«3. Medizinische Klinik und Poliklinik – Hämatologische Forschung Cks1 is a critical regulator of hematopoietic stem cell cycling, quiescence and ...»
3.16. RNA isolation RNA isolation was performed using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. In brief, pelleted cells were resuspended in RLT buffer containing guanidine-thiocyanate, the cells are thereby lysed and RNases are deactivated. Subsequently, the samples are homogenized by running through QIAshredder spin columns. The homogenized samples are mixed with ethanol to optimize binding conditions and loaded on RNeasy Mini spin columns where the RNA binds to the silica-based membrane. Contaminants were washed away by the use of the wash buffers RW1 and RPE and finally, the total RNA was eluted under low-salt conditions.
For gene expression analysis sorted LSK and progenitor cells, RNA was isolated with the Dynabeads mRNA DIRECT Micro Kit (Life Technologies), following the protocol given in the kit. In brief, pelleted cells were resuspended for cell lysis in Lysis/Binding buffer and connected with the Dynabeads which bind to the poly-adenine tails of the mRNAs. The Dynabeads are magnetic, therefore the samples were placed in a magnetic field where the Dynabeads with the bound mRNA congregated at one side of the tube, which allowed the removal of the supernatant with its contaminants. After two washing steps, the Dynabeads with the bound mRNAs were resuspended in 10 mM Tris/HCl pH
7.5 and directly used for reverse transcription.
3.17. Reverse Transcriptation RNA from BaF3 cells was transcribed to cDNA by the use of the Omniscript RT Kit (Qiagen).
For gene expression analysis of sorted LSK and progenitor cells, RNA was transcribed with the QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer’s instructions.
3.18. Real Time PCR Real Time PCR (RT PCR) is a method used to determine the expression levels of genes. It measures how many cycles are needed before the amount of amplified DNA reaches certain value.
For cDNA samples of sorted cells, RT PCR was performed using the StepOnePlus Real Time System (Applied Biosystems) and the Power SYBR Green PCR Master Mix (Applied Biosystems). The CYBR Green PCR Master Mix (Life Technologies) was used for samples of CML patients and cell culture.
When mature cells were analyzed, Ubiquitin was chosen for use as housekeeping gene for normalization. In the case of early hematopoietic cells, Rpl13a was used as housekeeping gene
3.19. Western blotting A gold standard to analyze protein levels in cell lysates is the SDS (sodium dodecyl sulfate) Polyacrylamide gel electrophoresis (SDS-PAGE). SDS is an anionic detergent, which is applied to denaturize proteins SDS in order to linearize and negatively charge the protein chains. The denaturized, negatively charged proteins are loaded on a polyacrylamide gel with an electric field applied, causing their transition to the positive electrode. This leads to the separation of the proteins according to their size. Small proteins are transferred to the lower part of the gel, whereas bigger proteins remain at the top of the gel.
The level of acrylamide in the gel determines the size of the micro pores. The proteins are then transferred to polyvinylidene difluoride (PVDF) membranes (western blot) where they can be detected using primary antibodies against the METHODS certain protein to be analyzed and secondary peroxidase conjugated antibodies, which bind to the primary antibody. The detection occurs through a reaction, product of which is a luminescence in proportion with the amount of protein.
Cell lysis Prior lysing, freshly isolated BM cells were depleted of lineage-positive cells and enriched for c-Kit using magnetic beads separation (Miltenyi Biotec). Cell lysis for immunoblotting was performed with lysis buffer containing 50mM HEPES (pH 7.5), 150mM NaCl, 1mM EDTA, 2.5mM EGTA, 0.1% Tween-20 (all from Sigma-Aldrich, Germany) and protease inhibitors (Roche, Germany) followed by sonification (SONOPLUS HD 2070 (Bandelin), 10 pulses). The lysates were centrifugated at maximum speed for 10 min. The supernatant was used to determine the protein concentration with the help of the Protein assay Kit from BioRad, according to the manufacturer’s instructions. The proper amount (15-60 µg protein) of the supernatant was mixed with 5x loading buffer (250 mM Tris/HCl (Carl Roth), pH 6.8, 50 % Glycerol (Sigma-Aldrich), 10 % SDS (SigmaAldrich), 0.5 M DTT (Sigma-Aldrich), 0,125 % Bromophenolblue (SigmaAldrich)), heated for 5 min at 95°C for denaturation and used for SDS-PAGE.
SDS-PAGE For polyacrylamide gels the gel electrophoresis system from BioRad was used.
The Precision Plus Protein Dual Color Standard (BioRad) was used as a molecular weight marker. The samples were loaded onto the gel and the gel was run with 120 V.
