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«MITOTICKÉ DISRUPCE U MNOHOČETNÉHO MYELOMU Diplomová práce Jakub Petrík VEDOUCÍ PRÁCE: prof. MUDr. Roman Hájek, CSc. Brno 2013 ...»

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MASARYKOVA UNIVERZITA

P írodovědecká fakulta

Ústav experimentální biologie

Oddělení genetiky a molekulární biologie

_______________________________________

MITOTICKÉ DISRUPCE U

MNOHOČETNÉHO MYELOMU

Diplomová práce

Jakub Petrík

VEDOUCÍ PRÁCE: prof. MUDr. Roman Hájek, CSc. Brno 2013

Bibliografický záznam

Bc. Jakub Petrík

Autor:

Přírodovědecká fakulta, Masarykova univerzita Ústav experimentální biologie Název práce: Mitotické disrupce u mnohočetného myelomu Studijní program: PřF N-EXB Experimentální biologie Studijní obor: PřF BIMG Molekulární biologie a genetika Vedoucí práce: prof. MUDr. Roman Hájek, CSc.

Akademický rok: 2013/2014 Počet stran: 89 Klíčová slova: mnohočetný myelom, mitotické disrupce, hyperdiploidie, centrozómové amplifikace, IgH disrupce Bibliographic entry Bc. Jakub Petrík

Author:

Faculty of Science, Masaryk University Department of Experimental Biology Title of thesis: Mitotic disruptions in multiple myeloma Degree programme: Experimental Biology Field of study: Molecular Biology and Genetics prof. MUDr. Roman Hájek, CSc.

Supervisor:

Academic year: 2013/2014 Number of pages: 89 Keywords: multiple myeloma, mitotic disruptions, hyperdiploidy, centrosome amplifications, IgH disruption Abstrakt Mnohočetný myelom (MM) je ve světě druhé nejčastější hematoonkologické onemocnění. Je charakterizován expanzí monoklonálních plazmatických buněk, které se akumulují v kostní dřeni a ovlivňují tam hematopoézu. S nástupem nových léků se MM změnil z neléčitelného na těžko léčitelný. Nicméně, pacienti stále relabují. Cílem léčby je proto dosažení kompletní remise, kdy vymizí klinické příznaky, a prodloužení této doby na maximum. Patogeneze MM dosud není zcela jasná. Zdá se, že roli by mohli hrát aberantní centrozomy. Centrozomy jsou organely, které mají funkci v organizování mikrotubulů v průběhu dělení buňky. Jejich počet je koordinován s buněčným cyklem a jejich aberantní počet může tento proces narušit.

Centrozomové amplifikace (CA) byly detekovány u všech stádií monoklonálních gamapatií.

Proto se předpokládá, že mohou představovat jeden z mechanizmů disrupce buněčného cyklu, co vede k chromozomální nestabilitě plazmatických buněk u MM. Cílem této diplomové práce bylo porovnat skupiny s CA a bez nich a zjistit, zda se mezi nimi nachází statisticky významné rozdíly v přítomnosti vybraných cytogenetických abnormalit a rozdíly v expresi vybraných strukturních genů centrozomu. Jednotlivá porovnání byla prováděna jednak na vzorcích z kostní dřeně pacientů s MM, i na lidských myelomových buněčných liniích.

Rozdíly byly nalezeny v přítomnosti disrupce IgH genu mezi CA pozitivními a CA negativními skupinami pacientů. V porovnání genové expresi byly mezi CA pozitivními a CA negativními skupinami nalezeny rozdíly u buněčných liniích v genech TUBG1 a CENT2 a u pacientů v genu TUBG1.

Abstract Multiple myeloma (MM) is the second most common hematooncological disease in the world. It is a malignant plasma cell (PC) disorder, characterized by PC infiltration into the bone marrow (BM), where alter physiological hematopoiesis, together with the production of a monoclonal immunoglobulin. Introduction of new drugs into the therapy has changed the disease from incurable into hard-to-treatable. However it is not curative, since most patients relapse. The aim of the treatment is to reach clinical remission, when no clinical features are present, and to extend this duration to maximum. Pathogenesis of MM is still unclear. One of the possible mechanisms, which trigger pathogenesis, could be aberration of centrosomes.

