Study Of Function Of OAZ Gene During Differentiation Of K562 Cells

The polyamines (putrescine,spermidine,and spermine) are essential for allliving cells. They are largely bound to negatively charged molecules such as DNAand RNA and play an important role in cell proliferation, gene expression, cellproliferation and differentiation. The absence of polyamines will lead cellproliferation stepping down, while rising of the polyamines level results incancerogenesis and apoptosis.Ornithine decarboxylase(ODC)is the rate-limitingenzyme in polyamine biosynthesis and catalyzes ornithine to putrescine. ODC whichhas a short half-life of several minutes to 1 hour(the shortest half-life of any knownenzyme) acts an important key enzyme in polyamine biosynthesis and its activity canbe regulated at level of transtription, translation and posttranslation. Ornithinedecarboxylase antizyme inhibits ODC and then directs its degradation by the 26Sproteasome. Overexpression of OAZ in cells not only decreases the level ofpolyamines, but also coincides with growth inhibition. Furthermore, OAZ candecrease the concentration of polyamine inside cell by inhibiting polyamine uptakeand stimulating polyamine excretion. There are at least three isoforms in OAZ family,in which the important isoform is OAZ1 composed of 227 amino acids. TheC-terminal domain of OAZ1 is sufficient for ODC binding whereas the N-terminal region is important to promote degradation of ODC. Isoform-2 of antizyme (OAZ-2)does not appear to stimulate ODC degradation at the proteaome but does maintain theability to negatively regulate polyamine transport. A third isoform,OAZ-3,is limitedto only one cell type, testis germ cells, where its expression occurs at a particularstage of spermatogenesis, with implications fertility. A unique ribosomal frameshiftmechanism is needed for the expression of functional antizymes. All antizymes havetwo overlapping open reading frames, a short ORF1 with a translational start codonand a stop codon at the frameshifl site and another ORF2, which encodes most of theprotein but lacks a discrete start codon. Therefore, translation of a functional,full-length antizyme requires a +1 ribosomal frameshift. The nucleotide sequencesurrounding the frameshif site is highly conserved which was particularly helpful inidentifying antizyme orthologues from various organisms. The +1 translationalframeshift is induced by polyamines via a mechanism that is not completelyunderstood. When polyamine level rises, it can promote the frameshift and induce theexpression of OAZ, which can reduce the polyamines in a feedback mechanism. Thismechanism serves to prevent extreme fluctuation in polyamine levels, which arethought to be toxic. Many experiments prove that OAZ has the effect of anti-tumorcharacteristic while the degradation of ODC is associated with its ability of growthinhibition of cells. Overproduction of OAZ in a variety of cell types, includingmalignant oral keratinocytes, heaptoma cell lines, and prostate cancer cells, coincideswith growth inhibition and cell cycle arrest in the G1 phase.Furthermore, overexpression of antizyme in mouse skin cancer models hasbeen shown to result in tumor suppression. Our goal is to gain the mutation OAZgene without the site of frame-shift using site-directed mutagenesis method. Then wetransfect the pEGFP-N1-OAZ-mutation into K562 cells after construction of therecombinant between mutation OAZ gene and pEGFP-N1 vector. Study of the biology effect of OAZ gene upon leukemia cells undergoes through the assay ofgrowth and differentiation of K562 as well as the expression of cyclin D1.K562 cell is highly malignant human immortalized erythroleukemia cellderived from a patient with chronic myelogenous leukemia(CML).Philadelphiachromosome(Ph) is the result of a t(9:22)reciprocal chromosomal translocation and isthe malignant clonal marker of CML. At the molecular level,Ph translocation leads aprotein tyrosine kinase(PTK),to a new position downstream of the gene bcr gene on22q11 and forms a new bcr-abl fusion gene which encodes a chimeric protein P210.The k562 cells are bipotential and can be induced differentiation by many inducers,which represent an important in vitro model for basic studies of leukemogenesis.K562 can be induced express embryonic and fetal hemoglobins, so elucidation of themechanism will do well to therapy sickle cell disease andβ-thalassemia syndromes.The concept of leukermia therapy has changed from the traditional method of killingtumor cells to induce leukermia cells differentiation. Side effect of apoptosis willoccur while the K562 cells are induced differentiation by many chemistry materials,such as retinoic acid, Phorbol ester, hydrea, which promote cells apoptosis when theyinduce K562 cells differentiation. Some experiments show that there is no connectionbetween inhibitions