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Blood, Vol. 109, Issue 8, 3509-3512, April 15, 2007
Rapamycin derivatives reduce mTORC2 signaling and inhibit AKT activation in AML
Blood Zeng et al.
109: 3509
Supplemental materials for: Zeng et al, Vol 109, Issue 8, 3509-3512
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 66 KB)
- Table S1. Clinical characteristics of patients (PDF, 38.8 KB) -
PTEN expression was detected by Western blot analysis using the PTEN antibody that detects only the endogenous level of unmutated PTEN (cat no. 9552; Cell Signaling, Beverly, MA). The expression level of PTEN was quantitated by densitometry and presented as a ratio to GAPDH expression. Wild-type PTEN was detected in all samples studied, consistent with previously published reports.12 PB indicates peripheral blood; BM, bone marrow; MDS, myelodysplastic syndrome; RAEB, refractory anemia with excess blasts; and RARS, refractory anemia with ringed sideroblasts.
- Table S2. Correlation of biological activity to mTORC2 signaling (PDF, 28.4 KB) -
For quantitation of Western blots, densitometric analyses were performed by calculating normalized ratios of phospho- to total AKT levels. Relative changes in expression were then calculated by comparing normalized protein expression after and prior to everolimus administration. denotes a less than 30% or no change; ↓ denotes 30% or more decrease; and ↑ 30% or more increase in normalized phospho-protein expression. Biologic activity was defined as either more than 50% decrease in peripheral blood absolute blast count (for acute leukemia patients) or absolute lymphocyte count (for CLL) for more than 1-week duration, or sustained increase in platelet count for more than 3-week duration. AML indicates acute myeloid leukemia; ALL, acute lymphocytic leukemia; CLL, chronic lymphocytic leukemia; MDS, myelodysplastic syndrome; and RAEB, refractory anemia with excess blasts.
- Table S3. AKT transcriptional activity in patients treated with mTOR derivatives (PDF, 48.1 KB) -
Quantitative changes in cyclin D1, cyclin D2, and glut-1 transcription before and after treatment with rapamycin derivatives (CCI-779 or RAD001). The relative amount of the transcripts of interest relative to that of  -2-microglobulin (2M) of each sample was calculated as  Ct. Data represent mean ± SEM of the Ct values from 9 patients’ samples with decreased pAKT (6 samples from patients treated with everolimus RAD001 and 3 from patients treated with temsirolimus CCI-779); and from 4 patients’ samples with no change or increase in pAKT (2 from patients treated with everolimus and 2 from patients treated with temsirolimus). The averaged Ct values before and after treatment were then compared using Wilcoxon pairwise nonparametric method.
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