Effect Of Ornithine Decarboxylase Antizyme1on The Erythroid Differentiation And Apoptosis Of CML Cell Line K562

Ornithine decarboxylase antizyme (OAZ), was first known as the negative regulator of Ornithine decarboxylase (ODC, rate-limiting enzyme of the polyamine biosynthetic pathway). Ectopic expression of OAZ in solid tumor reduce malignant proliferation, induce cell cycle arrest, demonstrate its anti-tumor ability and potential value as tumor therapeutic target. So far, the understanding of OAZ function mechanism mainly focus on the following three aspects. First, there is an unique frameshift mechanism during the translation process of OAZ, that is when ribosome come to the termination codon of ORF1(TCCTGATG), polyamine could regulate it continue the translation process by moving one base forward so as to translate into an active antizyme. Therefor, we modified the frameshift site (TCCTGATG→TCCGA TG) so as to eliminate the interference effect arose from allogenic materials. Second, OAZ function as a hub in regulating polyamine homeostasis. OAZ bind to ODC with high affinity and then target ODC to26S proteasome, induce its degradation in an ubiquitin-independent manner. OAZ may also regulate polyamine transport carrier on the membrane, reducing cell uptake of exogenous polyamine and promoting its discharge. The adjustment of polyamine homeostasis caused by OAZ finally participate in vital cell activities. Finally, the ubiquitin-independent degradation manner induced by OAZ is also occur in some other important cellular macromolecules, such as cyclin D1, Smadl and Aurora A kinase, suggesting it should be one key mechanism for OAZ to participate in regulating cell functions.Chronic myeloid leukemia(CML), a malignant cloning diseases derived from hematopoietic stem cell, characterized by the balanced reciprocal translocation of chromosomes9and22(t(9;22)(q34;q11)), generating an enhanced BCR/ABL tyrosine kinase oncoprotein. It interfere normal cell process by spontaneous function through multiple signal transduction, resulting in the inhibition of cell differentiation and the tolerance of cell apoptosis, it is responsible for the pathogenesis of CML. With the in-depth study of tumor disease, induced differentiation therapy has become a new tumor therapeutic strategy. The application of differentiation therapy in cute myelogenous leukemia and myelodysplastic syndrome has been proved with exact curative effect and is stepping into the clinical research stage, while for CML is still in its infancy. K562cell is a good model for in vitro study of leukemia, it can be induced to benign direction differentiation by a variety of inducers, previously study confirmed that hemin could induce it to the specific erythroid differentiation. The RD-PCR method,which was created by Ma Wen-li etc., was used to screen out the differential expression cDNA fragments in K562cells treated with hemin. The result showed that OAZ1was associated with the erythroid differentiation process, yet the exact function and mechanism were remind to be further study. In addition, we also found that OAZ1may involve in cell apoptosis. Therefore, this subject aimed to clear OAZ1’s function and molecular mechanism in inducing K562cell erythroid differentiation and apoptosis, provide the theoretical basis for OAZ1as the potential therapeutic target for CML treatment.Method:The lentivirus vector (pLVX-Neo-OAZ1-IRES-ZsGreen) was constructed, which the inserted gene was modified at the frameshift site (TCCTG ATG→TCCGATG) and was the artificially synthesized fragment(687bp). The packaged lentivirus were then used to transfect K562cells. We obtained the K562/OAZ1and K562/GFP stable cell lines after G418or FACS screening.OAZ1overexpression efficiency was assessed by Real-time RT-PCR and Western blot technology. Collecting three groups of cells, the test group K562/OAZ1, the negative control group K562/GFP and the untreated group K562, used for the following experimental analysis.We obtained the potential effect of OAZ1in inducing erythroid differentiation by detecting two surface markers (CD71and GPA) and also by the benzidine staining. To do a tentative exploration of its mechanism, the relevant genes (GATA1, BCR/ABL and TGFβ) which were bound up with the differentiation and canceration of leukemia were detected by Real-time RT-PCR, here we added the Hemin-induced group. We observed that it’s hard to maintain the overexpressing cell line, the green fluorescent rate and intensity decreased as the cells passaged, possibly related to antizyme’s inhibition ability, the apoptosis rate was analyzed by the Annexin V-PE/7-AAD kit. Moreover,OAZ1mRNA was measured by Real-time RT-PCR in43cases with CML and23controls so as to verify the previous in vitro cell experimental conclusion. The further understanding of its molecular mechanism in inducing K562cell erythroid differentiation and apoptosis were followed by the detection of the focused human leukemia PCR array. The human leukemia RT2profiler PCR array, which analyzes84key genes commonly involved in leukemia initiation and progression, it can be easily and reliably analyze the expression of the focused panel of genes with this array. Monitoring the expression of these genes that underly the overexpression of OAZ1in K562cells guide to a better understanding of its molecular mechanisms in leukemia. According to the results of chip,4favorable differentially expressed