Centrosomes are cellular microtubule organizing centers, whose normal function is implicated in cell division. Their number is coordinated with the cell cycle and any aberration in the count could be crucial. Centrosome amplifications (CA) have been detected in every stage of monoclonal gammopathies. CA, therefore, may represent a mechanism leading to disruption of the cell cycle, which subsequently leads to chromosomal instability of PC in MM. The aim of this diploma thesis was to compare CA positive and CA negative groups in the presence of selected cytogenetic abnormalities and in gene expression of selected structural centrosomal genes. Each comparison was, separately, done on human myeloma cell lines and on samples from patients with MM. Differences were found in the presence of IgH gene disruption between CA positive and CA negative subgroups of patients. In comparison of gene expression, differences were found in TUBG1 and CENT2 genes between CA subgroups of cell lines and in TUBG1 gene between subgroups of patients.

Poďakovanie Na tomto mieste by som rád po akoval svojmu školiteľovi prof. MUDr. Romanovi Hájkovi, CSc., za odborné vedenie a cenné pripomienky pri spracovaní tejto diplomovej práce a RNDr.

Sabine Ševčíkovej Ph.D., za konzultácie a poskytnuté rady. So štatistickými analýzami mi pomáhal MUDr. Fedor Kryukov, Ph.D., ktorému týmto tiež akujem. Tiež by som chcel po akovať MUDr. Elene Kryukovovej, Ph.D. a Renáte Kupskej za pomoc pri spracovávaní vzoriek. alej by som chcel ešte po akovať celému kolektívu Babákovej myelómovej skupiny pri Ústave patologickej fyziológie Lekárskej fakulty Masarykovej univerzity za vytvorenie príjemného pracovného prostredia.





Prehlásenie Týmto prehlasujem, že som svoju diplomovú prácu vypracoval samostatne s využitím dostupných informačných zdrojov, ktoré sú v práci citované.

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LIST OF ABBREVIATIONS

1. THEORETICAL BACKGROUND

1.1. Multiple myeloma

1.1.1. Epidemiology

1.1.2. Pathogenesis

1.1.3. Molecular pathogenesis

1.1.3.1 Hyperdiploid and non-hyperdiploid MM

1.1.3.2. Primary translocations

1.1.3.3. Secondary translocations and other late genetic events

1.1.4. Clinical features

1.1.5. Diagnostic criteria and staging systems for MM

1.1.6. Treatment

1.2. Mitotic disruptions in MM

1.2.1. Chromosomal instability (CIN)

1.2.2. Centrosome

1.2.2.1. Centrosomal structural genes

1.2.2.2. Centrosome cycle

1.2.2.3. Centrosome abnormalities

1.2.2.4. CA in MM

2. AIMS OF THE THESIS

3. MATERIALS AND METHODS

γ.1. Patients’ characteristics

3.2. Cell lines

3.3. Interphase fluorescent in situ hybridization (I-FISH)

3.3.1. Cell lines preparation

3.3.2. Preparation for I-FISH

3.3.3 I-FISH

3.3.4 I-FISH Evaluation

3.3.4.1. IgH disruption evaluation

3.3.4.2. Hyperdiploidy evaluation

3.4. Immunofluorescent staining of centrin

3.4.1. Evaluation of centrin immunofluorescent staining

3.5. RNA isolation

3.5.1. Quality control and RNA concentration measurements

3.6. Reverse transcription (RT)

3.7. Preamplification

3.8. Quantitative real-time polymerase chain reaction (qRT-PCR)

3.8.1. qRT-PCR evaluation

4. RESULTS

4.1. Cytogenetic characterization

4.1.1. IgH disruption

4.1.2. Ploidy category

4.2. CA characterization

4.3. qRT-PCR

4.4. Comparison of CA positive and CA negative groups

4.4.1. Comparison between CA positive and CA negative cell lines

4.4.2. Comparison between CA positive and CA negative patients

5. DISCUSSION

6. SUMMARY

7. SOUHRN

8. REFERENCES

9. APPENDICES

LIST OF ABBREVIATIONS

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BMPC bone marrow plasma cells CA centrosome amplifications Cdk cyclin dependent kinase CIN chromosomal instability DAPI 4,6-diamidino-2-phenylindole Dulbecco’s Modified Eagle Medium DMEM DSMZ Deutche Sammlung von Mikroorganismen und Zellkulturen

