Cloning of WWOX gene and its growth-inhibiting effects on ovarian cancer cells

Abstract

The growth-inhibiting and apoptosis-inducing effects of WW domain-containing oxidoreductase (WWOX) gene on ovarian cancer cell line A2780 were investigated. The full length cDNA of human WWOX gene was amplified from normal human ovary tissues. The correct cDNA of full length WWOX was subcloned into eukaryocytic expression vector pCMV. After introduction of WWOX gene into cancer cells with liposome, the WWOX mRNA and protein level in the cancer cells were detected by reverse transcription polymerase chain reaction (RT-PCR) and immunoblotting. The growth activities of cancer cells were detected by Trypan blue staining. The clone formation assay in soft agar was employed to observe the proliferation of the cancer cells. Apoptosis was examined by DNA ladder and acridine orange-ethidium bromide fluorescent staining. The results showed that 72 h after WWOX gene transfection, the WWOX expression was increased significantly (P<0.01). The growth of ovarian cancer cells was decreased by 16.41% to 38.49% (P<0.01). The clone formation abilities were reduced (P<0.01). Some cancer cells presented the characteristic morphological changes of apoptosis with obvious ladder bands on electrophoresis. The apoptosis rate was (20.7±6.0)% (P<0.01). It was concluded that over-expression of WWOX gene could induce apoptosis and inhibit the growth of ovarian cancer cells, which might be potentially useful in the gene therapy of ovarian cancers.

Keywords

gene expression / ovarian neoplasms / WW domain-containing oxidoreductase / apoptosis

References

[1] BednarekA.K., Keck-WaggonerC.L., DanielR.L., et al.. WWOX, the FRA16D gene, behaves as a suppressor of tumor growth. Cancer Res, 2001, 61(22): 8068-8073

[2] O’KeefeL.V., RichardsR.I.. Common chromosomal fragile sites and cancer: focus on FRA16D. Cancer Lett, 2006, 232(1): 37-147

[3] BednarekA.K., LaflinK.J., DanielR.L., et al.. WWOX, a novel WW domain-containing protein mapping to human chromosome 16q23.3–24.1, a region frequently affected in breast cancer. Cancer Res, 2000, 60(8): 2140-2145

[4] NunezM.I., RosenD.G., Ludes-MeyersJ.H., et al.. WWOX protein expression varies among ovarian carcinoma histotypes and correlates with less favorable outcome. BMC Cancer, 2005, 5(1): 1186-1471

[5] PaigeA.J., TaylorK.J., TaylorC., et al.. WWOX: A candidate tumor suppressor gene involved in multiple tumor types. Proc Natl Acad Sci USA, 2001, 98(20): 11417-11422

[6] NunezM.I., RosenD.G., Ludes-MeyersJ.H., et al.. WWOX protein expression varies among ovarian carcinoma histotypes and correlates with less favorable outcome. BMC Cancer, 2005, 5(1): 1186-1471

[7] GourleyC., PaigeA.J., TaylorK.J., et al.. WWOX mRNA expression profile in epithelial ovarian cancer supports the role of WWOX variant 1 as a tumour suppressor, although the role of variant 4 remains unclear. Int J Oncol, 2005, 26(6): 1681-1689

[8] ChangN.S.. A potential role of p53 and WOX1 in mitochondrial apoptosis. Int J Mol Med, 2002, 9(1): 19-24

[9] AqeilanR.I., PekarskyY., HerreroJ.J., et al.. Functional association between Wwox tumor suppressor protein and p73, a p53 homolog. Proc Natl Acad Sci USA, 2004, 101(13): 4401-4406