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.

Proliferating cell nuclear antigen immunoreactivity in the ovarian surface epithelium of mice of varying ages and total lifetime ovulation number following ovulation.

Everytime an oocyte is released at ovulation, the ovarian surface epithelium (OSE) is ruptured and must be restored by epithelial cell proliferation. Ovulation site closure was studied in mice of various ages along with total lifetime ovulation number to investigate the known association of these factors with the risk of epithelial ovarian cancer. Ovaries from Swiss Webster mice were collected at various time points postovulation from 3-mo virgin animals (estimated median total lifetime ovulation number, 92; n = 40 mice), 8-mo virgin animals subject to incessant ovulation (estimated median total lifetime ovulation number, 652; n = 15 mice), and 12-mo breeders (estimated median total lifetime ovulation number, 208; n = 35 mice). Diameters of ovulation sites were estimated by scanning electron microscopy. No differences were found in the rate of ovulation site closure between the groups. Sections of ovaries were analyzed using immunohistochemistry for proliferating cell nuclear antigen (PCNA). The highest density of immunoreactive cells was observed in all animal groups in the cuboidal cells around the rupture site the day after ovulation. Despite the similarity in ovulation site closure rates between groups, the total number of OSE cells that were positive for PCNA in both the 8- and 12-mo animals was significantly reduced, so the number of stained cells appeared to be insufficient to cover the ovulation site. These data suggest that other mechanisms, such as proliferation of the extraovarian mesothelium, may play a role in the re-epithelialization of the ovary.