least 30 cells were determined in duplicate cultures 14 d later. Coculture with human umbilical cord vascular endothelial cells. As described in detail previously , AML cells and endothelial cells were separated by a semipermeable membrane during 7 d of coculture . AML cell viability. This was analysed after staining with Annexin V propidium iodide as described in detail Survivin Signaling Pathway previously . Analysis of constitutive cytokine release Acute myeloid leukaemia cells were cultured in StemSpan medium in 24well culture plates for 48 h before supernatants were harvested. Soluble mediator levels were determined by Quantikine enzymelinked immunosorbent assay . Minimal detectable levels were CXCL8 35 pg ml, CXCL9 384 pg ml, CXCL10 17 pg ml, CXCL11 139 pg ml, vascular endothelial growth factor 50 pg ml, hepatocyte growth factor 40 pg ml, angiopoietin 1 345 pg ml, Ang2 83 pg ml, MMP 2 016 ng ml and MMP9 0156 ng ml.
Flow cytometric analysis of FLT3 and CD34 expression Cells were cultured for 24 h with or without intervention in standard conditions, then harvested and washed with PBS, before incubation for 10 min with 200 lg Pazopanib ml of human immunoglobulin . The cells were then washed and stained with specific monoclonal FLT3 CD135 or CD34 antibodies in PBS with 2% bovine albumin serum. Flow cytometric analyses were performed using Accuri C6 and further analyses performed by using flowjo software . Bioinformatical and statistical analysis Analysis were performed using the JExpress 2009 analysis suite . Concentrations were median normalized and transformed to logarithmic values before HSPs and cytokine levels were compared.
Unsupervized hierarchical clustering was performed with Pearson’s correlation as distance measure and average weighted linkage. All statistical analyses were performed using the Statistical Package for the Social Sciences version 15.0 and graphpad prism 4 . Correlation analyses were performed using Pearson’s correlation, for comparing paired data the tissues Wilcoxon’s signed rank test was used, while appropriate ttests or chisquare test were used to compare different patient groups. Pvalues <005 were generally regarded as statistically significant, but when multiple comparisons were done only Pvalues <001 were regarded as significant.
Results Intracellular HSP levels in primary human AML cells show a wide variation between patients The intracellular protein levels of HSP27 , HSP27 , HSP40, HSP60, HSP70 and HSP90a were quantified for AML cells derived from 75 consecutive patients. All patients showed detectable levels for all of the investigated HSPs , but there was a wide variation between individual patients for each HSP. HSP60 showed the highest levels followed by HSP70, whereas HSP90 and especially both HSP27 phosphorylated forms showed relatively low intracellular concentrations.We performed pairwise correlations between various samples by using the Pearson’s correlation test, and identified two major clusters, referred to as cluster I and II, respectively . The figure illustrates that there is a limited heterogeneity within each of the two clusters, and the intracellular protein levels of HSP40, HSP60, HSP70 and HSP90 were all significantly lower for cluster I than for cluster II . The two clusters did not differ with regard .