Western blot Afterwards, the proteins were blotted on PVDF membranes (0.45 µm, Millipore) The membrane was blocked with 5 % milk powder (Carl Roth) and 0.1 % Tween 20 in PBS for 1 hour and then transferred into 50 ml falcons containing the primary antibody diluted in blocking buffer and incubated overnight. The next day the membranes were washed twice with PBS with 0.1 % Tween 20 and then secondary antibody was added diluted in blocking buffer. The membranes were incubated for 1 hour at room temperature and then washed twice with PBS with 0.1 % Tween 20 and finally twice with PBS. Detection was METHODS
done with Super Signal West Dura or Femto solutions from Pierce.
3.20. Statistics Statistical analyses were performed using the statistical functions of Excel or GraphPad Prism (GraphPad Software, La Jolla, CA). The bars shown represent the standard error of the mean (SEM). All statistical analyses were t-tests. Only P values 0.05 were considered statistically significant.
4.1. Effects of Cks1 loss on CKI levels and expression of Cks1 in HSC/HPC Cks1 has been recognized as a rate-limiting component of the SCFSkp2 E3 ubiquitin ligase complex which regulates proteosomal degradation of cell cycle inhibitory CIP/KIP family members [128, 134, 135]. Thus, Cks1 is a significant component of the cell cycle regulatory machinery. Since cell cycle control is from a major importance in hematopoietic regulation, it was hypothesized that Cks1 plays an important role in hematopoiesis. In the beginning of this project the level of SCFSkp2 regulated proteins in the hematopoietic subsets of Cks1knockout mice was established. Also, the Cks1 transcript level in bone marrow (BM) cells of WT mice was analyzed.
4.1.1. Cks1 loss correlates with accumulation of CKI in early hematopoietic subsets To assess whether Cks1 deficiency leads to alterations in the expression of cell cycle inhibitors in early BM populations, Cks1-/- and WT control BM enriched for Lin- cKit+ cells and control Lin+ cells was analyzed for CKI protein levels using western blot (Fig. 5A, B). Indeed, there was a significant accumulation of all three CIP/KIP family members, p57, p27, p21 and also of another cell cycle regulator and known SCFSkp2 target, p130, in Cks1-/- hematopoietic cells as compared to their WT counterparts (Fig. 5B). Interestingly, this accumulation was more pronounced in the Lin- c-Kit+ subset, and Cks1 was stronger expressed just in the Lin- c-Kit+ fraction, suggesting a more prominent role for Cks1 in the control of CKI protein levels in earlier, c-Kit expressing HSC/HPC.
To track CKI protein changes in distinct HSC/HPC subsets, intracellular staining was used. Lineage-depleted BM cells from WT and Cks1-/- mice were stained with antibodies against p57, p27, p21 and p130. Accumulation of p21 and p27 was observed in all analyzed Cks1-deficient subsets and, consistent with the western blot analysis, was more pronounced in the early  CD150+ LSK (Fig.
5C, D). Interestingly, p57 and p130 protein levels were significantly increased only in the HSC including the CD150- and CD150+ LSK subsets (Fig. 5C, D).
RESULTS These data suggest a prominent function for Cks1 in the control of CKI protein levels and HSC homeostasis Figure 5: Loss of Cks1 results in accumulation of SCFSkp2 substrates.
(A) Efficiency of the isolation, analyzed by FACS. BM cells from 5 WT and 5 Cks1-/- mice were depleted of lineage-committed cells and positively enriched for c-Kit expression using immunomagnetic beads (Lin- c-Kit+). (B) Western blot analysis of the indicated proteins. (C) Quantification of intracellular staining of p21, p27, p57 and p130 in the CD150+ LSK population using flow cytometry. (D) Summarized results of intracellular staining of different subpopulations for the indicated SCFSkp2 targets (Cks1+/+ n=6, Cks1-/- n=5, 3 independent experiments).
4.1.2. High expression of Cks1 in early hematopoietic subsets Since CKI regulated through the SCFSkp2 E3 ubiquitin ligase complex were strongly accumulated in Cks1-/- LSK cells, the level of Cks1 in these populations needed to be tested in WT mice. For this purpose, flow cytometric cell sorting on WT BM cells was performed (Fig. 6A) and Cks1 transcript levels in HSC/HPC subsets were evaluated using real time PCR (Fig. 6B). In contrast to the intracellular staining, where the LT-HSC population was indicated by expression of CD150+ , the earliest subset in this case was defined as CD34- LSK . In accordance with the pronounced accumulation of SCFSkp2 regulated proteins in absence of Cks1 in early hematopoietic stages (Fig.5 C and D), Cks1 mRNA was expressed highest in the more immature CD34- LSK cells in WT mice (Fig. 6 B), confirming the suggestion that Cks1 might be an important regulator in early hematopoiesis.
