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Blood, Vol. 111, Issue 4, 1951-1961, February 15, 2008
The microtubule-targeting agent CA4P regresses leukemic xenografts by disrupting interaction with vascular cells and mitochondrial-dependent cell death
Blood Petit et al.
111: 1951
Supplemental materials for: Petit et al
Files in this Data Supplement:
- Figure S1. Pan-caspase inhibitors Z-VAD-fmk and Q-VD-OPh effectively block CA4P-induced caspase-3 activation (JPG, 33.8 KB)
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When pretreated with Z-VAD-fmk and Q-VD-OPh alone or in combination, a significant reduction (>90%) of activated caspase-3 positive cells is observed in KG1a leukemic cells treated with CA4P or staurosporine for 48 hours, confirming the efficacy of the two general caspase-inhibitors. Results are representative of three independent experiments.
- Figure S2. CA4P induces cytochrome c and ARTS translocation to the nucleus (JPG, 93.1 KB)
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Leukemic cells were incubated with CA4P (HL60 and K562: 2.5 nM, U937: 5 nM) for 48 hours and the localization of cytochrome c (A) and ARTS (B) was determined by immunofluorescent staining (green). Nuclei were visualized by DAPI staining (blue). Magnification 100×. Scale bar=5 µm.
- Figure S3. CA4P promotes Ca2+-induced calpain activation (JPG, 38.3 KB)
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Leukemic cells were incubated with CA4P at different concentrations for 48 hours. The intracellular level of Ca2+ was determined using the Ca2+ indicator FLUO-3 (A), and calpain activity was determined with cell-permeable calpain substrate Boc-Leu-Met-CMAC that fluoresces upon cleavage by calpain (B). Fluorescent signals were then assessed by flow cytometry. Results of three experiments in duplicate are expressed as the increase in cytosolic Ca2+ or calpain activity/control ± SEM (*p < 0.05 as compared with CA4P-untreated cells; n=3).
- Figure S4. CA4P induces AIF translocation to the nucleus (JPG, 90.7 KB)
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Leukemic cells were incubated with CA4P at different concentrations for 48 hours and the localization of AIF was determined by immunofluorescent staining (red). Nuclei were visualized by DAPI staining (blue). Magnification 100×. Scale bar=5 µm.
- Figure S5. CA4P decreases organ-specific leukemic cell infiltration with xenotransplanted U937 AML cells (JPG, 111 KB)
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On day 30 post-inoculation of GFP+ U937 cells, CA4P treated mice showed no evidence of leukemic cells in the spleen, liver and lung. In contrast, control mice had significant leukemic spread to the spleen, liver and lung. Magnification 40×, scale bar 50 µm.
- Figure S6. CA4P has no major toxicity on normal hematopoietic cells.
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8-week-old sex matched CD1 mice treated with CA4P at 25 mg/kg subcutaneously every other day for 4 weeks were regularly bled and WBC count (A), Platelet count (B), Hgb level (C) and absolute neutrophil count (ANC) (D) were examined. Mice displayed a mild decrease in WBC and ANC only, suggesting minimal bone marrow toxicity. (E, F) CA4P does not affect stem cell function. Survival of human CB CD34+ cells cultured in presence of CA4P after 48h was measured by annexin/PI staining (E) and ability to give rise to differentiated colonies was assessed in methylcellulose assay (F, n=3).
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