in lymphoma, where patients with intracranial lymphomas in particular are in desperate need of improved treatments because of their poor prognosis. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 47 Various glioblastoma and lymphoma cell lines were cultured in the presence of increasing concentration of different traditional NSAIDs, as well as selective COX-2 inhibitors. Amongst all the drugs tested celecoxib was the only drug which inhibited cell proliferation at a concentration lower that 100 pM. The rest of the drugs required a considerably higher concentration, even though in glioblastoma all the selective COX-2 inhibitors inhibited the production of PGE2 in a similar manner to celecoxib (Figure 3.6). Further more, we observed that cells with undetectable levels of COX-2 protein, such as A172 and all of the lymphoma cell lines (Figure 3.1), were inhibited by celecoxib in the same manner as other cells with high levels of COX-2. Also, we observed a miscorrelation between the decline of PGE2 levels (Figure 3.6) and the inhibitory effect of the various selective COX-2 inhibitors (Figures 3.8, 3.9). Finally, the addition of exogenous PGE2 to celecoxib treated cells could not protect the cells from celecoxib’s anti-proliferative effect. Thus, these findings strongly suggest that the inhibitory effect of celecoxib in cell growth is independent of its inhibition of the COX-2 enzyme. Other findings have been reported in different cell types that NSAIDs might act independently of COX-2 enzyme 5'22’2 5’31'7 5 '83. Clearly celecoxib is a much more potent inhibitor of glioblastoma and lymphoma cell proliferation than any of the traditional NSAIDs tested. These results correlate with the others investigating lung, breast, ovarian, and prostate carcinoma cells and showed that celecoxib was a much more potent Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 48 inhibitor than indomethacin 33. In studies that investigated flurbiprofen, acetaminophen, and ibuprofen on glioblastoma cell lines, high concentrations of these drugs had to be used to observe the growth inhibitory effects 7 39. Several selective COX-2 inhibitors are available, but with regards to their antiproleferation effect in vitro, they too are not as effective as celecoxib. In comparison none of the other drugs inhibited cell proliferation (Figures 3.2, 3.3), or the enzymatic activity of CDK (Figure 3.9), as effectively as celecoxib. These observations are consistent with others who showed an increased growth inhibitory efficacy of celecoxib in comparison to other selective COX-2 inhibitors in a variety of cancer cells 33'3 4 '37'80. in addition, Joki et a l.3 2 demonstrated using two different glioblastoma cell lines that 100 pM of NS-398, a selective COX-2 inhibitor, inhibited the cells by approximately 50% after four days, whereas, in our experiments using the same concentration of celecoxib, we observed growth inhibition over 95% after two days (Figure 3.2). In addition, the inhibitory effect of celecoxib does not seem to be restricted to tumor cells, since it is found to inhibit rheumatoid synovial fibroblasts 1 8 . It is unclear how and why celecoxib exerts more potent antiproliferative effects than the rest of the COX inhibitors. Therefore, to better understand the molecular processes that underlie its effect, we investigated the effects of celecoxib on the cell cycle machinery. Celecoxib was found to down-regulate cyclin dependant kinase (CDK) activity (Figures Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 49 3.10, 3.11). Considering that CDKs is the “cell cycle engine” which is required for the cell to under go proliferation 3 7 ,8 °, its inhibition by celecoxib is responsible for the cells’ growth arrest. The absence of cyclin A and cyclin B proteins (Figures 3.13, 3.14), which are the regulatory subunits CDK complexes, is a reason for the observed down-regulation of CDK activity. Further more, we show that the lack of expression of both proteins in glioblastoma is due to the transcription inhibition of the respective promoter (Figure 3.15). Thus, it seem that celecoxib treatment of glioblastoma cells results in the transcriptional shut-down of both cyclin A and cyclin B promoter activity, leading to the loss of CDK activity, and later a block in cell growth and proliferation. With regard to the common CDK inhibitors p21C ip 1 and p27K ip1, we observed a cell specific inhibitory mechanism in which celecoxib exerts is effect. In Raji cells, p21C ip 1 was undetectable, whereas p27K ip 1 was significantly elevated with the increasing concentration of celecoxib. Conversely, in A6876 and glioblastoma cells we did not observe any increase in the levels of p21C ip 1 and p27K ip 1 , although, in A6876 there seemed to be a small increase in p27K ip 1 levels at a low concentration, which does not change with the increase in celecoxib. This specificity is also observed in different cell types, it has been shown that the expression of both inhibitors has increased due to celecoxib treatment in colon cancer cell lines 22. