EXPRESSION OF HUMAN GLANDULAR KALLIKREIN, HK2, IN CELLS

The human kallikrein family consists of three members, hK1, hK2, and h K3 [prostate-specific antigen (PSA)]. PSA is a widely accepted marker for prostate cancer. The mRNAs for both hK2 and PSA are localized pred ominantly to prostate epithelium and are regulated by androgens. In ad dition, hK2 has 78% amino acid homology to PSA. Although similarities to PSA make hK2 a potential prostate cancer marker, they also impede b iochemical characterization of hK2 in those human tissues and body flu ids where PSA is abundant. To study the expression, biosynthesis, and processing of hK2, recombinant hK2 was expressed in the adenovirus-ind uced Syrian hamster tumor cell line AV12-664 (AV12-hK2). Expression of hK2 was analyzed by Western blots and ELISA using monoclonal antibodi es HK1G 464.3 [specific for prohK2 (phK2)] and HK1D 106.4 [specific fo r phK2 and mature hK2 (hK2)]. Western blot and ELISA analyses showed t hat phK2 was secreted into the media by AV12-hK2 cells on day 1 and wa s gradually converted to the mature form of hK2 by day 7. N-terminal a mino acid sequencing verified the Western blot and ELISA data. This de monstrates for the first time that hK2 is secreted as phK2 and convert ed to hK2 extracellularly. In addition, hK2 detected in day 4-7 AV12-h K2-spent media was enzymatically active. Recombinant hK2 was also expr essed in human prostate carcinoma cell lines, PC3 (PC3-hK2) and DU145 (DU145-hK2), that do not express endogenous hK2 or PSA. Similar to AV1 2-hK2 cells, both cell lines secreted phK2 that was converted to hK2 e xtracellularly. phK2 was the major form detected in the spent media of PC3-hK2 cells, even after 7 days, indicating a slow conversion of phK 2 to hK2. hK2 was the predominant form detected in the spent media of DU145-hK2 starting on day 1, indicating the rapid conversion of phK2 t o hK2. In this study, we demonstrate that hK2 exists in different farm s and is secreted as phK2. phK2 is then converted to enzymatically act ive hK2 extracellularly.