Characterization, isolation and expression cloning of a tumor-associated protein ( tNOX) that exhibits NADH:protein disulfide reductase activity with capsaicin -inhibition
Abstract
The purpose of this dissertation was to characterize, isolate and expression clone a tumor-associated protein (tNOX) that exhibits protein:disulfide reductase activity at the expense of NADH. tNOX activity in cancer cells is distinct from NOX activity in non-transformed cells in several manners. First of all, tNOX activity is no longer under regulation and is constitutively active. Second, tNOX activity in cancer cells is responsive to thiol reagents and is inhibited by antitumor drugs including sulfonylurea and capsaicin. Capsaicin preferentially inhibits both the tNOX activity in plasma membranes and the growth of transformed cells. This cancer-specific capsaicin inhibition in tNOX activity served as a criteria for isolation of tNOX protein from pooled sera of cancer patients. A 33.5 kD protein with capsaicin-inhibited tNOX activity was isolated and used for generation of a monoclonal antibody. Furthermore, a gene was cloned by screening a HeLa cDNA library using the monoclonal antibody. The open reading frame of the derived amino acid sequence encoded a protein with several functional motifs which suggested a possible candidate of tNOX including NADH and quinone binding sites. A possible protein disulfide isomerase motif also was found in the deduced amino acid sequence that might be involved in thiol-disulfide interchange activity of tNOX. The cloned gene has been reported by another group previously, two mistakes result in a shift in the open reading frame and the original report is not a full-length of cDNA. The findings in this dissertation suggest that tNOX is a unique protein and appears to better cancer-specific. The cDNA obtained in this study may be used as a potential marker for detection of cancer in patients by Northern blot analysis or PCR. The discovery of tNOX has opened a new era for cancer research and the exploration of tNOX may lead us to understand tumorgenesis at the molecular level. Ultimately, the studies of tNOX may result in improved preventative measures and treatments for cancer.
Degree
Ph.D.
Advisors
Morre, Purdue University.
Subject Area
Biochemistry|Molecular biology
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