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Blood, Vol. 110, Issue 7, 2674-2684, October 1, 2007
Inhibition of HMGcoA reductase by atorvastatin prevents and reverses MYC-induced lymphomagenesis
Blood Shachaf et al.
110: 2674
Supplemental materials for Shachaf et al, Vol. 110, Issue 7, 2674-2684
Files in this Data Supplement:
- Figure S1. The mevalonate pathway (JPG, 70.8 KB)
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The HMGcoA reductase enzyme mediates the production of many metabolites required for the post-transcriptional regulation and activation of proteins required to sustain tumorigenesis.
- Figure S2. Atorvastatin induced proliferative arrest in tumor cells but not in normal cells (JPG, 69.3 KB)
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Thymocytes from normal murine thymus and MYC-induced T-cell lymphoma were pulsed with BRDU (10 µM) and examined for cell proliferation by determining BRDU incorporation. Both thymocytes and the MYC-induced T-cell lymphoma were either not treated (MYC ON) or treated with doxycycline (20 ng/mL) to inactivate MYC transgene expression (MYC OFF). The cells were also treated with 100 µM mevalonate (Mev), 10 µM atorvastatin (AT) or 10 µM atorvastatin and100 µM mevalonate (AT + Mev). Cells undergoing specific stages of the cell cycle are boxed and labeled as the following; S-phase (upper box), G1 phase (lower left box) and G2 phase (lower right box) are indicated. The upper panel represents normal thymocytes. The lower panel represents MYC-induced T-cell lymphoma cells.
- Figure S3. Atorvastatin induced apoptosis in tumor cells but not in normal cells (JPG, 84.9 KB)
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Thymocytes from normal murine thymus (see Figure S2) and MYC-induced T-cell lymphoma were examined for apoptosis by AnnexinV-PE/7-Amino-actinomycin D (7AAD) staining. Both thymocytes and the MYC-induced T-cell lymphoma were either not treated (MYC ON) or treated with doxycycline (20 ng/mL) to inactivate MYC transgene expression (MYC OFF). The cells were also treated with 100 µM mevalonate (Mev), 10 µM atorvastatin (AT) or 10 µM atorvastatin and100 µM mevalonate (AT + Mev). During the early stages of apoptosis, cells become Annexin V positive (lower right quadrant). During the progression of apoptosis, cells incorporate 7AAD (upper right quadrant) and during the final stages of apoptosis, cells will no longer bind Annexin V but will incorporate 7AAD (upper left quadrant). The upper panel represents normal thymocytes. The lower panel represent MYC-induced T-cell lymphoma cells.
- Figure S4. Tumors expressing constitutively activated K-Ras (G12D) are not sensitive to atorvastatin treatment (JPG, 14.4 KB)
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Survival of mice transplanted with a K-Ras (G12D) induced lymphoma cell line. Mice were injected with tumor cells and when moribund with tumor were either not treated (square) or treated with atorvastatin (AT1 1 mg/kg) (triangle). Each cohort consisted of 5 mice. No significant difference in survival was found using Chi square test (AT 1mg/kg).
- Figure S5. Atorvastatin induced apoptosis in thymocytes overexpressing MYC (JPG, 86.8 KB)
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Thymocytes from mice overexpressing MYC (MYC ON) for prior to tumor development or not overexpressing MYC (MYC OFF) were examined for apoptosis by AnnexinV-PE/7AAD staining and FACS analysis, before treatment ((Nontreated) and following atorvastatin treatment (Atorvastatin (100mg/kg)).
- Figure S6. Kinetics of tumor elimination by bioluminescence imaging (BLI) (JPG, 70.7 KB)
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(A) Tumor cells expressing luciferase were injected into mice and imaged using BLI. Mice were either not treated (MYC ON) or treated with doxycycline (100 µg/mL) in their drinking water (MYC ON then OFF) or treated with atorvastatin (AT 100 mg/kg) three times weekly. (B) Representative images of mice prior to treatment and after 8 days of treatment are indicated.
- Figure S7. Schema (JPG, 91.6 KB)
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Signal transduction pathways affected by atorvastatin treatment that may result in MYC inactivation.
- Figure S8. Atorvastatin treatment of human Burkitt lymphoma results in the loss of phosphorylation of ERK1/2 and MYC (JPG, 22.1 KB)
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Human Burkitt lymphoma cell line ST486 was treated with atorvastatin (AT (10 µM)), mevalonate (Mev (100 µM)) or atorvastatin plus mevalonate (AT + Mev) and analyzed by Western blot analysis to assess the phosphorylation state of ERK1/2 and MYC.
- Figure S9. Atorvastatin treatment inhibits HMGcoA reductase expression in MYC-expressing tumors (JPG, 39.5 KB)
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(A) MYC regulated T-cell lymphomas were analyzed for the expression of HMGcoA reductase by Western blot analysis. Tumor cells overexpressed MYC (MYC ON) or not (MYC OFF) after doxycycline treatment (20 ng/mL). MYC-expressing tumors were treated with 10 µM atorvastatin (AT), 100 µM mevalonate (Mev), or 10 µM atorvastatin plus 100 µM mevalonate (AT + Mev), and (B) HMGcoA reductase gene expression was analyzed in MYC regulated T-cell lymphomas before and after MYC inactivation. The gene expression profile of the tumor cells before and after MYC inactivation was analyzed by microarray (data not shown). The gene expression intensity was normalized over all arrays in the experiment and centered by the mean of total intensity. The data represents 3 independent experiments. (C) HMGcoA reductase expression was measured by QT-PCR and normalized to the expression of ubiquitin C (ubc). Error bars are SD.
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