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«3. Medizinische Klinik und Poliklinik – Hämatologische Forschung Cks1 is a critical regulator of hematopoietic stem cell cycling, quiescence and ...»

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Nevertheless, Cks1 exhibit CDK independent functions in mammalian cells as a part of a complex, which is involved in degradation of CKI [128] 1.3.1. Ubiquitination and the SCFSkp2 complex Protein turnover through synthesis and degradation is of major significance for accomplishing the majority of cellular processes. Degradation of most of the intracellular proteins in eukaryotes is processed through the ubiquitin (Ub) proteasome pathway [129]. In order to be recognized and degraded by the 26S proteasome, proteins need to be labeled with covalently bound Ub molecules, a process called ubiquitination [129]. Ubiquitination involves three major enzymes E1 activating enzyme, E2 conjugating enzyme and E3 ubiquitin ligases [130, 131]. E3 ligases are responsible for transferring activated Ub molecules from the E2 conjugating enzyme yielding an Ub chain onto proteins targeted for degradation [130, 131]. Most of the E3 ligases contain so called RING finger domains and are either monomeric enzymes or many subunits containing complexes [129]. Cullin-RING ligases built the largest group of E3 enzymes [132] and a subgroup of cullin-RING ligases, the Skp1-Cul1-Fbox (SCF) complexes are the best understood ones [129]. The F-Box protein in the complex is a variable component which contains a substrate-binding motive and function as substrate recognition subunit [130, 133]. In many cases, phosphorylation of the substrate is needed in order to be recognized by the FBox subunit [130]. For instance, in the SCFSkp2 complex, T187 phosphorylated p27 is targeted by the F-Box protein Skp2 (Fig. 3 and [134]). Importantly, an additional protein in the complex was found to be essential for the recognition and the following ubiquitination of phosphorylated p27, namely Cks1 [128, 135].

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Figure 3: SCFSkp2 – induced p27 degradation (Modified from Bartek and Lukas 2001b [136]).

1.3.2. SCFSkp2-Cks1 and its role in cell cycle control Since regulation of CKI occurs mainly at post-translational level through control of protein stability, ubiquitination and subsequent degradation of these cell cycle regulators is of major importance in controlling the cycling process.

As part of the SCFSkp2 complex, Cks1 interacts with Skp2, inducing allosteric changes in the F-box protein and increasing its affinity to cycline A/E CDK2 kinase phosphorylated p27 [137, 138]. Thus, Cks1 enables the ubiquitination of p27 through the SCFSkp2 ubiquitin ligase complex [136]. Therefore, loss of Cks1 in mice results in reduced body size and decreased proliferation associated with an accumulation of p27 [128].

In addition to the regulation of p27 protein levels, SCFSkp2 has further been associated with the post-translational regulation of other CKI including p21 [139, 140], p57 [141], and the Rb family member p130 [142]. The SCFSkp2 subunit Cks1 is not only a crucial component of SCFSkp2- mediated ubiquitination but is also involved in regulation of CDK1 transcription [143]. Furthermore, loss of Cks1 results in reduced CDK2 kinase activity and defect S Phase entry [144].

INTRODUCTION

1.3.3. Regulation of Cks1 expression The SCFSkp2 ubiquitin ligase complex is involved in the cell cycle through targeting cell cycle regulators for degradation, hence transcriptional regulation of Skp2 and Cks1 is part of the cell cycle control. The forkhead box m1 (Foxm1) gene, which functions are important for the G1/S transition and especially for mitotic progression, regulates amongst others transcription of Skp2 and Cks1 [145]. Additionally, Cks1 is regulated through the cycle-dependent element and cell cycle gene homology region, the so called CDE/CHR tandem element in the Cks1 promoter [146]. Promoters containing CDE/CHR element control cell cycle genes and their expression depending on the cell cycle status and are typically activated through several CCAAT elements [146]. Trimeric nuclear factor-Y (NF-Y) binds to CCAAT boxes and acts as main activator, hence NF-Y could be observed as the main activator of Cks1 [146]. On the other hand, p53 downregulates the expression of Cks1, whereas its repression is not influenced by NF-Y [146]. Furthermore, Cks1 regulation occurs through TGF-β signaling [147]. As pointed out before (chapter 1.2.2), the TGF-β pathway plays an important role in regulating the cell cycle and is important for maintaining quiescence in HSC. Through downregulation of Cks1 and Skp2 TGF-β signaling also inhibits the SCFSkp2 ubiquitin ligase activity [147]. The oncogenic transcriptional factor Myc is inducing Cks1, Skp2 and Cul1 expression and thus initiates p27 degradation, cell proliferation and lymphomagenesis [148].

