Translated Abstract
Acute promyelocytic leukemia (APL) is a form of acute myeloid leukemia, which has been identifid as the M3 subtype. APL accounts for 7%~27% of acute myeloid leukemia in adults. A unique chromosome translocation t (15; 17)(q22; q21) found in 90%~95% of APL patients leads to the formation of the promyelocytic leukemia retinoic acid receptor α (PML-RARα) fusion gene. The fusion protein encoded by the PML-RARα gene polymerizes and combines with retinoid-X receptor. The resultant protein complexes enhance histone deacetylase, thus repressing the transcription of the gene and disrupting the retinoic acid signal pathway. This change results in the excessive growth of malignant promyelocytes and an inhibition of granulocyte differentiation. Except the clinical expressions of anemia, fever and infiltration, the hemorrhage rate for all patients was 72%~94%. All-trans retinoic acid (ATRA), as a successful model of differentiation therapy, has improved the curative effect and extended the survival time of patients with APL. However, fatal retinoic acid syndrome and ATRA resistance in the majority of patients ultimately leads to treatment failure. Arsenic trioxide (As2O3) and tetra-arsenic tetra-sulfide (As4S4) have been used widely for the treatment of newly diagnosed and relapse APL. As4S4, which exerts similar effectiveness and less toxicity, provides not only a better quality-of-life, but is also advantageous in cytogenetic remission and PML-RARα reversion for newly diagnosed and hematological relapse patients. The exact molecular mechanism of the drug action remains unclear and warrants further investigation.
The oncoprotein SET, also known as I2PP2A or TAF-1β, is involved in many cell processes, such as DNA replication, chromatin remodeling, gene transcription, differentiation, migration and cell-cycle regulation. In our previous proteomics study, we treated the ATRA-resistant APL cell line NB4-R1 with As4S4 and obtained an As4S4-induced differential protein expression profile. There was a significant decrease in the expression of the SET oncoprotein after the treatment with As4S4 for 24 h and a disappearance after treatment for 48 h compared to the vector control, which implies the importance of SET in the regulation of signal transduction pathways and in the mechanism of APL cell apoptosis. In this study we established SET gene up-regulation and down-regulation subcellular models to analyze the effect of SET on the cell function. Then the differential expression patterns of total proteins between CON-shRNA and SET-shRNA were analyzed using proteomics which was based on two-dimensional gel electrophoresis (2-DE) separation coupled with LC-MS/MS analysis. Furthermore we identified the differential expressed proteins and discussed their biological function. There are significant meanings in discovery and application based on tumor markers and therapeutic target for hematologic neoplasms.
Objectives:
1. To investigate the effect of As4S4 on the apoptosis of retinoic acid resistant APL and its potential mechanisms.
2. To construct a eukaryotic cell expression plasmid and transfect it into 293T human embryonic kidney cells to analyze the effect of SET on the cell function.
3. To compare the protein expression profiles between CON-shRNA and SET-shRNA. Then to provide more theoretical foundations for the molecule mechanism of SET involved in the signal transduction pathways process.
Methods:
1. The NB4-R1 were cultured in vitro and divided into control group and treatment group. The apoptosis rate and cell cycle were detected by flow cytometry. The apoptotic DNA fragments were analyzed by agarose gel electrophoresis. The changes of Bcl-2, Bax, caspase-3 and PARP were determined by western blot analysis.
2. The eukaryotic cell expression plasmid was constructed and transfected into 293T human embryonic kidney cells. The cell cycle was detected by flow cytometry. The SET and PP2A expression in the pEGFP-N1-SET transfected group compared with control was monitored by RT-qPCR and western blot. The changes of Bcl-2 and Bax were determined by western blot analysis.
3. The expression vector containing SET-shRNA were constructed and transfected into NB4-R1 cells. RT-qPCR and western blotting were used to assay the silencing efficiency of SET gene and the expression of PP2A. The cell cycle distribution was tested by flow cytometry.