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 50 The mechanism in which celecoxib exerts its effects on cyclin transcription is currently unclear and remains to be determined. It is possible that the drug targets signal transduction pathway(s) that affects the promoter region of the respective genes. Such as N F -k B, Stat3, and subtypes of peroxisome proliferator-activated receptor (PPAR) 3 8 - 47'4 8 77'7 9 82. In order to test celecoxib’s inhibitory effects in vivo, several experiments were conducted, in which the drug was interdicted in different stages of the tumor progression. The data from these experiments confirmed the efficacy of the COX-2 inhibitor in reducing the growth of the primary tumor. The drug was more effective when the treatment started immediately after implantation. Our study did not address the issue of mechanism by which celecoxib attenuates tumor growth in vivo. A number of studies have reported that stromally derived COX-2 is important for tumor growth 71. Angiogenesis, an important component of neoplastic growth, is also promoted by COX-2 activity, and its inhibition could play a curtail role in the antineoplastic action of celecoxib 42- 71'73. This may explain the different results obtained from both experiments, by treating the animals immediately after implantation celecoxib may interfere with the neovascularization, therefore, reducing tumor growth. However, this remains to be established. In addition, we investigated the role of COX-2 enzyme’s inhibition in celecoxib’s antiproliferative effect. Even though no detectable levels of COX- 2 enzyme were observed in lymphoma cells, the addition of purified PGE2to Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 51 treated cells could not over come the inhibitory effect of celecoxib (Figure 3.7). Furthermore, we examined the antiproleferative effect of DMC, a derivative of celecoxib that does not exhibit COX-2-inhibitory function 52. When compared to the other COX-2 inhibitors, DMC exhibited a strikingly similar resemblance to celecoxib, in which both drugs inhibited cell proliferation in a comparable fashion (Figure 3.17). Likewise, when CDK activity and cell cycle proteins were analyzed, similar results were obtained (Figures 3.18, 3.19). Both celecoxib and DMC induced reasonably identical results, although, DMC seemed to be slightly more potent. Because all responses that were investigated were comparable in response to either drug, we suspect that both drugs impinge on the same signal transduction pathway(s) to exert their anti-proliferative effect. Thus, these findings further suggest that celecoxib’s inhibitory effect on cell growth is independent of its inhibition of the COX-2 enzyme. Other findings have been reported in different cell types that NSAIDs might act independently of COX-2 enzyme 5,9,22,25,76,65 In brief, our data has demonstrated that in both lymphoma and glioblastoma cells, the selective COX-2 inhibitor celecoxib inhibit cell growth and proliferation independently of COX-2 protein. Therefore, it may be beneficial to further investigate both celecoxib and its derivative DMC in order to examine the potential benefits of these drugs in the management of these diseases. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 52 References 1. Abrey, L.E., L.M. DeAngelis, and J. Yahalom, Long-term survival in primary CNS lymphoma. J Clin Oncol, 1998. 16(3): p. 859-63. 2. American Cancer Society. Cancer facts and .gures, 2004. 3. Baron, J. A., Cole, B. F., Sandler, R. S., Haile, R. W., Ahnen, D., Bresalier, R., McKeown-Eyssen, G., Summers, R. W., Rothstein, R., Burke, C. A., Snover, D. C., Church, T. 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J Biol Chem 1999; 274:27307-14. 80.Yamazaki R, Kusunoki N, Matsuzaki T, Hashimoto S Kawai S Selective cyclooxygenase-2 inhibitors show a differential ability to inhibit proliferation and induce apoptosis of colon adenocarcinoma cells. FEBS Lett 2002; 531:278-84. 