Regulation of Cks1 also occurs on protein level through the ubiquitinproteasome pathway and in a cell cycle dependent manner [149]. Protein levels of the substrate recognition subunits of the SCFSkp2 ubiquitin ligase, Cks1 and Skp2 are also shown to decrease in late M phase and G1 and increase again in S phase [150]. Degradation of these proteins via the APC/C(Cdh1) ubiquitin ligase at the specific time point of the cell cycle prohibit degradation of SCFSkp2 targets, such as CKI, and premature entering in the next cell cycle phase [151].

1.3.4. Cks1 in cancer Cancer is caused by genetic defects and following failure to control cell proliferation [2]. Since Cks1 is an essential regulative element in the complicated organization of cell cycle control, its expression is disturbed in vast

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number of cancer types. In general, Cks1 is overexpressed and correlates with poor prognosis in cancer [152].

For instance, Cks1 is overexpressed in gastric carcinoma [153], non-small cell lung carcinomas [154], colorectal carcinoma [155], ovarian cancer [156] as well as in breast tumors [157]. Gene amplification and overexpression of Cks1 in breast tumors is associated with decrease in p27 levels, lymph node metastasis and poor prognosis [157, 158]. Furthermore, the resistance of esophageal squamous cell carcinoma against radiotherapy is increased in patients with high Cks1 protein levels [159].

Searching for a mechanism of action of Cks1 in cancer revealed that upregulated Cks1 as well as Cks2 levels induce uncontrolled cell division probably partly through disturbing the S Phase checkpoint by binding to CDK2 and hindering inhibitory phosphorylation [160]. Also, it was shown recently that inhibitors of the SCFSkp2 complex block estrogen induced growth and p27 degradation [161], providing a possible treatment in cancers characterized by SCFSkp2 induced p27 degradation.

Moreover, Cks1 controls p27 levels in premalignant Eμ-Myc B cells, and loss of Cks1 reduces proliferation and delays lymphoma development and dissemination of the disease [148]. Despite the fact that Cks1 is required for tumor cell proliferation, Cks1 on its own is not sufficient to induce hematopoietic malignances [162].

1.4. Aim of this thesis Cyclin/CDK complexes and their inhibitory CKI are important factors for the proper cell cycle progression in HSC/HPC. Considering the importance of SCFSkp2-mediated degradation of CKI family members in regulation of the cell cycle in HSC/HPC, this project investigates the role of Cks1, the rate limiting component of the SCFSkp2 complex in early hematopoiesis.

The aim of this project was to analyze the effects of Cks1 loss on the quality and quantity of HSC/HPC by using in vitro and in vivo methods. In addition, using double knockouts for Cks1 and p27, it was evaluated, which of the observed effects after Cks1 loss were p27 dependent. Furthermore, the regulation of the human CKS1B gene in the myeloproliferative disorder CML

INTRODUCTION

was studied and the role of Cks1 for BCR-ABL mediated clonogenic activity was examined.

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2. Materials

2.1. Mice All animal experiments were performed in accordance with the regional animal ethics committee approvals.

Cks1-/- mice [128] and control WT littermates were bred on a (129S x C57BL/6J) F1 (129B6, Ly5.2) background.

Cks1-/- mice (C57Bl6-129 mixed background) were bred to p27Kip1-/- mice (C57BL/6J) [28] for 6 generations to generate Cks1-/- p27Kip1-/- (DKO) and control animals on a mixed C57BL/6-129 background. Since loss of p27 leads to ovulatory effect and sterility [28], only p27+/- female mice were used in the breeding.

In transplantation experiments 129S2xC57BL/6.SJL/J (129Ly5.1) mice were used as recipients.

2.2. Cell lines

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2.3. Bacteria One Shot TOP10 Chemically Competent E. coli (Life Technologies)

2.4. Vectors MSCV-IRES-Bcr-Abl-p210-GFP was kindly provided from Prof. Dr. Nikolas von Bubnoff, Albert-Ludwigs-Universität, Freiburg

2.5. Antibodies Table1: Antibodies used for surface staining in flow cytometry

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Table 2: Antibodies used for intracellular staining in flow cytometry Table 3: Antibodies used for western blotting

Secondary Antibodies:

Straptavidine, eFluor®450 conjugated, eBioscience Anti-rabbit IgG, AlexaFluor®488 conjugated, Life Technologies Anti-rabbit IgG, Santa Cruz Anti-rabbit IgG, AlexaFluor®488 conjugated, Santa Cruz Anti-mouse IgG, AlexaFluor®488 conjugated, Santa Cruz Anti-rabbit IgG, HRP conjugated, GE Heatlthcare UK limited Anti-mouse IgG, HRP conjugated, GE Heatlthcare UK limited

