Prednisolone (PRED) response is highly prognostic of eventual outcome in childhood acute lymphoblastic leukemia (ALL). Based on day 8 blast count (>1000/µl), BFM investigators have consistently shown that good PRED responders have highly significantly superior outcome (EFS 80%) compared to poor PRED responders (EFS 38%). Using proteomic research tools, we investigated the intriguing mechanisms underlying PRED response using leukemia cell lines as an exploratory model, to identify potential biomarkers that can accurately reproduce the disease prognostication in childhood ALL.
Three clinically important cell lines: REH (TEL-AML1/t(12;21)), 697(E2A-PBX1/t(1;19)), RS4;11(MLL-AF4/t(4;11)) were used to examine the predictive power of early response to therapy using PRED alone. We determined that the PRED concentration of 1.0µg/ml for 48h provided the optimal separation of the three cell lines into resistant (REH) and sensitive (697, RS4;11) using MTT assay. Two dimensional gel electrophoresis (2DGE) and MALDI-TOF-TOF were used to separate and identify the proteins from pre-treated and post-treated cell lines that are differentially expressed; the PDQuest software (bio-rad) was applied to analyze the images of the three groups of gels. Then we verified the proteins of interests using western blot and RQ-PCR.
Using 2DGE and MALDI-TOF-TOF, more than 20 protein spots of interests were identified in each cell line. Most of these proteins identified were related to metabolomic, apoptosis and cell cycle regulation. Among them, eight proteins were found differentially expressed in at least two cell lines and we verified the protein of interests and its corresponding gene expression levels using western blot and RQ-PCR. These include cofilin1, PCNA, HSP27 and VDAC 1 etc. These proteins are critical partners in the steroid receptor and apoptotic pathways which provide a highly illuminating and exciting mechanism to elucidate the response to PRED in these cell lines and patient samples.
In conclusion, we have discovered several promising protein biomarkers, including cofilin1, PCNA, HSP27 and VDAC 1, that are involved in the steroid receptor and apoptotic pathways which may explain the highly prognostically importance of PRED resistance and thus disease prognosis in ALL .This will provide valuable information in elucidating the signal transduction pathway of the glucocorticoid induced apoptosis in leukemia cells and a protein signature of response.
98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA