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Blood, Vol. 107, Issue 10, 4109-4114, May 15, 2006
MDM2 antagonists activate p53 and synergize with genotoxic drugs in B-cell chronic lymphocytic leukemia cells
Blood Coll-Mulet et al.
107: 4109
Supplemental materials for: Coll-Mulet et al
SUPPLEMENTAL MATERIALS AND METHODS Western blot analysis of ATM protein
B-CLL cells were lysed by sonication in buffer containing 50 mM Tris-HCl (pH 7.5), 9 M urea, and 150 mM  -mercaptoethanol, and Western blot analysis was performed as previously described20 using antibodies against ATM (Ab78, Abcam, Cambridge, United Kingdom).Fluorescence in situ hybridization (FISH)
In order to detect prognostically relevant anomalies of chromosomal regions 11q, 13q and 17p, the following fluorescent-labeled DNA probes were used in interphase cytogenetic analyses: LSI ATM (11q23), LSI D13S319 (13q14) and LSI p53 (17p13.1); all probes purchased from Abbot Vysis, Stuttgart, Germany). Sample preparation and FISH were performed according to manufacturer’s protocol. MLPA for genomic alterations
DNA was isolated and analyzed by multiplex ligation–dependent probe amplification (MLPA) using SALSA MLPA KIT P037 and P038 from MRC-Holland (Amsterdam, The Netherlands). These kits were used to determine the loss of TP53 (17p13; 8 probes), the RB1 / DLEU / MIR15-16 region on 13q14 (12 probes) and the ATM gene on 11q23 (7 probes) in DNA samples obtained from chronic lymphocytic leukemia (B-CLL).
Files in this Data Supplement:
- Table S1. Characteristics of B-CLL Patients (PDF, 38.6 KB) -
Abbreviations: WBC indicates white blood cell count (109/L); CH, chlorambucil; F, fludarabine; FCM, fludarabine-chlorambucil-mitoxantrone; CVP, cyclophosphamide-vincristine-prednisone; CA, campath; RFC rituximab-fludarabine-cyclophosphamide; P, prednisone; R, rituximab; and ND, not determined.
∗According to Binet/Rai’s classification.
†Genomic alterations were determined by FISH except for:
‡determined by MLPA or
§determined by MLPA and FISH.
- Figure S1. Apoptosis-related gene expression profile induced by MDM2 antagonists (JPG, 50.2 KB)
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Cells from B-CLL patients (patients 3 and 22) were untreated or treated with 5 µM nutlin-3a for 24 hours. Cells were lysed and expression of apoptosis-related genes was analyzed by RT-MLPA as described in “Patients, materials, and methods.” The results are shown as fold induction relative to untreated cells.
- Figure S2. Apoptosis-related gene expression profile induced by chemotherapeutic drugs (JPG, 95.2 KB)
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Cells from B-CLL patients with wild-type p53 (patients 20, 3 and 15; open bars) and mutant p53 (patients 16 and 17; filled bars) were untreated or treated with 0.8 µM doxorubicin (A) or 3 µM fludarabine (B) for 48 hours. Cells were lysed and expression of apoptosis-related genes was analyzed by RT-MLPA as described in “Patients, materials, and methods.” The results are shown as fold induction relative to untreated cells.
- Figure S3. Western blot of ATM protein in B-CLL samples (JPG, 14.9 KB)
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Cells were lysed and ATM was analyzed by Western blot as described in “Supplemental Materials and Methods.”
- Figure S4. Synergism between MDM2 antagonists and chemotherapeutic drugs in patients with ATM alterations (JPG, 39.3 KB)
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Cells from patient 14 or patient 25 were untreated (C) or treated with 0.2 µM doxorubicin (D), 2.5 µM chlorambucil (CH) or 0.4 µM fludarabine (F) without (open bars) or with (filled bars) 1 µM nutlin-3a for 48 hours. Viability was measured as nonapoptotic CD3—/CD19+ B cells as described in “Patients, materials, and methods” and expressed as the percentage of the viability of untreated cells.
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