The Study On The Binding Of Upstream Of P16 Gene And The Nuclear Matrix Protein In Acute Leukemia Cells

In eukaryotic organism, cell cycle is respectively regulated by different cyclins(CYC) and cyclinc dipendent kinase(CDK) in Gl/S and G2/M checkpoints. The free of the Gl/S and G2/M checkpoint control contributed to the tumor. Those cells which have genetic defect and selective proliferation of malignant cell can result in tumorigenesis. Protein 16 is an inhibitor of CDK4 coded by p16 gene. Naturally, Protein16 and CYC-D alternately inhibit activity of CDK4 and maintain cell proliferation and differentiation. The factors which reduce pl6 gene expression can accelerate tumorigenesis. Gene expression was regulated by transcription factors recognizing and binding to cis-acting element. Matrix attached regions(MARs) is a DNA element that binds specifically to the nuclear matrix and as a novel cis-acting element had been increasingly thought much of. Eukaryotic genomes bind to nuclear matrix by MARs to maintain DNA loop stabilization. Nuclear matrix play an important role in replication, transcription and post-transcriptional processing and closely correlated with tumor development. Recent reports have shown that nuclear matrixproteins closely correlated with leukemia, with the ultratechnique development, nuclear matrix was regarded as an important molecular basis in leukemia pathogenesis.The reports on correlation between pl6 gene in leukemia concentrated on the structural sequences. The correlation between leukemia nuclear matrix proteins and pl6gene has not been reported. This research aimed to detect the change of nuclear matrix proteins in acute leukemic cells and the correlation between the upstream of pl6 gene and nuclear matrix proteins in acute leukemia, and determine whether the -869bp fragment in the upstream of pl6gene include matrix attached region. Materials and MethodsThe 890bp fragment (including -869 fragment in the upstream of exon 1 of pl6 gene) was collected by agarose gel electrophoresis from the pi 6 promoter-luc-pGL2-Basic plasmid treated by EcoR I and Hindlll.The 890bp fragment as probe was labeled by random priming with digoxigenin. The probe's sensitivity was detected by DNA dot blot. Twenty-two samples were obtained from the patients in the Department of Hemotology of the First Affiliated Hospital of Zhengzhou University, 5 cases were ANLL-M2, 1 case was ANLL-M3, 6 cases were acute ALL, HL-60 cells were provided by Department of Histology and Embryology of Zhengzhou Medical University, 10 cases in control group were patients with normal bone marrow. The purified nuclei were collected by sucrose gradient centrifugation. Nuclear matrix proteins were prepared by high-salt extraction methods and separated by SDS-PAGE. The purified NMPs were electrotransferred onto nitrocellulose membranes for DNA-Protein binding(Southwestern blot). The bands were analyzed by Genetools software. Results were analyzed by student T-test and One-way ANOVA using a software SPSS10.0 and the standard of statistic significance was established as a =0.05.Resultsl.The probe labeled with digoxigenin was highly sensitive with a sensitivity at lpgM. Therefore, the probe was qualified for the requirement of the experiment..2.The nuclei obtained by sucrose gradient centrifugation were dyed and observed, there is a few cell fragments. Taking count of 200 cells, the number of purified nuclei without cytoplasm contamination was more than 190. The rate of purified nuclei was more than 95%.3.The composition of NMPs between acute leukemia group and control group was significantly different. Nuclear matrix proteins separated by SDS-PAGE, among all the protein bands, the band with MW35IQK 40KIK 60KD were the more heavy bands.Compared with control samples, the 40KD protein band was significantly reduced in the primary acute leukemic cells, and the 60KD protein band was significantly increased in primary acute leukemic cells and HL-60 cells. The protein level at 35KD was continued in all samples. There was no significant difference at protein level of bands with 40KD and 60KD between acute leukemic cells from ANLL and ALL patients to HL-60 cells.4.The binding of upstream of pl6gene and NMPs between acute leukemia group and control group was significantly different. The major bands were 40KEK 50KEK 60KDMW proteins in control group, while 60KDn 70KDMW proteins in acute leukemia group, the number of probed protein bands in acute leukemia group was lesser than that in control group. The labeled protein bands at 40KEK 60KD were significantly lighter than that in control group. There was no significant difference in 40KIK 60KD protein bands between ANLL> ALL and HL-60cells.Conclusions1. The composition of NMPs is significantly different in acute leukemic cells and nomal bone marrow cells. This imply that the changes of NMPs may be correlate with acute leukemia.2. The -869bp fragment in upstream of exonl of pl6gene can bind to NMPs. This indicate that the fragment include MAR sequence and can regulate pl6gene expression.3. The changes of NMPs in acute leukemia can affect their binding with MAR sequence included in upstream of pl6gene. This result may be inhibit pl6geneexpression and accelerate acute leukemia development.