Prepared And Clinical Application Of Low Density Protein Microarray For Detecting Leukemia Prognosis Related Markers

ObjectiveDrug resistance in leukemia cell is often mediated by more than one resistance-related protein. It is the result of various kinds of resistance-related proteins co-expression. The combined detection of various kinds of resistance-related proteins has positive effects on predicting the drug resistance and the prognosis evaluation in leukemia. The protein microarray technology is based on the coding micro-probes, to realize accurate, quick detection and analysis with large information. It provides a powerful tool for the high throughput parallel analysis of proteins within a single experiment.To investigate the application value of the protein microarray technology in detecting drug resistance related proteins in leukemia,we fabricated low density protein microarrays to detect the cell lysate of leukemia cell lines and bone marrow mononuclear cells from leukemia patients.The aim was to lay a foundation for the further research of the low density protein microarrays which can provide a new idea for prognosis prediction and provide individual treatment guidance for leukemia patients.Methods1. Fabrication of the protein microarrays:We fabricated the protein microarrays by antigen-antibody binding mode and chose 11 proteins as detection index which were closely related to drug-resistance of leukemia cell,including P-glycoprotein(P-gp),lung resistance protein/major vault protein(LRP/MVP),BCL-2,topoisomeraseâ…¡A, (Topoâ…¡A),topoisomeraseâ…¡B(Topoâ…¡B),glutathione-S-transferase-Ï€(GST-Ï€),vascular endothelial growth factor(VEGF),CXCR4,proliferating cell nuclear antigen(PCNA),lymphocyte function-associated antigen-1 (LFA-1),TNF-related apoptosis inducing ligand-receptor (TRAIL-R). 2. Using the protein microarrays to detect the the expression of resistance-related proteins in leukemic cell line:The cell lysate which were from human leukemic cell line HL60,K562, HL60/VCR,K562/ADM,Jurkat,Burkitt lymphoma cell line were detected by the prepared protein microarrays.3. Using the protein microarrays to detect the the expression of resistance-related proteins in leukemia patients'bone marrow mononuclear cells:The bone marrow mononuclear cells were collected from 48 acute leukemia patients and 15 control group cases.We used the prepared protein microarrays to detect the cell lysates of the bone marrow mononuclear cells.Results1. We successfully fabricated the low density protein microarrays which can simultaneously detect 11 resistance-related proteins.2. In the myeloid leukemic drug-resistant cell lines,the expression of P-gp,LR/MVP,GST-Ï€and VEGF were much higher than non-drug resistant cell lines. In the lymphocytic leukemic cell lines,P-gp and CXCR4 were highly expressed.3. According to therapeutic results the 48 acute leukemia patients were classified as refractory AML group,AML continuing remission group,refractory ALL group,ALL continuing remission group. Compared with the control group,the expression of P-gp,LRP/MVP,GST-Ï€,PCNA were significantly increased in refractory AML group(P<0.05),in refractory ALL group,the expression of P-gp,LRP/MVP,GST-Ï€,PCNA,LFA-1,CXCR4 were significantly increased(P<0.05),and the expression of PCNA was elevated in both AML continuing remission group and ALL continuing remission group(P<0.05).In refractory AML group the P-gp,GST-Ï€were highly expressed compared with AML continuing remission group,and in refractory ALL group the P-gp,GST-Ï€,LFA-1,CXCR4 were highly expressed than ALL continuing remission group(P<0.05).Compared with refractory AML group,the expression of LFA-1,CXCR4 were increased in refractory ALL group(P<0.05).We defined the cutoff value based on the detection results of control group.Taking the drug resistance judging standard as two markers higher than the cutoff value,the prediction accuracy was 81%.Patients who had 3 markers higher than the cutoff value were all belonged to the refractory and relapse group(100%).ConclusionIt is feasible to develop a new protein microarray technique for the detection of multidrug resistance-related proteins,with high throughput,simple procedure,low cost,fast detection and needed less samples. Many data can be obtained in only one experiment. Combined detection of more than one drug resistance is helpful to study the drug resistance mechanism of leukemia and predict the chemotherapy efficacy and prognosis more accurately in acute leukemia.