D. James Morre, Ziaoyu Tang, Dorothy M. Morre, Kenneth L. Pennington, Heather Hignite, Helen Orvis, Douglas J. Schwartzentruber, Department of Medicinal Chemistry and Molecular Pharmecology, Purdue University, West Lafayette, IN 47907, NOX Technologies, Inc., West Lafayette, IN 47906, Department of Foods and Nutrition, Purdue University, West Lafayette, IN 47907 and Center for Cancer Care, Goshen, IN 46526.
A novel hydroquinone and NADH oxidase with protein disulfide-thiol interchange activity (designated tNOX), is associated exclusively with the outer leaflet of the plasma membrane at the surface of cancer cells. Also present in sera of cancer patients, it is absent from the surface of non-cancer cells and from sera from healthy individuals. Nevertheless, tNOX mRNA has approximately the same abundance in both normal and cancer cells. Our research demonstrates alternative splicing as the basis for the cancer specificity of tNOX expression at the cell surface. We have developed probes to detect by RTPCR a 150 bp product indicative of tNOX splice variant mRNA in the blood of cancer patients. Venous blood was collected into 7 ml vacutainers containing potassium EDTA. The specimens were snap frozen and stored at -80° C until RNA extraction. Total RNA from whole peripheral blood was extracted using a Fast Lane Cell cDNA kit (Qiagen). Purity and amounts of total RNA were assessed by electrophoresis in Tris-Acetate-EDTA buffer on 1% agarose gels, with the DNA bands visualized by ethidium bromide staining. We evaluated 40 patients with histologic diagnosis of breast, lung, or ovarian cancer and 20 healthy volunteers. The specificity and sensitivity of the procedure was established initially using human cancer cell lines in culture (HeLa cervical carcinoma, BT-20 mammary carcinoma, MCF-10 mammary non-cancer.) Splice variant-specific tNOX primers were designed according to published sequence information (Available from GenBank under Accession No. AF 207881). Each RT-PCR run included a positive control (tNOX-positive cell line or blood donor) and blood from a negative donor. Under the assay conditions developed, 70 % of patients with cancer were positive for tNOX slice variant mRNA. Samples from individuals free of disease or with disorders other than cancer were negative. Our expectation is that this assay will provide a tool for detecting cancer cells in the circulation. Larger cohorts will be required in future studies to define the utility of this technology to detect splice variant tNOX mRNA in blood borne cells in cancer management.