tNOX is the designation for a subset of the ECTO-NOX (NADH oxidase) proteins involved in growth that is unique to the surface of cancer cells and is inhibited specifically by a variety of quinone site inhibitors all of which have anticancer activity (Genbank Accession No. AF20788; Chueh et al., 2002; Morre and Morre, 2003). These family members are designated as members of the ENOX2 group to distinguish them from the constituitive ECTO-NOX proteins of both cancer and non-cancer cells designated as CNOX or ENOX1. The NTI diagnostic development program is based on the discovery of these proteins at Purdue University by the research team of D. James and Dorothy Morre.
As are all of the ECTO-NOX (ENOX) proteins, the tNOX proteins are hydroquinone oxidases that also oxidize externally supplied NADH, hence NOX Figure1. They are oxidases with a di-copper center that lack flavin. They carry out protein disulfide interchange (protein disulfide isomerase like activity) but are unique in that they also lack the CXXC motif common to most if not all, of the classic protein disulfide isomerase family members. A number of tNOX isoforms have been identified, each common to a particular form of human cancer Figures 2-4. tNOX proteins isoforms appear to be splice variants of a single tNOX gene located on chromosome X and comprised of at least thirteen exons. The proteins are shed into the circulation. They are protease resistant and accumulate to serve as circulating markers of cancer presence. tNOX isoforms are associated with all forms of human cancer and are absent from non-cancer cells, tissues and sera and from sera of subjects with diseases other than cancer.
The presence of tNOX in serum is due to shedding from the patient’s cancer. This provides a readily available, non-invasive serum or blood diagnostic marker. The antitumor drug-responsive tNOX activity is the first cell surface change reported to be associated with most, if not all, forms of human cancer. However, while tNOX presence provided a non-invasive approach to cancer detection, until recently it offered no indication as to cancer type or location. Initially, detection utilized a pan-tNOX recombinant single chain variable region (scFv) antibody carrying an S tag that cross reacted with all known tNOX isoforms from solid tumors of human origin and did not differentiate among different kinds of cancer. However, analyses using this antibody when combined with two-dimensional gel electrophoretic separations revealed specific tNOX isoforms, each characteristic of a particular form of cancer Figures 2-4. This discovery makes possible ELISA-based detection protocols that will distinguish among non-small cell lung cancer and small cell lung cancer for example and among all of the major epithelial cancers (lung, breast, ovarian, prostate, colon) Figures 2-4. The isoform patterns may have the potential to predict therapeutic response and/or disease severity. tNOX proteins are robust and are highly resistant to heat and protein degradation, making them powerful, non-invasive markers for cancer diagnosis.
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