A Cell-Based Assay System for Monitoring NF-B Activity in Human HaCaT Transfectant Cells

Authors: Moon K-Y.¹; Hahn B-S.¹; Lee J.²; Kim Y.S.¹

Source: Analytical Biochemistry, Volume 292, Number 1, May 2001 , pp. 17-21(5)

Publisher: Academic Press

Abstract:

A cell-based assay system for monitoring NF-B activity was developed to determine the influence of activated NF-B in human HaCaT cells. The pNF-B-SEAP-NPT plasmid that permits expression of the secretory alkaline phosphatase (SEAP) reporter gene in response to the NF-B activity and contains the neomycin phosphotransferase (NPT) gene for geneticin resistance in host cells was constructed and transfected into the human keratinocyte cell line HaCaT. Human HaCaT transfectant cells were demonstrated to secrete the SEAP enzyme into the culture medium in a time-dependent manner until 72 h. NF-B activities were measured by the SEAP reporter gene assay using a fluorescence detection method. HaCaT cell transfectants treated with antioxidants [e.g., N-acetyl-l-cysteine and vitamin C] showed reduction of NF-B activity in a time- and concentration-dependent manner, whereas phorbol 12-myristate 13-acetate known as a stimulator of NF-B expression increased NF-B activity in a time- and concentration-dependent manner. This assay system could be used to determine the quantitative measurement of NF-B activity in the human skin and allow the screening of anti-inflammatory agents for dermatological purpose from various synthetic chemicals and natural products. Copyright 2001 Academic Press.

Keywords: NF-B activity; human HaCaT transfectant cells; cell-based assay; secretory alkaline phosphatase

Language: English

Document Type: Research article

Affiliations: 1: Natural Products Research Institute, Seoul National University, 28 Yeonkun-Dong, Jongno-Ku, Seoul, 110-460, Korea 2: Wonkwang University, 344-2 Shinyong-dong, Professional Graduate School of Oriental Medicine, Iksan, Chonbuk 570-749, Korea

Publication date: 2001-05-01

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