Suppression Of Cyclin B1 Expression By Full-length Cyclin B1 Antisense CDNA Induces Cell Cycle Arrest And Inhibits Tumorigenicity

Background and Objective: Cyclin B1, a cell cycle protein in mitosis phase, connecting with p34 kinase (CDK1) to form muturior promoting factor (MPF), is necessary for mitotic initiation in eukaryotic cells and it plays an important role in cancer development; cyclin B1 is over-expressed in a variety of tumors and is thought to be an indicator of the malignant potential of tumors. Antisense cDNA is a simple, easy and influential method to down-regulate the expression of target genes. It remains to be clarified whether down-regulation of cyclin Bl with full-length antisense cDNA of cyclin B1 (AS-CLB1) in tumor cells is an effective strategy for cancer therapy.Methods: A recombinant plasmid containing the full-length antisense cDNA of mouse cyclin Bl (pAS-mCLBl) and pcDNA3.1 empty vector were constructed and introduced into LL/2 and CT26 cells to establish AS-mCLB1 (Ac) and pcDNA3.1 (Pc) stable transfectants respectively. The cell cycles and apoptosis of Ac, Pc and untransfected (Nc) cells were determined using flow cytometry. mRNA and protein content of cyclin Bl were tested by two-step semi-quantitative RT-PCR and western blot assay respectively. The activity of cell proliferation was measured by MTT and cell growth assay.Tumorigenicity and survival were observed in the mice which were implanted with Ac, Pc and Nc cells respectively. The proteomics of cells and animal serums were compared between the AC and NC groups. In the tumor tissues, the expression of mCyclin B1 was detected with immunohistochemistry and DNA Fragmentation Detection Kit was used to test DNA fragmentation associated with the apoptotic cells.Result: Prominent G1 arrest and apoptosis were shown and abnormal morphology with inhibition of cell growth appeared in the Ac cells in which persistent and evident down-regulation of mCyclin B1 expression was induced. Moreover, after implanting, tumors in the Ac group developed on day 9 versus day 5 in the controlled groups (Nc and Pc) in LL/2-bearing mice. In CT26-loaded mice, tumors in the Ac group developed on day 6 versus day 3 in the controlled after inoculation. Therefore, survival benefits might be achieved through the inhibition of tumorigenicity in the mice of Ac groups. Both in cells and animal serums, the differences of proteomics were apparent. The expression of mCyclin B1 protein was decreased and cell apoptotic rate was increased in the tumor tissues of Ac groups.Conclusion: To our understanding, this is the first report concerning the biological activities of cyclin Bl knockdown tumor cells using AS-mCLB1. These results suggested that the suppression of cyclin Bl by AS-CLBl could induce G1 arrest and apoptosis of tumor cells obviously, which involved in inhibiting the activity of tumor cells in vitro and in vivo efficiently. So AS-CLB1 might be a viable method for tumor biotherapy.