81. Yin MJ, Yamamoto Y Gaynor RB The anti-inflammatory agents aspirin and salicylate inhibit the activity of l(kappa)B kinase-beta. Nature 1998; 396:77-80. 82.Zhang L, Yu J, Park BH, Kinzler KW Vogelstein B Role of BAX in the apoptotic response to anticancer agents. Science 2000; 290:989-92. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 60 83.Zhang X, Morham SG, Langenbach R Young DA Malignant transformation and antineoplastic actions of nonsteroidal antiinflammatory drugs (NSAIDs) on cyclooxygenase-null embryo fibroblasts. J Exp Med 1999; 190:451-59. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 61 Appendix A: List of Companies Company name Amersham Pharmacia Biotech Becton Dickinson Labware (Falcon) BioRad Laboratories Cellgro Corning Inc Gibco BRL Molecular Probe NeoMarkers Inc Osmonics Pierce Promega Roche Santa Cruz Biotechnology Inc Sigma VWR Scientific Inc Zymed Laboratories Location Buckinghamshire, England Franklin Lakes,NJ Hercules, CA Herndon, VA Corning, NY Carlsbad, CA Eugene, OR Fremont, CA Kent, WA Rockford, IL Madison, Wl Indianapolis, IN Santa Cruz, CA St. Louis, MO West Chester, PA San Francisco, CA Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Appendix B: Antibodies and Plasmids 62 Antibodies Anti-human antigens Ig Isotype Company Cdk1 (C-20) Rabbit Polyclonal IgG Santa Cruz BT Cdk2 (H-298) Rabbit Polyclonal IgG Santa Cruz BT Cdk4 (H-22) Rabbit Polyclonal IgG Santa Cruz BT COX-1 (C-20) Rabbit Polyclonal IgG Santa Cruz BT COX-2 (C-20) Rabbit Polyclonal IgG Santa Cruz BT Cyclin A(C-19) Rabbit Polyclonal IgG Santa Cruz BT Cyclin B1 (H-433) Rabbit Polyclonal IgG Santa Cruz BT Erk1 (K-23) Rabbit Polyclonal IgG anta Cruz BT P21 (C-19) Rabbit Polyclonal IgG Santa Cruz BT P27 (C-19) Rabbit Polyclonal IgG Santa Cruz BT PP2A (C-20) Rabbit Polyclonal IgG Santa Cruz BT Plasmids • CycA-luc: Contains the promoter of cyclin A gene directing expression of the luciferase (luc) reporter gene. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. • CycB-luc: Contains the promoter of cyclin B gene directding expression of the luciferase (luc) reporter gene. • 3200cdc2-luc: Contains 3200 bp of the promoter of cdc2 (Cdk1) gene directs expression of the luciferase (luc) reporter gene. • PP2A-luc: Contains the promoter of protein phosphatase type 2A catalytic subunit gene expression of the luciferase (luc) reporter gene. • CMV-luc: Contains the promoter of cytomegalovirus expression of the luciferase (luc) reporter gene. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 64 Appendix C: Buffers and Solutions Cell Lysis RIPA Detergent Lysis Buffer 150 mM Sodium Chloride 1.0% NP-40 0.5% Sodium deoxycholate 0.1%SDS 50 mM Phenylmethylsulfonyl fluoride 1 tablet of Complete Proteinase Inhibitor Cocktail (Roche) per 8 ml SDS PAGE 5X SDS Sample Buffer 60 mM Tris HCI pH 6.8 25% Glycerol 2% SDS 14.4 MM 2-Mercaptoethanol 0.1%Bromphenol blue Electrophoresis Buffer 25 mM Tris 192 mM Glycine 0.1% SDS Coomassie Gel Stain 1 mg/ml Coomassie Blue R-250 45% Methanol 10% Glacial acetic acid Coomassie Gel Destain 10% Methanol 10% Glacial acetic acid Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 65 Semidry Transfer Bjerrum Schaefer-Nielsen Transfer Buffer 48 mM Tris 39 mM Glycine 20% Methanol Western Blot Tris Buffered Saline with Tween20 (TBST) 10 mM Tris-HCI Ph 7.5 150 mM NaCI 0.1% Tween20 Plasmid Preperation LB medium 1 % Bacto trypton 1% NaCI 0.5% Yeast Extract pH adjusted to 7.5 50 pg/ml Ampicillin Lysozyme Solution 2 mg/ml Lysozyme 50 mM Glucose 10 mM EDTA 25 mM Tris pH 8.0 TE 50 mM Tris-CI 1 mM EDTA Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 6 6 Transfection 2X HEPES buffered saline (HeBS) 280 mM Sodium Chloride 50 mM HEPES acid 1.5 mM Sodium Phosphate pH adjusted to 7.05 filter sterilized Calcium Chloride 2.5 M Calcium Chloride filter sterilized Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

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Asset Metadata

Creator Kardosh, Adel (author)

Core Title The effect of cyclooxygenase inhibitors on cell cycle regulation and proliferation of glioblastoma and lymphoma cell lines

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, cell,health sciences, oncology,Health Sciences, Pharmacology,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-321795

Unique identifier UC11337550

Identifier 1424246.pdf (filename),usctheses-c16-321795 (legacy record id)

Legacy Identifier 1424246.pdf

Dmrecord 321795

Document Type Thesis

Rights Kardosh, Adel

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA