Monoclonal antibody to a cancer-specific and drug-responsive hydroquinone (NADH) oxidase from the sera of cancer patients.
Monoclonal antibodies were generated in mice to a 34-kDa circulating form of a drug-responsive hydroquinone (NADH) oxidase with a protein disulfide-thiol interchange activity specific to the surface of cancer cells and the sera of cancer patients. Screening used Western blots with purified 34-kDa tNOX from HeLa cells and the sera of cancer patients. Epitopes were sought that inhibited the drug-responsive oxidation of NADH with the sera of cancer patients, but which had no effect on NADH oxidation with the sera of healthy volunteers. Two such antisera were generated. One, designated monoclonal antibody (mAb) 12.1, was characterized extensively. The NADH oxidase activity inhibited by mAb 12.1 also was inhibited by the quinone site inhibitor capsaicin (8-methyl- N-vanillyl-6-noneamide). The inhibition was competitive for the drug-responsive protein disulfide-thiol interchange activity assayed either by restoration of activity to scrambled RNase or by cleavage of a dithiodipyridine substrate, and was uncompetitive for NADH oxidation. Both the mAb 12.1 and the postimmune antisera immunoprecipitated drug-responsive NOX activity and identified the same 34-kDa tNOX protein in the sera of cancer patients that was absent from sera of healthy volunteers, and was utilized as immunogen. Preimmune sera from the same mouse as the postimmune antisera was without effect. Both mouse ascites containing mAb 12.1 and postimmune sera (but not preimmune sera) slowed the growth of human cancer cell lines in culture, but did not affect the growth of non-cancerous cell lines. Immunocytochemical and histochemical findings showed that mAb 12.1 reacted with the surface membranes of human carcinoma cells and tissues.
Cho
NaMi
N
Department of Medicinal Chemistry and Molecular Pharmacology, Hansen Life Sciences Research Building, Purdue University, West Lafayette, IN 47907, USA.
Chueh
Pin-Ju
PJ
Kim
Chinpal
C
Caldwell
Sara
S
Morré
Dorothy M
DM
Morré
D James
DJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D017209:Apoptosis; D015153:Blotting, Western; D004220:Disulfides / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D005260:Female; D006367:HeLa Cells; D006801:Humans; D006825:Hybridomas / Q000378:metabolism; D007150:Immunohistochemistry; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D009097:Multienzyme Complexes / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009369:Neoplasms / Q000097:blood*; D010100:Oxygen / Q000378:metabolism; D011233:Precipitin Tests; D013997:Time Factors
D000911:Antibodies, Monoclonal; D004220:Disulfides; D000939:Epitopes; D009097:Multienzyme Complexes; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D010100:Oxygen