changes in protein profiles in bortezomib applied multiple myeloma ...

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ABSTRACT CHANGES IN PROTEIN PROFILES IN BORTEZOMIB APPLIED MULTIPLE MYELOMA CELLS Multiple Myeloma is a malignant B-cell neoplasm that is characterized by the accumulation of malignant plasma cells in the bone marrow. Over the recent years, several novel agents have been introduced in the treatment of this disease. Bortezomib is the first of a new class of agents known as proteasome inhibitors. The main objective of the project was basically to both determine the cytotoxic and apoptotic effects of Bortezomib on Multiple Myeloma U-266 cells and compare and explore the differences between Bortezomib applied Multiple Myeloma cells and control group Multiple Myeloma cells, by proteomics studies. In order to achieve our aims in the project, variety of multidisciplinary subjects were come together. Cancer research techniques, biochemical studies at protein level and proteomics were combined in our studies. In this study, our experimental results demonstrated that Bortezomib has antiproliferative and apoptotic effects on MM U-266 cells. On the other hand, the responsible proteins for the effect mechanism of anti-cancer agent on cells were determined by MALDI-TOF-TOF Mass Spectrometry for the first time. According to the mass spectrometric analysis, 37 protein spots were differentially expressed. Among them, five proteins were newly formed, ten proteins lost, twelve proteins were up- regulated and ten proteins were down-regulated as compared to control group (untreated cells).These differential expressed proteins in response to Bortezomib have different important functions ranging from cell signaling transduction, cell cycle regulation, apoptosis to immunity and defense mechanism. In conclusion, it was identified which proteins have a central role behind the effect of Bortezomib on MM U-266 cells. The identified proteins may let to be possible to treat other cancer types by same anticancer agent. The data obtained by this study may also be helpful for medical schools and drug designers and may also provide new treatments. iv
ÖZET BORTEZOMĠB UYGULANAN MULTĠPL MĠYELOMA HÜCRELERĠNĠN PROTEĠN PROFĠLLERĠNDEKĠ DEĞĠġĠKLĠKLER Multilpl Miyelom, bağıĢıklık sisteminin bir parçası olan B hücrelerinin, hasarlı olarak kemik iliğinde çoğalması ve birikmesiyle oluĢan hematolojik bir kanserdir. Son yıllarda, bu hastalığın tedavisi için birçok yeni kimyasal ajan tıp dünyasınin hizmetine sunulmuĢtur. Bortezomib, Multipl Miyelom tedavisinde kullanılan ve tedavi sırasında proteazom inhibitörü olarak görev yaptığı bilinen bir anti-kanser ajandır. Projenin esas amacı, hem Bortezomibin Multipl Miyeloma hücreleri üzerindeki sitotoksik ve apoptotik etkilerini belirlemek hemde Bortezomib uygulanan bu hücreler ile control grubu hücreleri arasındaki farklılıkları kıyaslamak ve keĢfedebilmekti. Bu projede, hedeflerimize ulaĢmak için, çeĢitli interdisipliner konular biraraya getirilmiĢtir. Bu hedef doğrultusunda, kanser araĢtırma teknikleri, protein bazlı biyokimyasal çalıĢmalar ve proteomik yaklaĢımlar kombine edilerek kullanılmıĢtır. Bu çalıĢmadaki deneysel sonuçlarımız, Bortezomibin, MM U-266 hücreleri üzerinde antiproliferatif ve apoptotic etkilere sahip olduğunu gösterdi. Öte yandan, bu anti-kanser ajanın hücreler üzerindeki etki mekanizmasında görev alan proteinler MALDI-TOF-TOF Kütle Spektrometresi ile ilk defa belirlenmiĢtir. Kütle spektrometresi sonuçlarına gore, otuz yedi protein spotunda farklılık gözlenmiĢtir. Bunlardan beĢ spot yeni oluĢan ve on spot kaybolan proteinlere karĢılık gelmiĢtir. Ayrıca, on iki spotun seviyesinde artıĢlar gözlenirken, on ayrı spotun da ekspirasyon seviyelerinde azalma gözlenmiĢtir. Bu proteinler hücre içerisinde, sinyal iletiminden, hücre bölünmesine, apoptozdan vücudun savunma mekanizmasına kadar değiĢen önemli fonksiyonlara sahipler. Sonuç olarak Bortezomibin MM U-266 hücreleri üzerindeki etki mekanizmasında hayati öneme sahip proteinler tanımlanmıĢtır. Farklılık gösteren bu proteinlerin ve fonksiyonlarının belirlenmesi sayesinde aynı kemoterapatik ajan ile aynı proteinlerin rol aldığı diğer kanser türlerinin de tedavi edilebilirliği mümkün olabilir. Ayrıca buradan elde edilen bulgular, yeni tedavi yöntemleri konusunda tıp fakültelerine ve ilaç dizaynı üzerine çalıĢan araĢtırmacılara yeni ufuklar açabilir. v
