[*OPCML gene transferred by recombinant lentiviruses in vitro and its inhibition to ovarian cancer cells]

Abstract

Objective: To study the inhibition of OPCML on ovarian cancer cell lines using a lentiviral vector system for efficient gene transduction.

Methods: The murine OPCML cDNA was amplified by PCR from CD1 murine brain cDNA using gene specific primers, and subcloned into the lentiviral vector, pWPI-GFP, to generate the lentiviral expression vector, pWPI-OPCML. Recombinant lentiviruses were produced by 293T cells following the co-transfection of pWPI-OPCML, with the packaging plasmids pCMV-dR8.74 and pMDG. The resulting recombinant lentiviruses which carried OPCML or control viruses (only carrying GFP), were then used to infect the human ovarian cancer cell lines A2780 and OCC1 in addition to normal CD1 mouse ovarian surface epithelial cells. The infected cells were then characterized by cell proliferation assays, cell aggregation assays, cell cycle analysis by flow cytometry and tumorigenicity assays following injection into nude mice.

Results: (1) The efficiency of infection of the cell lines using the lentiviral vectors was almost 100% allowing the stable expression of OPCML in nearly all cells. Stable expression of OPCML (60 000) and GFP (27 000) proteins was confirmed by western blot analysis. (2) A2780 cells expressing OPCML [(7.6 +/- 1.0) x 10(5)] grew slowly compared to A2780 parental [(20.0 +/- 2.6) x 10(5)] or control virus infected cells [(18.1 +/- 1.7) x 10(5), P < 0.01], but the expression of OPCML had no effect on the proliferation rates of OCC1 and the normal CD1 cells when compared to their respective parental or controls (P > 0.05). (3) Flow cytometry based cell cycle assays showed that the expression of OPCML could arrest A2780 cells (G(0) approximately G(1) 67% vs 75%, P < 0.05); but not OCC1, CD1 cells. (4) The rate of aggregation of single cell suspensions was measured and found to be increased in all cell lines expressing OPCML indicating the increased cell surface adhesion mediated by OPCML. (5) A2780 cells expressing OPCML only formed a single tumor in 1/4 mice (10 mg) which was significantly smaller than controls [4/4; A2780 (280 +/- 53) mg and A2780/pWPI (677 +/- 323) mg; P < 0.01]. Expression of OPCML in tumor was confirmed by immunohistochemistry.

Conclusions: The use of lentiviral vectors allowed the efficient expression of OPCML in nearly 100% of target cells. Expression of the OPCML cDNA resulted in an increase of cell adhesion in all cell lines tested, and decreased the proliferation and tumorigenicity of the A2780 ovarian cancer cell line. This indicates that the OPCML may be a new tumor suppressor gene.