Alternative splicing as the basis for specific localization of tNOX, a unique hydroquinone (NADH) oxidase, to the cancer cell surface
A novel disulfide-thiol interchange protein with both hydroquinone and NADH oxidase activity (designated tNOX) is exclusively associated with the outer plasma membrane leaflet at the surface of cancer cells and in sera of cancer patients and is absent from the surface of non-cancer cells and sera from healthy individuals. Nevertheless, tNOX mRNA has approximately the same abundance in both normal cells and tumor cells. Our research suggests alternative splicing as the basis for the cancer specificity of tNOX. Four splice variants were found. Of these, the exon 4 minus form and exon 5 minus form were present in cancer cell lines and were absent in non-cancer cell lines. Transfection of COS cells with exon 4 minus cDNA resulted in overexpression of mature 34 kDa tNOX protein at the cell surface. Site-directed mutagenesis of methionine 231 codon prevented the translation of exon 4 minus cDNA and the appearance of a 34 kDa mature tNOX at the cell surface. The unprocessed molecular weight of 47 kDa of the exon 4 minus cDNA translated from methionine 231 corresponded to the molecular weight of the principal native tNOX form found in the endoplasmic reticulum. A relationship of tNOX to unregulated growth of cancer cells was provided by data where growth of HeLa cells was inhibited by transfection with the Exon 5 antisense oligonucleotides. Cell surface expression of tNOX and inhibition by the green tea catechin tNOX inhibitor (-)-epigallocatechin-3-gallate (EGC-) were similarly blocked. Exon 5 antisense oligonucleotides decreased both the ability of HeLa cells to invade Matrigel and their anchorage-independent growth. The findings suggest a direct relationship between cell-surface expression of tNOX and the unregulated growth of cancer cells that correlates specifically with the presence of the alternatively-spliced exon 4 minus tNOX mRNA.^
Dorothy M. Morre, Purdue University.
Biology, Cell|Health Sciences, Nutrition