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1 Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, 600 University Ave., Toronto, Ontario, M5G 1X5 Canada.
2 Department of Laboratory Medicine and Pathobiology, University of Toronto, 100 College St., Toronto, Ontario, M5G 1L5 Canada.
3 Department of Urology, University Hospital Charite, Humboldt University of Berlin, 100498 Berlin, Germany.
4 National Center of Scientific Research Demokritos, IPC, Athens, Greece 15310.
aAuthor for correspondence. Fax 416-586-8628; e-mail ediamandis{at}mtsinai.on.ca.
Background: Human kallikrein 4 (gene, KLK4; protein, hK4), a recently discovered member of the kallikrein gene family, shares many characteristics with prostate-specific antigen, the best available marker for prostate cancer. Because the protein has not been detected in any human tissue, we attempted to develop immunologic methods for hK4 analysis and use them to detect hK4 in healthy and cancerous tissue extracts and biological fluids.
Methods: We extracted total RNA from 20 pairs of matched (healthy–cancer) prostate tissue samples. KLK4 cDNA was amplified by reverse transcription-PCR (RT-PCR) and cloned in a pPICZ
A expression vector. We then transformed the construct product into Pichia pastoris yeast strains and induced secreted recombinant protein production by addition of methanol. We purified the recombinant protein by nickel ion-affinity chromatography and used it as an immunogen in rabbits and mice to generate polyclonal anti-hK4 antibodies. These antibodies were used to develop a sandwich-type immunoassay suitable for hK4 quantification in biological fluids and tissue extracts.
Results: The immunoassay had a detection limit of 0.1 µg/L. We detected hK4 in 10 of 21 matched (healthy–cancer) prostate tissues, and hK4 was frequently higher in healthy tissues. In one matched-sample pair, the hK4 content was relatively high in both the healthy [4.62 µg/g of total protein (TP)] and the cancerous (1.22 µg/g of TP) prostate tissue. Among tissue extracts, we found the highest concentrations of hK4 in healthy (0.0–4.62 µg/g of TP) and cancerous (0.0–1.72 µg/g of TP) prostatic extracts and in placental extracts (0.0–0.05 µg/g of TP). We also detected traces of hK4 protein immunoreactivity in amniotic fluid (<0.1–0.6 µg/L), human breast milk (<0.1–0.75 µg/L), and seminal plasma (0.2–0.9 µg/L). Immunohistochemical studies showed cytoplasmic staining for hK4 protein in both malignant and benign epithelial cells of the prostate. However, we did not detect hK4 in cerebrospinal fluid, healthy and cancerous ovarian tissue extracts, and many other human tissue extracts.
Conclusions: hK4 protein is present in some prostatic tissue extracts but at relatively low concentrations, although KLK4 mRNA is readily detectable by RT-PCR. We propose that the protein either is not synthesized efficiently or is degraded very quickly.
The following articles in journals at HighWire Press have cited this article:
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  G. J. Mize, W. Wang, and T. K. Takayama Prostate-Specific Kallikreins-2 and -4 Enhance the Proliferation of DU-145 Prostate Cancer Cells through Protease-Activated Receptors-1 and -2 Mol. Cancer Res., June 1, 2008; 6(6): 1043 - 1051. [Abstract] [Full Text] [PDF]
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 T. I. Klokk, A. Kilander, Z. Xi, H. Waehre, B. Risberg, H. E. Danielsen, and F. Saatcioglu Kallikrein 4 Is a Proliferative Factor that Is Overexpressed in Prostate Cancer Cancer Res., June 1, 2007; 67(11): 5221 - 5230. [Abstract] [Full Text] [PDF]
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 M. H. Slagter, A. Scorilas, L. J.G. Gooren, W. de Ronde, A. Soosaipillai, E. J. Giltay, M. Paliouras, and E. P. Diamandis Effect of Testosterone Administration on Serum and Urine Kallikrein Concentrations in Female-to-Male Transsexuals Clin. Chem., August 1, 2006; 52(8): 1546 - 1551. [Abstract] [Full Text] [PDF]
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M. H. Slagter, L. J.G. Gooren, W. de Ronde, A. Soosaipillai, A. Scorilas, E. J. Giltay, M. Paliouras, and E. P. Diamandis Serum and Urine Tissue Kallikrein Concentrations in Male-to-Female Transsexuals Treated with Antiandrogens and Estrogens Clin. Chem., July 1, 2006; 52(7): 1356 - 1365. [Abstract] [Full Text] [PDF]
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 T L Veveris-Lowe, M G Lawrence, R L Collard, L Bui, A C Herington, D L Nicol, and J A Clements Kallikrein 4 (hK4) and prostate-specific antigen (PSA) are associated with the loss of E-cadherin and an epithelial-mesenchymal transition (EMT)-like effect in prostate cancer cells Endocr. Relat. Cancer, September 1, 2005; 12(3): 631 - 643. [Abstract] [Full Text] [PDF]
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C. V. Obiezu, S. J.C. Shan, A. Soosaipillai, L.-Y. Luo, L. Grass, G. Sotiropoulou, C. D. Petraki, P. A. Papanastasiou, M. A. Levesque, and E. P. Diamandis Human Kallikrein 4: Quantitative Study in Tissues and Evidence for Its Secretion into Biological Fluids Clin. Chem., August 1, 2005; 51(8): 1432 - 1442. [Abstract] [Full Text] [PDF]
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J. P. Simmer, J. D. Bartlett, and F. Saatcioglu Kallikrein 4 Is a Secreted Protein Cancer Res., November 15, 2004; 64(22): 8481 - 8483. [Full Text] [PDF]
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V. Vaisanen, S. Eriksson, K. K. Ivaska, H. Lilja, M. Nurmi, and K. Pettersson Development of Sensitive Immunoassays for Free and Total Human Glandular Kallikrein 2 Clin. Chem., September 1, 2004; 50(9): 1607 - 1617. [Abstract] [Full Text] [PDF]
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C. A. Borgono, I. P. Michael, and E. P. Diamandis Human Tissue Kallikreins: Physiologic Roles and Applications in Cancer Mol. Cancer Res., May 1, 2004; 2(5): 257 - 280. [Abstract] [Full Text] [PDF]
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T. Kishi, A. Soosaipillai, L. Grass, S. P. Little, E. M. Johnstone, and E. P. Diamandis Development of an Immunofluorometric Assay and Quantification of Human Kallikrein 7 in Tissue Extracts and Biological Fluids Clin. Chem., April 1, 2004; 50(4): 709 - 716. [Abstract] [Full Text] [PDF]
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C. A. Borgono, L. Grass, A. Soosaipillai, G. M. Yousef, C. D. Petraki, D. H. C. Howarth, S. Fracchioli, D. Katsaros, and E. P. Diamandis Human Kallikrein 14: A New Potential Biomarker for Ovarian and Breast Cancer Cancer Res., December 15, 2003; 63(24): 9032 - 9041. [Abstract] [Full Text] [PDF]
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