Tohoku J. Exp. Med., 2001, 194 (1)

The Mechanism of Cell Death in Human Cultured Colon Adenocarcinoma Cell Line COLO 201 Induced by b-D-N-Acetylglucosaminyl-p-Nitrophenol

JUNKO KUKIDOME, IKUKO KAKIZAKI, KEIICHI TAKAGAKI, AKIHIKO MATSUKI,¹ AKIHIRO MUNAKATA¹ and MASAHIKO ENDO

Department of Biochemistry, and ¹First Department of Internal Medicine, Hirosaki University School of Medicine, Hirosaki 036-8562

COLO 201, human colon adenocarcinoma cells were incubated with artificial primers, p-nitrophenyl-glycoside derivatives at 1.0 mmol (mM) in the medium containing 10% fetal bovine serum to detect sugar chain elongation. However, when p-nitrophenyl-b-N-acetylglucosamine (b-GlcNAc-PNP) was added, the medium changed color to yellow and the cells were dead. To explain this finding, the cells were incubated with 1.0 mM each of b-GlcNAc-PNP and 4-methylumbelliferyl-b-N-acetylglucosamine, then the number of living cells was measured in a time course. In b-GlcNAc-PNP, the living cells were decreased at 24 hours. The cells were survived with N-acetylglucosamine, whereas in the presence of p-nitrophenol (PNP) the living cells were decreased. It was suggested that PNP released from b-GlcNAc-PNP induced the cell death. Activity of b-D-N-acetylglucosaminidase was detected in fetal bovine serum. It was shown that PNP induced the cell death in time-and-dose dependent manner. Genomic DNA from COLO 201 analyzed by agarose gel electrophoresis was fragmentated. PNP analogues were tested for toxicity, and the results suggested that the phenolic OH-group linked to benzene ring and nitro-group linked to the structure in para-form (PNP) was the most effective.

Keywords —— apoptosis; cell culture; COLO 201; b-D-N-acetylglucosaminidase; PNP

© 2001 Tohoku University Medical Press

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Tohoku J. Exp. Med., 2001, 194, 23-34

Address for reprints: Prof. Masahiko Endo, Department of Biochemistry, Hirosaki University School of Medicine, Hirosaki 036-8562, Japan.

e-mail: endo-m@cc.hirosaki-u.ac.jp

All glycosides presented in the text are D-forms.