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Blood, Vol. 113, Issue 12, 2791-2794, March 19, 2009
PKCβ is essential for the development of chronic lymphocytic leukemia in the TCL1 transgenic mouse model: validation of PKCβ as a therapeutic target in chronic lymphocytic leukemia
Blood Holler et al.
113: 2791
Supplemental materials for: Holler et al
Mouse cell preparation
Mice were sacrified by CO2 when visibly ill or at specific time-points when pre-tumor phenotypes were needed to be analyzed. Single-cell suspension of spleen, liver, peripheral lymph nodes (paired auxiliary), mesenterial lymph nodes (if enlarged) and thymus were generated by gently meshing the organs through a sieve and filtering through a nylon pre-separation filter (Miltenyi Biotec). Bone marrow cell suspensions were prepared by flushing bilateral femurs with KDS-BSS buffer using a 26-gauge needle. Peritoneal cavity cells were obtained by injecting the peritoneal cavity with 2–3 ml KDS-BSS, and recovering the buffer. Peripheral blood was taken from heart ventricles during dissection. 30µl 0.1M EDTA was added to avoid coagulation. Volume of blood was ascertained to determine white blood cell count (WBC). Blood erythrocytes were lysed using erythrocyte lysis buffer. All organs altered in shape and size were fixed in 37% formalin (diluted in PBS) over night, washed in PBS and handed over to the Department of Pathology at the SALK for paraffin embedding. For BCR crosslinking experiments, murine splenocytes were diluted to a density of 1 × 106 cells/ml in Advanced RPMI-1640 (Invitrogen), supplemented with 10% heat-inactivated fetal calf serum (FCS, PAA Laboratories), 2 mM L-glutamine (Sigma-Aldrich, St. Louis, MO), HEPES pH7.0 (Sigma-Aldrich), mM b-mercaptoethanol (Gibco BRL, Invitrogen), and 100 U/mL penicillin/streptomycin (Biochrom AG, Berlin, Germany). BCR crosslinking
96 well plates (Costar #3590, Szabo-Scandic) were coated with F(ab′)2 goat anti mouse IgM (Jackson ImmunoResearch, Dianova) diluted in PBS. Plates were incubated for 2h at 37°C. Unbound F(ab′)2 fragments were aspirated and plates were washed with PBS. 200 µl murine spleen cell suspension (1 × 106 cells/ml) of different genotypes were added. Apoptosis for B cells and T cells was measured after 24h per FACS by staining 4 × 105 cells with CD19-PC7, Annexin V-FITC, and 7-AAD. Quantitative RTD-PCR analysis of target gene expression
For the analysis of expression level of BCL2, c-MYC, and ARF-1 in CLL tumour samples from spleen and blood of TCL1 (N=4) and PKC+∕−TCL1 (N=7) mice, total RNA was isolated from spleen cells and blood leukocytes using the RNAqueous™ kit (Ambion). The I-Script cDNA synthesis kit (Bio-Rad) was used to reverse transcribe 500 ng of total RNA in a final reaction volume of 20 µl. The cDNA reaction was then diluted to a final volume of 100 µl for use in RTD-PCR reactions. Quantitative RTD-PCR was performed in duplicate in 96-sample plates separately for each target gene. The cDNA equivalent of 25 ng of total RNA (5 µl of the diluted iScript reaction) was used per 20 µl reaction containing 10 µl 2× TaqMan® PCR Gene Expression Master Mix, 4 µl dH2O, and 1 µl Taqman gene expression assay for either ARF (Mm01257348_m1), Bcl-2 (Mm00477631_m1), c-Myc (Mm00487804_m1), or the endogenous control GAPDH (RefSeq: NM_008084.2mRNA) (all from Applied Biosystems). All reactions and detection were carried out on the ABI7500 Real-Time PCR System (Applied Biosystems) according to the following program: 1 cycle of a 10 min denaturation at 95°C, and 40 cycles of 15 sec denaturation at 95°C followed by 1 min annealing-elongation at 65°C. The expression levels for ARF, Bcl-2, and Myc in PKCβ+∕− TCL1 mice relative to TCL1 mice were calculated using the following formula: RQ = 2-(dCT(PKCβ+∕− TCL1) − dCT(TCL)), where dCT is the mean CT value of the target gene minus the mean CT value for the endogenous control. Data are presented as fold induction. Cell culture and viability assay
