The Experimental Research Of P21, P27, P16, P15 Gene (Cyclin-denpendent Kinase Inhibitors) Clone And STI571 To Treat Chronic Myeloid Leukemia

Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder due to an acquired abnormality in a pluripotential hematopoietic stem cell. CML is cytogenetically marked by the Philadelphia (Ph) chromosome, which originates from a reciprocal translocation between chromosome 9 and 22 and is molecularly marked a chimeric bcr-abl gene, resulting from juxta-positive of the abl proto-oncogene on chromosome 9 with the bcr gene, which is normally located on chromosome 22. The chimeric bcr-abl gene expression an 8.5kb hybrid mRNA transcript giving rise to a 210-KD fusion protein (P210bcr-abl) with increased tyrosine kinase activity. P210bcr-abl plays a key role in the pathogenesis of CML. The continuous cell line K562 was established from the pleural effusion of a 53-year-old female with CML in terminal blast crisis, and was a human erythroleukemia line, contained Ph chromosome. K562 cell line was attained widespread use in vitro target for CML assay.For the reason of Bcr-abl tyrosine kinase activity enhanceing myeloproliferation, to specially inhibit its function has become emphasis in the therapy of CML. STI571, a tyrosine kinase inhibitor used in clinical, was reported to inhibit the tyrosine kinase of BCR-ABL with high selectivity, so it had optimal therapy function to K562 cell line and set up an epoch era in molecule target therapy. Its function to treat CM1 is just as follows: specially inhibits the tyrosine kinase of c-abl, induces apoptosis and diffentiation of bcr-abl+ cell, changes the expression of downstream gene of bcr-abl, affects the adherent function of tumor cell andregulates the expression of cyclin proteins. But multidrug resistance, recurrance and poor curative effect to CML acute phase were its major problem, so to investigate combined therapy is a goal for future development wayRecently, scholars find that it had close relation between leukemia and the malajustment of cell cycle. Cell cycle is regulated by cyclin/CDK and CKIs. Cyclin/CDK has positive funtion to cell cycle and CKIs has negative function to cell cycle. CKIs has two groups, one is INK4 family, including pl6,pl5,pl8 and pl9,the other is KIP family including p21,p27 and p57. They can inhibit the function of cyclin/CDK. In all these p21, p27, pl6and p15 are the most important gene, and regarded as tumor suppressor genes. Its deletion and mutation have important effect to inducing cell's milagant proliferation. We know already that deletion of pl6 gene and function absence of p21 in K562 cell, so keeping up on the four gene' function in CML has more important meaning. In the research, we had contructed express vectors of the four genes, and the vector was transfected into K562 cell, then STI571 was united to each express vector differently. By that we could understand its function to K562 cell in cell cycle, proliferation and apoptosis. That is a new experiment in CML therapy.The four genes were obtained by RT-PCR, and its sequence was approved to be correct, then P21-pcDNA3.1, P27-pcDNA3.1 P16-pcDNA3.1 and P15-pcDNA3.1 vector were constructed and transfected into K562 cell line. After selected with G418, P21-pcDNA3.1-K562, P27-pcDNA3.1-K562, P16-pcDNA3.1-K562 and P15-pcDNA3.1-K562 cell clone that stably expression P21, p27, pl6 and pl5 protein were constructed. Proteins expression was identified by western blot. The cell' surviving rate were tested by MTT, cell cycle and apoptosis were tested by Flow cytometry. Meanwhile research of gene clone was united with STI571.The results and conclusions of research is just as follows:1. STI571 can inhibit K562 cell proliferation, induce apoptosis, and cell is arrested in G0/G1 phase.The surviving rate of k562 cell was tested by MTT. With prolonging of time or increasing of concentration of STI571, the surviving rate of k562 cell declined. It means the surviving rate relying on time or concentration. Cell cycle and apoptosis were tested by Flow cytometry. Conparing to control, after K562 cell affected by STI571, the cell in Gl phase increased and in S phase decreased appare...