The Study Of P53 Gene Therapy Hematology Tumor Cell Line K562 In Vitro

Objective: p53 tumor-suppressor gene is critical to induce cell cycle arrest in G1 and apoptosis. It plays a role in maintaining the genetic stability of DNA. The loss of p53 function has been implicated as an important event in tumor initiation, progression and therapy. p53 is one of the most frequently mutated genes in human cancers. Point mutations and/or p53 gene deletion occur in approximately 50% malignant tumors. Many researches focused on p53 gene therapy in tumor. There were also some clinical trials involved in p53 gene, and some proofs showed p53 gene therapy clinical validity. But these clinical trials were related to such solid tumor therapy as head and heck squamous cancer and non-small cell lung cancer. The epidemiological surveys showed that p53 gene mutations and/or deletions of different degrees occurred in all kinds of leukemia, and were associated with a low complete remission rate, early relapse and poor survival. Resent research showed that Jurkat (null p53 protein) deriving from malignant lymphatic system tumor could be induced apoptosis after p53 gene transfer, but some inconsistent results were reported in K562 after p53 gene transfer. In our research, we would transfer p53 gene to K562 cell line (null p53 protein), which come from chronic myeloid leukemia, observe K562 with p53 tumor suppressor protein biological changes before and after vp-16-213 treatment and hope to find a new approach to remedy present leukemiatreatment methods.Methods:We select monoclonal K562 cell with limited dilution, transfer p53 gene with electroporation to monoclonal K562 cells and detect p53 gene transcription and expression with RT-PCR and immunocytochemical staining in p53-K562. After affirmation of transfection successfully, we compare proliferation in p53-K562 with wild type K562 and neo-K562 by 3H-thymidine (3H-TdR) incorporation assay, apoptosis in p53-K562 with wild type K562 and neo-K562 by TUNEL, Annexin V and hypodiploid apex before and after treatment with vp-16-213 and p53-K562 cell cycle distribution with neo-K562 and wild type K562 by cell cycle assay. Results:1. p53-K562 could express p53 mRNA and protein successfully after transfection 6 hours, and the expression could last over 72 hours.2. p53 protein could significant suppress K562 cell line proliferation by 24.17% with 3H-thymidine (3H-TdR) incorporation assay.3. The distribution of G1 cell in p53-K562 group is 48.76±3.43% (after p53 transfection 24 hours) and 50.33±3.56% (after transfection 48 hours), but 42.59±3.38% (after electroporation 24 hours) and 45.40±1.00% (after electroporation 48 hours) in control group-wild type K562. There were significant differences between two groups (p<0.05).4. p53 protein could not induce significant apoptosis compared to neo-K562 and wt-K562 with TUNEL, Annexin V and hypodiploid assays.5. After vp-16-213(1μg/ml) treated p53-K562 6 hours and 24 hours, we could not find apoptosis increase, compared to neo-K562 and wild type K562.Conclusions:1)p53-pREP9 plasmid could be transfected and overexpressed successfully inK562 cell line.2)p53 protein inhibit K562 cell line proliferation by 24.17%.3)p53 protein could induce K562 G1 arrest.4)p53 protein could not induce significant apoptosis.5)p53-K562 cell line treated with vp-16-213(1μg/ml) was not observed more significant apoptosis than wild type K562 treated with vp-16-213.