Page 1 and 2: CHANGES IN PROTEIN PROFILES IN BORT
Page 3: ACKNOWLEDGEMENTS First of all, with
Page 7 and 8: 2.1.4.1.2. Two-Dimensional Gel Elec
Page 9 and 10: LIST OF FIGURES Figure Page Figure
Page 11 and 12: LIST OF TABLES Table Page Table 1.1
Page 13 and 14: When normal cells in the body begin
Page 15 and 16: Figure 1.2. Development of Cancer S
Page 17 and 18: the myeloma cells does not help fig
Page 19 and 20: A rare form of MM called Nonsecreto
Page 21 and 22: 1.2.3. Risk Factors for MM A risk f
Page 23 and 24: of the mineralizations by adjusting
Page 25 and 26: To measure the levels of red cells,
Page 27 and 28: The levels of blood urea nitrogen (
Page 29 and 30: By using the system introduced by D
Page 31 and 32: 1.2.7.2. Biology of The Proteasome
Page 33 and 34: Figure 1.10. Ubiquitin-Proteasome P
Page 35 and 36: Figure 1.12. Effect of Bortezomib o
Page 37 and 38: encompasses the investigation of pr
Page 39 and 40: of modification. Ubiquitin is a sma
Page 41 and 42: the given cells. On the other hand,
Page 43 and 44: is-acrylamide (N,N'-methylene-bisac
Page 45 and 46: 2.1.4.2. Detection of Protein Spots
Page 47 and 48: ange of 600-2500 Da. This is the id
Page 49 and 50: ions resolved by the mass analyzer
Page 51 and 52: 2.1.4.5.2. Mass Analyzers Figure 2.
Page 53 and 54: There is no doubt that different ma
Page 55 and 56:
mask low-quantity proteins that may
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3.1. Materials 3.1.1. Medias CHAPTE
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3.2.5. Cell Proliferation Assay Ant
Page 61 and 62:
After 72 hour incubation period at
Page 63 and 64:
eceptors of the Tumor Necrosis Fact
Page 65 and 66:
The preparation of stock BSA Standa
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3.2.6.3. Detection of the Loss of M
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centrifugation, the supernatant was
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selected symmetrically to load the
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approximately 35 ml, the volume of
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After electrophoresis was finished,
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7 Day Two: Reduction, Alkylation, a
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CHAPTER 4 RESULTS AND DISCUSSION 4.
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% Changes in Cytoplasmic/Mitochondr
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Table 4.1. Magnified Segments of Di
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Table 4.1. Magnified Segments of Di
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After labeling, black and red color
Page 89 and 90:
78 7 8 Y2S Y3S Table 4.2. Results o
Page 91 and 92:
80 12 S4Ca Table 4.2. Results of Di
Page 93 and 94:
82 27 Y5S Table 4.2. Results of Dif
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Spot 4, Ubiquitin-conjugating enzym
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is a transcriptional repressor, but
Page 99 and 100:
activity and loss of mitochondrial
Page 101 and 102:
distributed, but correlate with nat
Page 103 and 104:
DeMartino, G. N. and Slaughter, C.
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proteomic analyses of a systematica
Page 107 and 108:
Mahindraa, A., Hideshimab, T. and A
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Richardson, P. G., Hideshima, T. an
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Terpos, E. 2008. Bortezomib Directl
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A.1. RPMI-1640 Growth Medium APPEND
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Table B.1. Chemicals and Reagents U
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B.4.3. Standard Curve for BSA Table
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Urea (8M) 4.8 g CHAPS (%2) 0.2 g DT
Page 121 and 122:
E. F. G. STOCK REAGENT PREPARATION
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