Kumar A, Goel A S, Hill T M, Mikolajczyk S D, Millar L S, Kuus-Reichel K, Saedi M S
Hybritech Incorporated, San Diego, California 92196-9006, USA.
Cancer Res. 1996 Dec 1;56(23):5397-402.
The human kallikrein family consists of three members, hK1, hK2, and hK3 [prostate-specific antigen (PSA)]. PSA is a widely accepted marker for prostate cancer. The mRNAs for both hK2 and PSA are localized predominantly to prostate epithelium and are regulated by androgens. In addition, hK2 has 78% amino acid homology to PSA. Although similarities to PSA make hK2 a potential prostate cancer marker, they also impede biochemical characterization of hK2 in those human tissues and body fluids where PSA is abundant. To study the expression, biosynthesis, and processing of hK2, recombinant hK2 was expressed in the adenovirus-induced Syrian hamster tumor cell line AV12-664 (AV12-hK2). Expression of hK2 was analyzed by Western blots and ELISA using monoclonal antibodies HK1G 464.3 [specific for prohK2 (phK2)] and HK1D 106.4 [specific for phK2 and mature hK2 (hK2)1. Western blot and ELISA analyses showed that phK2 was secreted into the media by AV12-hK2 cells on day 1 and was gradually converted to the mature form of hK2 by day 7. N-terminal amino acid sequencing verified the Western blot and ELISA data. This demonstrates for the first time that hK2 is secreted as phK2 and converted to hK2 extracellularly. In addition, hK2 detected in day 4-7 AV12-hK2-spent media was enzymatically active. Recombinant hK2 was also expressed in human prostate carcinoma cell lines, PC3 (PC3-hK2) and DU145 (DU145-hK2), that do not express endogenous hK2 or PSA. Similar to AV12-hK2 cells, both cell lines secreted phK2 that was converted to hK2 extracellularly. phK2 was the major form detected in the spent media of PC3-hK2 cells, even after 7 days, indicating a slow conversion of phK2 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 to hK2. In this study, we demonstrate that hK2 exists in different forms and is secreted as phK2. phK2 is then converted to enzymatically active hK2 extracellularly.
人类激肽释放酶家族由三个成员组成，即hK1、hK2和hK3[前列腺特异性抗原（PSA）]。PSA是前列腺癌广泛认可的标志物。hK2和PSA的mRNA主要定位于前列腺上皮，且受雄激素调节。此外，hK2与PSA有78%的氨基酸同源性。尽管与PSA的相似性使hK2成为潜在的前列腺癌标志物，但在PSA丰富的人体组织和体液中，这些相似性也阻碍了hK2的生化特性鉴定。为了研究hK2的表达、生物合成及加工过程，重组hK2在腺病毒诱导的叙利亚仓鼠肿瘤细胞系AV12 - 664（AV12 - hK2）中表达。使用单克隆抗体HK1G 464.3[对前体hK2（phK2）具有特异性]和HK1D 106.4[对phK2和成熟hK2（hK2）具有特异性]，通过蛋白质免疫印迹法和酶联免疫吸附测定法分析hK2的表达。蛋白质免疫印迹法和酶联免疫吸附测定法分析表明，AV12 - hK2细胞在第1天将phK2分泌到培养基中，并在第7天逐渐转化为成熟形式的hK2。N端氨基酸测序验证了蛋白质免疫印迹法和酶联免疫吸附测定法的数据。这首次证明hK2以phK2形式分泌，并在细胞外转化为hK2。此外，在第4 - 7天AV12 - hK2消耗培养基中检测到的hK2具有酶活性。重组hK2也在不表达内源性hK2或PSA的人前列腺癌细胞系PC3（PC3 - hK2）和DU145（DU145 - hK2）中表达。与AV12 - hK2细胞类似，这两种细胞系都分泌phK2，并在细胞外转化为hK2。phK2是在PC3 - hK2细胞消耗培养基中检测到的主要形式，即使在7天后也是如此，这表明phK2向hK2的转化缓慢。从第1天开始，hK2就是在DU145 - hK2消耗培养基中检测到的主要形式，这表明phK2向hK2的转化迅速。在本研究中，我们证明hK2以不同形式存在，并以phK2形式分泌。然后phK2在细胞外转化为具有酶活性的hK2。