of cell growth by OAZ with apoptosis phenomenon. This gives anew thinking about induction differentiation of K562 cells.Study methods: Up-regulation of OAZ has been identified in erythroiddifferentiation of K562 cells induced by Hemin through the method of fluorescencequantify PCR. The result indicates up-expression of OAZ is associated with K562erythroid differentiation. According to the dates in Genebank, we find out the wholecDNA sequence of OAZ, and design the primers for amplifying the fragments ofORF2 and whole sequence of OAZ. The corresponding restriction site has beenmodified on the 5'end of the primers so that we can use the restriction endonuclease to cut the PCR products and recombine them to expression vector of pET32a andpEGFP-N1. The recombination gene of OAZ and vectors were named ofpET32a-OAZ-ORF2 and pEGFP-N1-OAZ after sequencing. Ecoli BL21 wastransformed with pET32a-OAZ-ORF2, and the OR2 protein was expressed afterIPTG induced. After purification of the combination protein, we further immunizerabbit to prepare polyclonal antibody. Then the T base at the site of frameshiftTCCTGATG was deleted by site base mutation method so that there was no overlappingbetween the two ORFs within OAZ fragment. The OAZ protein can occur withoutany spermine induced which can decrease the interfere of experiment result byxenobiotic added, pEGFP-N1-OAZ-mutation was named after sequencing andtransfected into K562 cells by lipfectin2000. K562 cell line later named K562_OAZwhich expresses OAZ protein stably was gained by filtration of G418. At the sametime, we designed 27 mer siRNA which was to be transfected into K562 andK562_OAZ to inhibit the expression of OAZ. In our experiment we use the method offluorescent quantitation PCR to detect the efficiency of interference of siRNA. Weforced the target OAZ gene up-regulation and down-regulation in K562 cells, growthassay, differentiation assay, cell cycle detection and expression of Cyclin D1 analysiswere performed to discuss the function of OAZ in K562 cell growth anddifferentiation.Results: When treated the K562 cells with 50mmol/L Hemin for 24, 48 and72 hours, we detect stepping down of the cell growth, increasement of BZ(+) cellsand hemoglobin. These dates show that K562 cell has been undergone erythroiddifferentiation. The expression of OAZ mRNA rises after hemin treatment using themethod of fluorescent quantification PCR. The expression of OAZ rises of 18.67％,51.12％and 92.11％respectively when treated the K562 cells with hemin for 24,48and 72 hours. To further study the biology effect of OAZ on K562, we construct recombination DNA of mutation OAZ and eukaryotic expression vector pEGFP-N1.The recombination DNA was transfected into K562 cells after sequencing. Whentreated with OAZ, we detect stepping down of the cell growth, increase of BZ(+)cells. And we find that these k562 cells were arrested in G1 phage, coincidence withthe low expression of cyclin D1. To test the confidence of experiment, we designed27met siRNA of target gene OAZ and transfect the siRNA into K562_oaz to inhibit theexpression of OAZ. The inhibition of OAZ expression in K562_OAZ treated withsiRNA for 24,48 and 72 hours was 46.04％,55.41％和50.07％respectively, whichindicates the efficiency of siRNA was credible. Positive rate of benzidine staining inK562_OAZ decreased to 4.3％±0.5％(24h),4.1％±0.7％(48h) and 4.6％±0.9％(72h) from6.6％±1.0％(0h) following with the down expression of OAZ. Furthermore, thegrowth of K562 cells rose, which indicated K562 cells had the tendency of recoveringits malignant characteristic. The same time we detected the up-expression of cyclinD1 by RT-PCR, which indicated the inhibition of cell cycle was dismissed. K562 celltreated with siRNA had the tendency of recovering its malignant proliferation,coincidence with the decrease of OAZ and the increase of cyclin D1.These dateshowed that OAZ had the ability to influence K562 cells.Conclusions: (1)we first use the method of fluorescent quantification PCR todetect the expression of OAZ in K562 cells erythroid-induced by hemin.The dateshows that the up-expression of OAZ is coincident with the erythroid differentiationof K562 cells.(2)Prokaryotic expression vector pET32a-OAZ-ORF2,eukaryoticexpression vectors pEGFP-N1-OAZ and pEGFP-N1-OAZ-mutation are constructedand the ORF2 protein was expressed in Ecoli BL21,while the whole OAZ proteinexpressed in K562 cells.(3)we first use the 27 mer siRNA to interfere the expressionof OAZ and confirmed the inhibition of interferce by fluorescence quantification PCR,which gives us a new choice to study the function of OAZ. (4)The results show that there is a dependability about OAZ gene and differentiation of K562 cells. Based onthe ability of inhibition of tumour cells'malignacy growth, OAZ has the tendency topromote the K562 cells undergoing erythroid differentiation.