4. Proteome expression maps of NB4-R1 cells in CON-shRNA or SET-shRNA group were established respectively by 2-DE techniques, and differentially expressed protein pots among the maps were screened by image analysis software and artificial comparison. These differential protein pots were identified with LC-MS/MS and classified among NCBInr and SwissProt databases with Mascrot software.
Results:
1. A time-dependent increase in cell death and DNA cleavage was observed following As4S4 treatment. Changes in Bcl-2 and Bax accompanied by the activation of caspase-3 and cleavage of PARP were observed as actions of As4S4. As4S4 induced an accumulation of NB4-R1 cells in the S and G2/M phases。
2. The transient transfection of these cells led to high level expression of the SET oncoprotein. The relative PP2A mRNA and protein expression in the pEGFP-N1-SET transfected 293T cells was down-regulated compared with the pEGFP-N1 transfected cells. Overexpression of SET increased the cells percentage in S and G2/M phase. An increase in Bcl-2 and a decrease in Bax protein expression were observed in pEGFP-N1-SET transfected cells compared with the controls. As4S4 disturbed the balance between Bcl-2 and Bax.
3. The recombinant lentiviral vectors were successfully constructed and led to the SET silence in NB4-R1 cells. Flow cytometry showed that the cell cycle progression was stuck at the G0/G1 phase and sub-G1 phase. Moreover SET silence increased the expression of PP2A in mRNA and protein levels.
4. There were 13 protein pots that the quantity of differentially expressed was more than two times among 2-DE maps screened by PDQuest software. Then the differential protein spots were successfully identified by LC-MS/MS, including 5 up-regulated proteins as T-complex protein 1 subunit gamma (TCPG), Vacuolar protein sorting-associated protein 37A (VP37A), Pyruvate dehydrogenase E1 component subunit alpha (ODPA), Rho GDP-dissociation inhibitor 2 (GDIR2), Nucleoside diphosphate kinase A (NDKA), 7 down-regulated proteins as Tubulin alpha-1B chain (TBA1B), Ribose-phosphate pyrophosphokinase 1(PRPS1), Annexin A1 (ANXA1), Proliferation-associated protein 2G4 (PA2G4), Inositol monophosphatase 1 (IMPA1), Serine/arginine-rich splicing factor 9 (SRSF9), Transcription factor BTF3 (BTF3) and 1 disappeared protein as Tropomyosin alpha-3 chain (TPM3).
Conclusion:
1. As4S4-mediated apoptosis in NB4-R1 cells involves a mitochondria-dependent pathway.
2. Overexpression of the SET could promote the synthesis of the DNA and inhibit the expression of PP2A. The increase in the proportion of the Bcl-2/Bax could be disturbed by As4S4.
3. The silencing gene using SET-shRNA lentiviral vector in NB4-R1 cells could increase the expression of PP2A and interfere with the cell cycle.
4. Thirteen differential expression proteins were iditenfied successfully, in which 5 proterins were up-regulated, 7 protenis were down-regulated, 1 proterin was disappeared. Through the functional analysis, most proteins are closely associated with tumorigenesis and development.
5. Eleven of these differential expression proteins are related to carcinogenesis. They are VP37A, ODPA, GDIR2, NDKA, TBA1B, TCPG, ANXA1, PA2G4, SRSF9, BTF3, TPM3.
6. TPM3, TBA1B, NDPKA, ANXA1 and VPS37A are correlated with growth, differentiation and metastasis. PA2G4 and GDIR2 take part in regulation of proliferation and apoptosis. ODPA、TCPG、SRSF9 and BTF3 are related to the adjustment of cell anabolism. Among them, expressional and functional regulation of target proteins VPS37A, GDIR2, NDPKA, ANXA1, PA2G4、SRSF9 and BTF3 might be the protential novel therapeutic target for APL.
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