A Genomic and Proteomic Research for the Regulatory Networks of ATRA-Induced Differentiation of Acute Promyelocytic Leukemia Cells.

The treatment of acute promyelocytic leukemia (APL) with all trans retinoic acid (ATRA) has set a model for the differentiation induction-based new therapy. Although a substantial body of data related to ATRA-induced differentiation mechanisms has been obtained, the molecular events down stream of RA receptor complexes remain obscure. With the goal to get further insights into regulatory networks underlying ATRA-induced differentiation in APL, we applied 2DG, MS and cDNA microarray combined with bioinformatics analysis such as self-organizing map(SOM) to profile NB4 cells treated with ATRA over 3 time points (0h, 12h, 48h). Our results showed that cell cycle was arrested and proliferation was suppressed. For instance, ;the Origin recognition complex subunit 3 involved in initiating DNA replication that make the cell switch to S phase was downregulated significantly at protein (67.55 times) and mRNA levels; Breast cancer type 2 susceptibility protein (BRCA2) that can interact with BRAF335 as the checkpoint of G2 / M phase was upregulated while mRNA expression unchanged. Our results showed that the expression of many proteins associated with Ca²⁺ signal pathway such as IP3 receptor isoform 1 and phospholipase C-β changed upon induction with ATRA, implying that Ca²⁺ signal pathways may play an important role during ATRA-induced APL cell differentiation. Our results also indicated that the cell apoptosis was inhibited in the process of ATRA-induced differentiation. For example, BFL1, one of the BCL-2 family and the main apoptosis repressor in neutrophils, was upregulated at protein and mRNA levels. BCL2-like13 is one of the BCL-2 families and its overexpression initiated the cell apoptosis pathway by activating caspase-3. It was downregulated at protein level. Our results revealed that the phosphopentose pathway and ribonucleotide synthesis were repressed obviously. The protein expressions of the key enzyme of phosphopentose pathway - Glucose-6-phosphate dehydrogenase and rate-limiting enzyme of purine nucleotide de novo synthesis - AICAR methylferase and adenylosuccinate synthetase were all downregulated remarkably. Moreover, the protein expression of nitricoxide synthase 2A was upregulated 236 times after 12 hours of treatment with ATRA. The dramatic changes imply that the effect of NO on the differentiation induced by ATRA deserves more investigation. In brief, the data presented above indicated the expression of a large number of genes and proteins is modulated upon effect of ATRA during ATRA-induced APL cell differentiation, resulting in a complex and well-organized network to ensured cell reprogramming.