Unless otherwise stated, peripheral blood mononuclear cells were cultured at a concentration of 1 × 106 cells/mL in complete medium which consisted of RPMI-1640 (PAA Laboratories, Pasching, Austria) supplemented with 10% heat-inactivated fetal calf serum (FCS, PAA Laboratories), 2 mM L-glutamine (Sigma-Aldrich, St. Louis, MO) and 100 U/mL penicillin/streptomycin (Biochrom AG, Berlin, Germany). All cells were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2. For viability assays using Enzastaurin, lymphocytes of 19 patients were cultured in complete medium in the absence or presence of Enzastaurin (1µM–50µM) for 24h. Enzastaurin (LY317615) was diluted in DMSO and added to cells in culture at the indicated concentrations. Cell viability was assessed by staining cells for 15 min with a mixture of FITC-conjugated Annexin V (Alexis Pharmaceuticals), 7-AAD (Beckman-Coulter) and fluorochrome-labelled monoclonal antibodies to CD19 and CD5 (all from Beckman-Coulter). Samples were immediately analyzed by flow cytometry. Annexin-FITC was detected on FL1, CD19-PE on FL2, 7-AAD on FL4, and CD5-PC7 on FL5 using an FC500 Beckmann Coulter flow cytometer. Enzastaurin inhibition of AKT activation.
PBMCs from a B-CLL patient (5 × 106 cells/well) were incubated with the indicated concentrations of the PKCβ inhibitor enzastaurin or the PI3K inhibitor LY294002 for 1 hour prior to the addition of 100nM PMA. Following a 20 min stimulation with PMA, cells were harvested and washed twice in ice-cold PBS. Cells were lysed in Triton X-100 lysis buffer (20mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) supplemented with protease and phosphatase inhibitors. Equivalent amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes. Activated Akt was detected using antibodies specific for pS(473)-AKT (#9271, Cell Signaling Technology) and standard Western blotting techniques. Membranes were then stripped and re-probed for total AKT (#9272, Cell Signaling Technology). Imaging and densitometric analyses was performed using UVP BioImaging AutoChemi™ System and LabWorks™ Image Acquistion and Analysis software.
Files in this Data Supplement:
- Table S1. Patient characteristics (PDF, 27.9 KB)
- Table S2. P values for comparisons of pre-tumor phenotype as depicted in Fig. 1B (PDF, 19.0 KB)
- Figure S1. Enzastaurin inhibits PMA-induced AKT activation in B-CLL cells (JPG, 47.3 KB)
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PBMCs from a B-CLL patient, containing 95% CD5+ CD19+ B-CLL cells, were pre-treated with Enzastaurin or LY294002 for one hour and then stimulated with PMA. The phosphorylation status of AKT in whole cell lysates was then analyzed by Western blot. The ratio of phospho-AKT to total AKT in each setting is indicated below each lane. PMA stimulation induces a rapid activation of AKT that can be blocked by pre-incubation with the PKCβ inhibitor Enzastaurin, but not the PI3K inhibitor LY294002. This suggests that PMA-induced PKC activation results in the downstream activation of AKT that is mediated by the β isoform and not PI3K. The blot shows representative data from one of three patients.
- Figure S2. Loss of one PKCβ allele in TCL1 mice delays but does not prevent the development of CLL (JPG, 57 KB)
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(A) Graphs showing the percentage of CD5/CD19 double positive cells as means +∕− standard deviation analyzed from the spleen, peripheral blood, bone marrow, and lymph nodes of TCL1 and PKCβ+∕− TCL1 mice, as indicated. Values of WT mice are given for comparison to normal levels. P values are indicated in the figure. (B) Analysis of gene expression of c-myc comparing expression levels in spleen of mice with CLL disease of indicated genotypes, as outlined in supplementary materials and methods. P-value by unpaired T-test was 0,037. Comparisons of gene expression for c-myc in blood, as well as ARF and Bcl-2 in blood and spleen did not show statistical differences between genotypes (all p values were >0,15).
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