Effect of adenovirus-mediated p27 gene expression on the proliferation and apoptosis of HL-60 and Raji cell lines

Background p27 is an essential mediator of cell cycle control,which plays a key negative role in the proliferation and tumorigenesis of certain cell types. Here, we designed this study to explore the possible effects of p27 on the proliferation and apoptosis of HL-60 and Raji cell lines.Methods HL-60 and Raji cells were transfected with p27 via an adenovirus-mediated approach. The efficiency of Adp27 infection and the expression of p27 mRNA and protein were evaluated by X-gal staining, RT-PCR, and flow cytometry. The proliferation and apoptosis of HL-60 and Raji cells were estimated by means of trypan blue staining, MTT assay, Annexin V/PI, and DNA ladder electrophoresis. Results The infection efficiencies in HL-60 and Raji cells were 40.3% and 32.0%, respectively. RT-PCR and flow cytometry showed that there was significant expression of p27 mRNA and protein in HL-60 and Raji cells infected with Adp27; on the other hand, uninfected HL-60 cells showed faint traces of p27 mRNA and protein and Raji cells showed nearly no signs of p27 mRNA and protein. As demonstrated by a cell growth curve and by an MTT assay, strong time-dependent proliferation inhibition was apparent in HL-60 and Raji cells infected by Adp27. After 72 hours of infection, the Annexin V+/PI- apoptotic cell rates in HL-60 and Raji cell lines were 46.9% and 35.7%, respectively, significantly higher than in the control groups (4.7% and 5.6%, respectively). Typical DNA ladder bands were detectable in HL-60 and Raji cells after 48 hours of Adp27 infection. Conclusions Adenoviral vector-mediated p27 gene transfection of HL-60 and Raji cells leads to the inhibition of cellular proliferation and the promotion of cell apoptosis. This technique may provide an approach to gene therapy for leukemia or lymphoma.

Inhibition of vascular smooth muscle cell proliferation by in troduction of retinoblastoma gene via a recombinant adenovirus vector

This study was supported in part by grant from National Natural Science Foundation of China (No. 39570775). Objective To investigate the vascular smooth muscle cell (SMC) growth suppression by recombinant adenovirus vector expressing a retinoblastoma (Rb) protein and to explore a gene therapy approach for vascular proliferative disorders including atherosclerosis and artery restenosis. Methods A replication deficient adenovirus vector encoding a wild type Rb and AdCMVRb, was constructed and transfected into cultured rabbit aortic SMC. The efficiency of gene transfection and expression was detected by immunochemical staining and polymerase chain reaction. The role of Rb in regulating vascular SMC proliferation was observed by cell counting, thymidine incorporation, and flow cytometry. Results Wild type Rb gene transfected effectively into the cultured SMC with AdCMVRb can suppress growth factor stimulated cell proliferation through regulation of DNA synthesis and cell cycle progression. Conclusion The results demonstrate the potential of adenovirus mediated Rb gene therapy for atherosclerosis and artery restenosis after balloon angioplasty.

Growth inhibiting effects of antisense eukaryotic expression vector of proliferating cell nuclear antigen gene on human bladder cancer cells

Objective To explore the growth inhibiting effects on human bladder cancer by antisense RNA targeting the proliferating cell nuclear antigen (PCNA) gene. Methods The eukaryotic expression vector for antisense PCNA cDNA was constructed and transferred into a bladder cancer EJ cell line. The PCNA expression in the cancer cells was detected by RT-PCR and Western blotting assays. The in vitro proliferation activities of the transferred cells were observed by growth curve,tetrazolium bromide (MTT) colorimetry,tritiated thymidine ( 3H-TdR)incorporation, flow cytometry and clone formation testing,while its in vivo anti-tumor effects were detected on nude mice allograft models.Results After the antisense vector,pLAPSN,was transferred,cellular PCNA expression was inhibited at both protein and mRNA levels. The growth rates of EJ cells were reduced from 27.91% to 62.07% ( P <0.01),with an inhibition of DNA synthesis rate by 52.31% ( P <0.01). Transferred cells were blocked at G 0/G 1 phases in cell-cycle assay,with the clone formation ability decreased by 50.81% ( P <0.01). The in vivo carcinogenic abilities of the transferred cancer cells were decreased by 54.23% ( P <0.05). Conclusions Antisense PCNA gene transfer could inhibit the growth of bladder cancer cells in vitro and in vivo,which provided an ideal strategy for gene therapy of human cancers.