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© The Rockefeller University Press, 0021-9525/1999//1 $5.00
The Journal of Cell Biology, Volume 144, Number 1, , 1999 1-9

Article

Detection by Epitope-defined Monoclonal Antibodies of Werner DNA Helicases in the Nucleoplasm and Their Upregulation by Cell Transformation and Immortalization



Miwa Shiratori*, Sakae Sakamoto*, Noriyuki Suzuki*, Yoshiki Tokutake*, Yoichi Kawabe{ddagger}, Takemi Enomoto{ddagger}, Masanobu Sugimoto*, Makoto Goto§, Takehisa Matsumoto*, and Yasuhiro Furuichi*

* AGENE Research Institute, Kamakura, Kanagawa 247, Japan; {ddagger} Faculty of Pharmaceutical Sciences, Tohoku University, Aoba-ku, Sendai 980, Japan; § Tokyo Metropolitan Otsuka Hospital, Toshima-ku, Tokyo 170, Japan

We prepared several monoclonal antibodies (mAbs) specific for the NH2- and COOH-terminal regions of the DNA helicase (WRN helicase) responsible for Werner's syndrome known as a premature aging disease. With these antibodies, we detected by immunoblot analysis the endogenous WRN helicase of a relative mass of 180 kD in several lines of cultured cells, but not in patient cells with a defined mutation. Immunocytochemical staining of proliferating fibroblasts and tumor cells showed that the major part of WRN helicase is in the nucleoplasm and not in the nucleolus. Similar experiments with a rat mAb specific to the mouse homologue of human WRN helicase yielded an identical conclusion. Although this nucleoplasmic staining was evident in cells in interphase, the condensed chromatin structure in metaphase was not stained by the same mAbs, suggesting that WRN helicases exist perhaps in a soluble form or bound to the unfolded chromatin structure. From quantitative immunoblot analysis, higher levels of WRN helicase were observed in all transformed cells and tumor cells examined than those of normal cells. The expression of WRN helicase was enhanced consistently in fibroblasts and B-lymphoblastoid cells by transformation with SV-40 and Epstein-Barr virus, respectively, suggesting that rapidly proliferating cells require a high copy numbers of WRN helicase.

Key Words: Werner's syndrome • RecQ DNA helicase • genetic instability • aging • nucleoplasmic localization

Abbreviations used in this paper: aa, amino acids; EBV, Epstein-Barr virus; EGFP, enhanced green fluorescent protein; NLS, nuclear localization signals; PDL, population doubling levels; WS, Werner's syndrome.

Address correspondence to Yasuhiro Furuichi, AGENE Research Institute, 200 Kajiwara, Kamakura, Kanagawa, 247 Japan. Fax: 81 467 48 6595. E-mail: furuichi{at}agene.co.jp


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