title: Detection of human kallikrein 4 in healthy and cancerous prostatic tissues by immunofluorometry and immunohistochemistry.
abstract: 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 pPICZalphaA 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 microg/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 microg/g of total protein (TP)] and the cancerous (1.22 microg/g of TP) prostate tissue. Among tissue extracts, we found the highest concentrations of hK4 in healthy (0.0-4.62 microg/g of TP) and cancerous (0.0-1.72 microg/g of TP) prostatic extracts and in placental extracts (0.0-0.05 microg/g of TP). We also detected traces of hK4 protein immunoreactivity in amniotic fluid (<0.1-0.6 microg/L), human breast milk (<0.1-0.75 microg/L), and seminal plasma (0.2-0.9 microg/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.
authors.affiliation: Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, 600 University Ave., Toronto, Ontario, M5G 1X5 Canada.
authors.forename: Christina V
authors.initials: CV
authors.lastname: Obiezu
authors.forename: Antoninus
authors.initials: A
authors.lastname: Soosaipillai
authors.forename: Klaus
authors.initials: K
authors.lastname: Jung
authors.forename: Carsten
authors.initials: C
authors.lastname: Stephan
authors.forename: Andreas
authors.initials: A
authors.lastname: Scorilas
authors.forename: David H C
authors.initials: DH
authors.lastname: Howarth
authors.forename: Eleftherios P
authors.initials: EP
authors.lastname: Diamandis
mesh_terms: D000818:Animals; D000906:Antibodies; D014408:Biomarkers, Tumor / Q000032:analysis*; D001826:Body Fluids / Q000737:chemistry; D005453:Fluorescence; D006801:Humans; D007150:Immunohistochemistry; D007610:Kallikreins / Q000032:analysis* / Q000096:biosynthesis / Q000302:isolation & purification; D008297:Male; D051379:Mice; D011467:Prostate / Q000737:chemistry*; D011471:Prostatic Neoplasms / Q000737:chemistry* / Q000175:diagnosis; D012333:RNA, Messenger / Q000032:analysis; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification; D012016:Reference Values; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D000906:Antibodies; D014408:Biomarkers, Tumor; D012333:RNA, Messenger; D011994:Recombinant Proteins; D007610:Kallikreins; C120801:kallikrein 4