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Immunocytochemistry for Drugs Containing an Aliphatic Primary Amino Group in the Molecule, Anticancer Antibiotic Daunomycin as a Model

Kunio Fujiwara, Hironori Takatsu and Kazuhiro Tsukamoto

Department of Applied Life Science, Faculty of Engineering, Sojo University, Kumamoto, Japan (KF); and Department of Pharmacotherapeutics, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan (HT,KT)

Correspondence to: Kunio Fujiwara, Department of Applied Life Science, Faculty of Engineering, Sojo University, Ikeda 4-22-1, Kumamoto 860-0082, Japan. E-mail: fujiwara{at}life.sojo-u.ac.jp

An immunocytochemistry (ICC) for the anticancer antibiotic daunomycin (DM) was developed using a combination of anti-DM serum produced against N-(gamma-maleimidobutyryloxy)succinimide (GMBS)-conjugated DM, and DM-uptake human melanoma BD cells. The antiserum was demonstrated to be specific for DM and the structurally related analogs adriamicin and epirubicin by an ICC model system of the enzyme immunoassay (EIA) using glutaraldehyde (GA)-conjugated DM as a solid phase antigen. No cross-reaction occurred with any of the other antibiotics tested such as bleomycin, pepleomycin, and mitomycin C. Successful DM ICC required a series of processes prior to the immunocytochemical reaction: the cells were first fixed with GA, then reduced with NaBH₄, treated with hydrochloric acid, and finally digested with protease. The cell specimens were then subjected to immunoreaction with anti-DM serum followed by peroxidase-labeled goat anti-rabbit IgG/Fab', and in both immune reagents the detergent Triton X-100 was contained as well. The present ICC covering all these processes successfully stained for DM in the nucleus and in the perinuclear Golgi region of the cytoplasm of the BD cells, consistent with the results obtained by the DM autofluorescence method. This ICC was found to be three times as sensitive as the cytofluorometric method and applicable to the paraffin sections of the liver of rats 24 hr after an IV injection of DM. The principle used in the present study for developing DM ICC might be applied to other drugs containing the primary amino group(s) in the molecule. Thus, these ICCs for drugs are direct, precise and easy new methods that should have potential for pharmacology and toxicology studies of drugs, revealing the localization of a drug in cells and tissues. (J Histochem Cytochem 53:467–474, 2005)

Key Words: daunomycin • immunocytochemistry • subcellular localization

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