2.6. Primer

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METHODS

3. Methods

3.1. Genotyping The polymerase chain reaction (PCR) was used to determine the genotype of the bred mice (wild type (WT), Cks1 knockout (Cks1-/-), p27 knockout (p27-/-) and double knockouts for both Cks1 and p27 (DKO)). Small pieces of the ear (from ear clipping) were used as samples. 50 µl lysis buffer ((100 mM Tris/HCl (Carl Roth), pH 8.3, 500 mM KCl (Sigma-Aldrich), 0.1 mg/ml gelatine (Carl Roth), 1% NP40 (Carl Roth), 1% Tween 20 (Carl Roth)) and 200 µg/ml Proteinase K (Life Technologies) was added to the samples. In order to decompose the tissue, the samples were incubated at 50°C with constant shaking overnight. The next day, the Proteinase K was deactivated by the incubation of the samples at 95°C for 5 min, centrifuged at 15 000 g for 10 min and the supernatant, containing the DNA, was used for a PCR analysis.

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For the mutant allele a fragment of approximately 200 bp should be amplified and for WT allele a fragment of approximately 150 bp should be amplified (Fig.

4A).

p27 genotyping Master Mix:

1 μl DNA 0,3 μl Primer 1 [100 pmol/μl] 0,3 μl Primer 2 [100 pmol/μl] 2 μl10x Buffer 4 μl5x Q Solution 0,7 μl dNTPs Mix (10 mM each) 0,3 μl Taq polymerase 11,4 μl H2O Primer Mix for WT allele: primer 1: mgK3, primer 2: mcK5.

Primer Mix for mutant allele: primer 1: mgK3, primer 2. p27-Neo

p27 genotyping PCR program:

preheating 95 °C 30 sec. 96°C 30 sec.57°C 4x 2 min 65°C 30 sec. 93°C 30 sec. 57°C 36x 2 min. 65°C 2 min. 65°C 4°C forever The reaction for WT allele should amplify a fragment of 1 kB.

The reaction for mutant allele should amplify a fragment of 0,5 kB (Fig. 4B).

5 µl of loading buffer (Quiagen) was added to the samples before loading on a 1% agarose gel made with NaB buffer (0,01 M di-sodium tetraborate (SigmaAldrich)) and ethidium bromide (0.5 µg/ml, Carl Roth). The gel was run with 300 V and analyzed with a BioRad Gel-Doc XR Imaging System.

METHODS

Figure 4: Genotyping of (A) Cks1-/- and (B) p27-/- mice.

3.2. Preparation of murine tissues In this project, the organs to be analyzed were bone marrow, spleen, lymph nodes and blood.

For the bone marrow analysis, the 4 long bones (femurs and tibia) of both hind legs were extracted and flushed with HF2 buffer (HBSS (Life Technologies), 10 mM HEPES (Life Technologies), 2 % FCS (PAA), 100 U Penicillin, 100 µg streptomycin (Life Technologies)). The cell suspension was homogenized with a blunt needle and filtered through a 30 µm filcon (BD Biosciences).

For an analysis of spleen or lymph nodes, the organs were squished, and after homogenizing filtered through 30 µm filcons.

Homogenized bone marrow or spleen samples were centrifuged at 300 g for 5 min; the pellets were resuspended in 2 ml of ACK lysing buffer (Life Technologies) and incubated on ice for 5 min in order to lyse the erythrocytes.

Samples were then centrifuged at 300 g for 5 min, resuspended in HF2 buffer and filtered again through a 30 µm filcon in order to remove chunk that developed after the erythroid lysation.

Blood was obtained from mice by punctuating the facial vein and collected in EDTA-coated vials. Blood samples were mixed with 10 x volume of ACK lysing buffer and incubated on ice for 15 min in order to lyse the erythrocytes.

Samples were then centrifuged at 300 g for 5 min and resuspended in HF2 buffer and filtered through a filcon.

Fetal liver (FL) cells from WT and Cks1-/- 14,5 days old embryos were used in a colony forming assay. FL were squished, and after homogenizing filtered through 30 µm filcons. The cells were than directly plated in methylcellulose medium. The heads were used to genotype the embryos.

METHODS

3.3. Transplantation assays A transplantation assay is a fundamental method for studying hematopoietic differentiation and specifically HSC self-renewal potential when performed as serial transplantations [12]. The recipients are being lethally irradiated, which leads to ablation of most of the hematopoietic tissue and allows the transplant to engraft. Whole bone marrow samples or single sorted hematopoietic population are transplanted into the recipients in order to replenish the hematopoietic system. Serial transplantations, using donor cells from the primary recipients provide information about the self-renewal capacity of the HSC from the primary donor, since these cells sustain and repopulate the new donor only if they are able to self-renew in the previous recipient. Serial transplantations can be performed until the HSC from the primary donor are exhausted and no longer capable to repopulate an irradiated host [12]. In order to distinguish donor and recipient cells throughout the transplantation assays, mice with congenic expression of different isotypes of the hematopoietic CD45 marker were used.



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