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Blood, Vol. 112, Issue 6, 2439-2449, September 15, 2008
Interruption of the Ras/MEK/ERK signaling cascade enhances Chk1 inhibitor–induced DNA damage in vitro and in vivo in human multiple myeloma cells
Blood Dai et al.
112: 2439
Supplemental materials for: Dai et al
Files in this Data Supplement:
- Figure S1. The FTI R115777 blocks UCN-01-induced Ras → ERK1/2 activation, leading to increased γH2A.X expression and lethality in MM.1S cells and their dexamethasone-resistant counterparts (MM.1R), as well as H929 cells (JPG, 120 KB)
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(A) Cell lysates (200µg protein per each) from untreated U266, RPMI8226, MM.1S or MM.1R cells were loaded with 100µM GTPγS or 1mM GDP for 30min at 30°C, and subjected to a Ras activation assay as described in “Methods.” Ras activity is reflected by amount of Ras-GTP pulled down by Raf-1 RBD. (B) MM.1S and MM.1R cells were exposed to 50-150 nM UCN-01 for 24h, after which Ras activation assays and Western blot analysis were performed to monitor Ras activity and ERK1/2 phosphorylation, respectively. (C) MM.1S and MM.1R cells were treated with UCN-01 (100 nM) ± R115777 (5 µM) for 24h, after which Western blot analysis was performed to monitor expression of total and phospho-ERK, as well as γH2A.X. (D) MM.1S and MM.1R cells were treated with 100 nM UCN-01 (UCN or U) ± 5 µM R115777 (R115 or R) for 24h, after which the percentage of annexin V+ cells was determined by flow cytometry. (E) H929 cells were exposed (24h) to 100 nM UCN-01 or 2 µM Chk1i in the absence or the presence of 5 µM R115777, after which cells were lysed and subjected to Western blot analysis using the indicated primary antibodies. (F) Alternatively, after 48h-treatment, the percentage of apoptotic cells was determined by flow cytometry. For panels S1A-C and S1E, Protein input per condition = 400 µg (for Ras activation assay) or 30 µg (for Western blot analysis). Blots were then stripped and reprobed with antibodies directed against β-actin to ensure equivalent transfer and loading. Two additional experiments yielded equivalent results. For panels S1D and S1F, results represent the means ± S.D. for three separate experiments performed in triplicate. * = significantly greater than values for treatment with Chk1 inhibitors alone (* P<0.05 and ** P<0.01).
- Figure S2. Co-treatment with R115777 and Chk1 inhibitors induces a dramatic increase in γH2A.X nuclear foci formation in U266 cells, while largely spares CD138− bone marrow cells (JPG, 127 KB)
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(A) U266 cells were treated with 150 nM UCN-01 or 2 µM Chk1i ± 5 µM R115777 for 24h, after which cytospin slides were stained with Alexa Fluor 488-conjugated γH2A.X antibodies. Images were captured at 20×/0.50. Results of a representative experiment are shown; two additional studies yielded equivalent results. Arrows indicated cells with γH2A.X nuclear foci. (B) CD138− cells were separated from the bone marrow of a patient (#1) with MM. Cells remained untreated or were exposed (16hr) to 150 nM UCN-01 (U) or 2 µM Chk1i ± 5 µM R115777 (R115). Cells were then stained with Alexa Fluor (AF) 488-conjugated phospho-H2A.X (Ser139) antibodies to monitor phospho-H2A.X foci formation and photographed at 60×/1.40 under oil.
- Figure S3. Co-administration of Chk1 inhibitors with R115777 or PD184352 increases γH2A.X expression in primary CD138+ MM cells, but not in CD138−bone marrow cells (JPG, 73.3 KB)
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(A, B) CD138+ and CD138− cells were separated from the bone marrow of a patient (#2) with MM as described in Materials and Methods. Cells were then either untreated or exposed (6hr for CD138+ or 24hr for CD138−) to 150 nM UCN-01 (UCN) or 2 µM Chk1i in the absence or the presence of either 5 µM R115777 (R115) or 5 µM PD184352 (PD184). After treatment, cells were harvested and stained with Alexa Fluor (AF) 488-conjugated phospho-H2A.X (Ser139) antibody for flow cytometric analysis. Values to the right of the histograms represent the percentage increase, for each condition in relation to untreated (UT) control cells, in cells displaying relatively high fluorescent intensity (M1-gated population, percentage of total events).
- Figure S4. The R115777/UCN-01 regimen suppresses tumor growth in a murine bioluminescent RPMI8226 xenograft model (JPG, 76.4 KB)
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(A) Nude mice were inoculated subcutaneously into the right rear flank with 5 × 106 RPMI8226 cells stably transfected with a luciferase gene. After tumors were visible, 50 mg/kg R115777 ± 0.5 mg/kg UCN-01 were administrated i.p. daily for 10 days. Tumor growth was monitored by sequential scanning after i.p. D-luciferin (150 mg/kg) using an IVIS 200 Imaging System (Xenogen, Hopkinton, MA) every two days during drug treatment. Images were captured 4hr after the final dose. Results are representative for each group (n = 4).
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