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ISS international staging system I-FISH interphase fluorescent in situ hybridization MACS magnetic activated cell sorting MGUS monoclonal gammopathy of unknown significance M-Ig monoclonal immunoglobulin

–  –  –

PCM pericentriolar material pRb retinoblastoma protein qRT-PCR quantitative real-time polymerase chain reaction RT reverse transcription

1. THEORETICAL BACKGROUND

1.1. Multiple myeloma Multiple myeloma (MM) is a malignant plasma cell (PC) disorder characterized by PC infiltration into the bone marrow (BM) together with the production of a monoclonal immunoglobulin (also called paraprotein) (M-Ig or M-protein) in the serum and/or urine (Minter et al., 2011). The invasion of PC into the bone causes bone lesions which results in pain and bone fractures. MM is the most common cancer which affects the bone (The bone and cancer foundation, 2008) and bone pain is the most common symptom. The mass production of M-Ig often leads to renal failure. MM is a heterogeneous disease with patients dying in a range between a few weeks and more than 10 years. The reason is the combination of interactions between host factors and features of the disease (IMWG, 2009).

1.1.1. Epidemiology According to the Agency for Research on Cancer, there were approximately 103 000 people across the world diagnosed with MM in 2008 (http://globocan.iarc.fr). It was about 1% of all diagnosed cancers and 12% of all hematological cancers (Ferlay et al., 2010). MM is the second most common hematological malignancy in western countries (Jemal et al, 2010). It is a disease of elderly people - the median age at diagnosis is 65 years, and the occurrence increases with age. Patients younger than 40 years account for 2.2% of all MM (Goel et al., 2011). The median overall survival is 3 to 4 years, ranging from a few weeks to more than 10 years (IMWG, 2003). The incidence in the Czech Republic is 4 cases per 100 000 inhabitants (Hájek et al., β011). The incidence in European countries is compared in Figure 1.

Figure 1: European incidence rates of MM in 2008 (source: http://www.cancerresearchuk.org) 1.1.2. Pathogenesis Normal B cell (BC) development leads to the production of non-dividing PC. They are terminally differentiated and secrete antigen-specific antibodies. The production of PC happens in two pathways (depicted in Figure 2). It could be triggered by T cell-independent antigenic exposure or by antigenic exposure in cooperation with antigen-specific T helper cells (Calame et al., 2003).

In T cell-independent way, antigen, in combination with other signals, initiates naive BC in the splenic marginal zone and circulating mature follicular BC to proliferate and differentiate into short-lived PC. Short-lived PC produces low affinity IgM and serves as the first antibody response to the pathogen (Shapiro-Shelef & Calame, 2004). In T cell-dependent way, antigen and antigen-specific T helper cells activate naive follicular BC. Some activated BC form a germinal center (GC), where they undergo specific reactions such as: proliferation, somatic hypermutation, affinity maturation, antigen selection and immunoglobulin (Ig) classswitch recombination. Those reactions produce long-lived PC with high affinity antibody secretion (e.g. IgG, IgA, IgE). These PC become long-lived if they receive survival signals (e.g. interleukin-6), mainly in the BM and then can live for months to years (Manz et al., 2005). Normally, the frequency of PC does not exceed 0.5% of all mononuclear cells in the BM (Brieva et al., 1991).

Figure 2: BC maturation (adopted from Anderson & Carrasco, 2011): After interaction with antigen, naive BC differentiates into PC. Most PC are short-lived and mature without the help of the T cells.

Some of the BC are activated in T cell dependent pathway and form a GC. Post-germinal PC develops through a memory BC stage or develops directly from germinal center BC. If post-germinal PC finds survival niches, they become long-lived. The survival niche is mainly located in the BM.

MM cells are exclusively long lived post-germinal PC, which undergone the PC maturation process in GC after antigen exposure. MM cells proliferate at a low rate, generally less than 1% (Dimopoulos & Terpos, 2010). The Ig gene sequences are somatically hypermutated and remain constant through the course of the disease (Bakkus et al., 1992).

MM cells produce M-Ig of a single heavy chain and light chain (also called the paraprotein).

The most abundant are IgG and IgA; IgD, IgE and IgM are less common (Karia & Ruparelia, 2011). But in one fifth of the patients, there is only free light chain production (kappa or lambda); in rare cases (about 5%), there is no monoclonal protein secreted by the clonal PC population (this state is called non-secretory MM).



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