Telomerase expression and telomere length in immortal leukocyte lines from channel cat®sh Katherine S. Barker a , Sylvie M.A. Quiniou a , Melanie R. Wilson a , Eva Bengten a , Tor B. Stuge a , Greg W. Warr b , L. William Clem a , Norman W. Miller a, * a Department of Microbiology, University of Mississippi Medical Center, Jackson, MS 39216, USA b Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA Received 18 October 1999; received in revised form 6 February 2000; accepted 18 February 2000 Abstract Normal channel cat®sh leukocytes readily undergo spontaneous in vitro immortalization yielding functionally active diploid cell lines. Since telomerase activation appears to be a critical step in the establishment of immortal mammalian cells, studies were undertaken to determine if and when telomerase expression occurs during the in vitro immortalization process of channel cat®sh leukocytes. To this end, freshly isolated peripheral blood leukocytes (PBL) from normal ®sh were shown to exhibit low to undetectable levels of telomerase activity and within four days after culture initiation showed dramatic increases in telomerase activity which typically remained high for at least four weeks. This activity then declined, concomitant with decreases in cellular proliferation and increases in cell death. Cells which escaped this culture ``crisis'' re-expressed high levels of telomerase activity inde®nitely. Although telomerase activity was expressed early in the immortalization process, clonal cell lines derived from these cultures had relatively short telomeres. These results suggest that telomerase expression in cat®sh leukocytes is activation- induced, and its expression does not necessarily stabilize telomere length until a critically, albeit ill-de®ned, short length is reached. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Telomerase; Telomere; Cell immortalization; Channel cat®sh; Leukocytes 1. Introduction Previous studies have shown that normal chan- nel cat®sh leukocytes can undergo continuous in vitro proliferation and frequently yield long-term cultures that can be maintained inde®nitely with routine passage, i.e. appear immortal. Many of these long-term cultures have been successfully Developmental and Comparative Immunology 24 (2000) 583±595 0145-305X/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S0145-305X(00)00021-5 www.elsevier.com/locate/devcompimm * Corresponding author. Tel.: +1-601-984-1719; fax: +1- 601-984-1708. E-mail address: nmiller@microbio.umsmed.edu (N.W. Miller). Abbreviations: PBL; peripheral blood leukocytes; LPS; lipopolysaccharide; ConA; concanavalin A; AL; AIM-V + L- 15; CFS; cat®sh serum; PMA; phorbol-12- myristate 13-acet- ate; TRAP; telomere repeat ampli®cation protocol; TRF; telo- mere restriction fragment; FISH; ¯uorescent in situ hybridization; hTERT; human telomerase reverse transcrip- tase.