Figure 6: High Cks1 transcript levels in the CD34- LSK subset of WT mice.
(A) Representative FACS-Sort diagram for (B) real time PCR analysis of Cks1 mRNA expression in the indicated WT FACS-sorted subgroups. Shown are the results from n=2 independent experiments. For each experiments the pooled bone marrow of n=6 mice was used.
4.2. Role of Cks1 in steady state and stress hematopoiesis Taken together, the initial experiments revealed that Cks1 expression is highest in early LSK populations and loss of Cks1 results in a pronounced accumulation of CKI (p21, p27 p57) and p130 in these cells compared to MPP and more mature hematopoietic populations. Based on these results, it was hypothesized, that Cks1 is involved in the regulation of early hematopoiesis.
The next part of this study focused on experiments comparing WT and Cks1deficient mice in order to study the role of Cks1 in hematopoiesis.
4.2.1. Normal steady state adult hematopoiesis in Cks1-deficient mice Mice lacking Cks1 are viable and in normal health condition . Though, they are abnormally small (10-20% smaller than their littermates of the same gender) and Cks1-/- -derived mouse embryonic fibroblasts proliferate poorly and accumulate p27 . Hematopoiesis in Cks1-deficient mice has not been studied in detail.
In the beginning of this part of the study blood and bone marrow of the knockout mice was analyzed and compared to WT. For the blood analysis blood from WT and knockout mice was measured with blood counter and analyzed by FACS after staining with the cell surface markers as follows: CD45 for leukocytes, combination of CD4 and CD8a for T cells, B220 for B cells and Gr1 and CD11B to distinguish granulocytes and monocytes. No significant differences were observed either in the blood cell counts (Fig. 7A), nor in the different mature blood cell populations (Fig. 7B, C). Hematoxylin-Eosin stainings (Collaboration with PD Dr. Leticia Quintanilla-Fend) showed no abnormalities in the bone marrow structure of the knockout mice (Fig. 7D).
Interestingly, the absolute cell numbers in the hematopoietic tissues BM an fetal liver (FL) of the Cks1-/- mice was strongly reduced (Fig. 7E). It is known that Cks1-/- mice are smaller in size [144, 172] and respectively have smaller organs. Though the reduction of the absolute cell number was enhanced (36compared to the reduction of body size (10-20%), hinting towards a pronounced role of Cks1 in hematopoiesis.
Figure 7: Normal steady state adult hematopoiesis in Cks1-deficient mice.
(A) Blood counts (WBC: white blood cells; Hgb: hemoglobin; PLT: platelets) in WT and Cks1-/mice, n=6. The differences are not statistically significant. (B) Representative flow cytometry analysis of the indicated surface markers for adult hematopoietic cells, gated on CD45+ cells.
(C) Percentage of B cells (B220+), T cells (CD4/8a+), granulocytes (Gr1++ CD11B+) and monocytes (Gr1+ CD11B+) in the periferal blood of wild type and knock out animals at steady state (Cks1+/+ n=5; Cks1-/- n=6). The differences are not statistically significant. (D) Hematoxylin-Eosin staining of bone marrow section from mice of the indicated genotype (Collaboration with PD Dr. Leticia Quintanilla-Fend). Representative sections are shown. (E) Absolute cell counts of bone marrow (n=30) and fetal liver (Cks1+/+ n=10; Cks1-/- n=8).
2. Decreased absolute HPC numbers and increased relative HSC numbers in Cks1-/- mice The absolute cell number was drastically decreased in FL as well as in the BM of Cks1-/- animals (Fig.7E), suggesting altered hematopoiesis in absence of Cks1. To gain further insight into the early hematopoietic subsets of Cks1-/mice, FACS analysis with WT and Cks1-/- BM cells was performed. Figure 8 demonstrates the gating strategy which was used based on earlier studies [31, 43, 44, 46]. A biotinylated cocktail for the lineage marker Terr119 (erythroid cells), CD11B (myeloid cells), Gr1 (myeloid cells), CD3 (lymphoid T-cells), B220 (lymphoid B cells) stained secondary with straptavidine-conjugated antibody was used for the lineage gating (Fig. 8A). c-Kit and Sca1 were used to differentiate the lineage negative cells into MPP and LSK (Fig. 8A). The MPP were then distinguished into GMP, CMP and MEP by the expression of CD16/32 and CD34 (Fig. 8B) and LSK were further gated either into CD34- or CD34-CD150+ LSK (Fig. 8C).
Figure 8: BM analysis in WT and Cks1-/-mice. Representative FACS Analysis.
(A) Lineage, MPP, and LSK gating. (B) CMP, GMP and MEP gating. (C) CD34- LSK and CD34CD150+ LSK gating.