genes were verified by Real-time RT-PCR and the possible regulated mechanism caused by OAZ1were discussed.Result:The recombinant lentivirus vector was validated by double enzyme digestion and sequencing identification, proven to be successfully constructed. The overexpressing cell line were also proven to be successfully constructed by Real-time RT-PCR and Western blot analysis. The positive rate of double labeling (CD71+/GPA+) was (11.22±2.09)%in K562/OAZ1group, which was significantly higher than that in untreated cells (4.07±1.04)%or in cells treated with empty virus (1.79±2.36)%(P<0.01). The positive rate of benzidine staining was (14.037±0.083)%, which was also significantly higher than the other two groups (P<0.01). These results indicated that OAZ1could significantly increase the erythroid differentiation of K562cells. Quantitative results indicated that OAZ1had more significant function on TGFβ than GATA1or BCR/ABL. In conclusion, OAZ1could effectively induce the erythroid differentiation of K562cells and this induction mechanism is possible related to TGFβ signaling pathway. The apoptosis rate detected by the Annexin V-PE/7-AAD dual staining kit showed that K562/OAZ1was (23.54±2.23)%, enhanced significantly than those of the K562(8.21±2.18%) and K562/GFP (15.91±2.23%)(p<0.01), indicated that OAZ1could increase the apoptosis of K562cells. OAZ1, TGFβ mRNA level were significant down-regulation in CML patients, revealing the potential antitumor effect of OAZ1and confirming the gene expression correlation between OAZ1and TGFβ, which were coincidence with the result gained from in vitro cell experiment. We compared expression profiles of the OAZ1overexpression cells with the negative control by using the human leukemia PCR array. Out of84genes on the leukemia array,19genes showed differentially expression with two fold or higher changes, with10up-regulated genes and9down-regulated genes. Although not all of the changes were developed toward a favorable direction, the differentially expressed genes caused by OAZ1 overexpression in K562cells finally led to a significantly elevate in cell erythroid differentiation and apoptosis. Based on the array results, four favorable differentially expressed genes (CXCL10, DAPK1, CDH13and IKZF3) were selected and verified by Real-time RT-PCR. Consistent with the results obtained from the leukemia array, CXCL10, DAPK1and IKZF3were up-regulated significantly except for CDH13.TGFβ (transforming growth factor β), could gather the hemoglobin in K562cells, coordinating with other factors to participate in cell differentiation, it’s confirmed that TGFβ/Smads signal transduction system involve in cell growth inhibition during hematopoiesis, the high expression of TGFβ could effective regulate the malignant proliferation and differentiation of leukemia cell. CXCL10, a chemokine of the CXC subfamily which binds to CXCR3receptor results in pleiotropic effects, including chemotaxis, apoptosis, proliferation and mediation of angiostatic. DAPK1, is a death-associated protein kinase which was found with aberrant methylation of CpG island in CML. IKZF3, a member of the Ikaros family of zinc-finger proteins, function as a transcription factor that is important in the regulation of B lymphocyte proliferation and differentiation.OAZ1through direct or indirect function to regulate these genes expression level, breaking the pathological balance of cell and restoring the normal hematopoiesis, thereby result in the erythroid differentiation and apoptosis of leukemia cells, whereas the regulation mechanism remains to be further proved.Polyamine, a common multifunctional organic cation in organism(including the spermine, spermine and putrescine), could combine with negatively charged biological macromolecules, such as DNA, RNA and phospholipids in the cell. The lack of polyamine lead to cell growth inhibition, while excessive of it lead to carcinogenesis or cytotoxic, it’s significance to maintain polyamine homeostasis. Yamamoto D etc. found that the ectopic expression of OAZ in hamster oral cancer cells resulted in global hypomethylation of genome DNA, along with the regain expression of some tumor suppressor genes, growth or differentiation related genes. We know that, there is a metabolic pathway coupling between polyamine synthesis and methylation reaction. dcSAM is an aminopropyl donor of polyamine biosynthesis but it also acts as a competitive inhibitor of SAM virtually in all methylation reactions. Inhibition of ODC activity with high expression of OAZ1leads to polyamine depletion and dcSAM accumulation, which inhibits methylation reactions, resulting in global hypomethylation of genome DNA. Our results demonstrate that several genes (or even more genes) directly or indirectly up-regulate after OAZ1overexpression, whether if the mediation of polyamine and genomic methylation state caused by OAZ1done to them remain to be confirm.Conclusion:(1) OAZ1could effectively induce the erythroid differentiation of K562cells (2) OAZ1overexpression could effectively increase the apoptosis rate of K562cells, it may involve in the induction of cell apoptosis.(3) OAZ1mRNA has been proven to be down-regulation in CML patients, verifying the potential antitumor effect of OAZ1.(4) OAZ1overexpression in leukemia K562cells cause a large number of genes expression changes, we verified the difference expression of the focused genes TGFβ,CXCL10, DAPK1and IKZF3.