Compartmentalized expression of kallikrein 4 (KLK4/hK4) isoforms in prostate cancer: nuclear, cytoplasmic and secreted forms.

The prostate-specific antigen-related serine protease gene, kallikrein 4 (KLK4), is expressed in the prostate and, more importantly, overexpressed in prostate cancer. Several KLK4 mRNA splice variants have been reported, but it is still not clear which of these is most relevant to prostate cancer. Here we report that, in addition to the full-length KLK4 (KLK4-254) transcript, the exon 1 deleted KLK4 transcripts, in particular, the 5'-truncated KLK4-205 transcript, is expressed in prostate cancer. Using V5/His6 and green fluorescent protein (GFP) carboxy terminal tagged expression constructs and immunocytochemical approaches, we found that hK4-254 is cytoplasmically localized, while the N-terminal truncated hK4-205 is in the nucleus of transfected PC-3 prostate cancer cells. At the protein level, using anti-hK4 peptide antibodies specific to different regions of hK4-254 (N-terminal and C-terminal), we also demonstrated that endogenous hK4-254 (detected with the N-terminal antibody) is more intensely stained in malignant cells than in benign prostate cells, and is secreted into seminal fluid. In contrast, for the endogenous nuclear-localized N-terminal truncated hK4-205 form, there was less difference in staining intensity between benign and cancer glands. Thus, KLK4-254/hK4-254 may have utility as an immunohistochemical marker for prostate cancer. Our studies also indicate that the expression levels of the truncated KLK4 transcripts, but not KLK4-254, are regulated by androgens in LNCaP cells. Thus, these data demonstrate that there are two major isoforms of hK4 (KLK4-254/hK4-254 and KLK4-205/hK4-205) expressed in prostate cancer with different regulatory and expression profiles that imply both secreted and novel nuclear roles.

Identification and characterization of KLK14, a novel kallikrein serine protease gene located on human chromosome 19q13.4 and expressed in prostate and skeletal muscle.

The kallikreins are a subfamily of serine proteases encoded in human, mouse, and rat by highly conserved tightly clustered multigene families. Here we report the identification and characterization of KLK14, a novel kallikrein gene located within the human kallikrein locus at 19q13.4. KLK14 is approximately 5.4 kb in length spanning seven exons and, by Northern blot analysis, transcribes two alternative transcripts present only in prostate (1.5 kb) and skeletal muscle (1.9 kb). The protein product, K14, predicted to be a 251-amino-acid secreted serine protease with trypsin-like substrate specificity, is translated in vitro with a molecular mass of approximately 31 kDa. In situ hybridization revealed that, in prostate, KLK14 is expressed by both benign and malignant glandular epithelial cells, thus exhibiting an expression pattern similar to that of two other prostatic kallikreins, KLK2 and KLK3, which encode K2 and prostate-specific antigen, respectively.

Reappraisal of vesicular types in the syncytial tegument of the Echinococcus granulosus protoscolex.

The structure, abundance, and distribution of tegumentary vesicles was compared among Echinococcus granulosus protoscoleces that had been prepared for electron microscopy using four processing schedules: a conventional method, alternative fixations using uranyl acetate or osmium tetroxide-potassium ferricyanide, and a freeze-substitution method. Four morphologically distinct types of vesicles were found in the somal region. The morphology of the first form, with moderately electron-opaque contents, and the second form, with similar size and shape but containing an electron-opaque core, varied little among the preparation methods. Two additional forms of vesicles, with characteristic intensely electron-opaque contents, were revealed only after freeze-substitution. These elongate vesicles were also found in the scolex tegument where they were most conspicuous, and appeared markedly increased in number after freeze-substitution. Large, spherical vesicles with an electron-lucent core embedded in a dense matrix of fibrillar strands were the dominant vesicle forms in the scolex region after all methods of preparation. Fixation by osmium tetroxide-potassium ferricyanide revealed the presence of spherical vesicles with amorphous electron-opaque contents and a few inclusions. This form of vesicle was also observed after freeze-substitution, but the inclusions in the vesicular lumen were more numerous. The variation in the distribution of vesicle forms among the body regions strongly implies a variety of vesicle functions. In addition, our observations suggest that comparative studies of different fixative methods are necessary to demonstrate the detailed vesicular morphology of the tegument of E. granulosus and other cestodes.