Biochemical characterization of prostate-specific membrane antigen from canine prostate carcinoma cells.

BACKGROUND: Prostate-specific membrane antigen (PSMA) remains an important target for diagnostic and therapeutic application for human prostate cancer. Model cell lines have been recently developed to study canine prostate cancer but their PSMA expression and enzymatic activity have not been elucidated. The present study was focused on determining PSMA expression in these model canine cell lines and the use of fluorescent small-molecule enzyme inhibitors to detect canine PSMA expression by flow cytometry. METHODS: Western blot and RT-PCR were used to determine the transcriptional and translational expression of PSMA on the canine cell lines Leo and Ace-1. An endpoint HPLC-based assay was used to monitor the enzymatic activity of canine PSMA and the potency of enzyme inhibitors. Flow cytometry was used to detect the PSMA expressed on Leo and Ace-1 cells using a fluorescently tagged PSMA enzyme inhibitor. RESULTS: Canine PSMA expression on the Leo cell line was confirmed by Western blot and RT-PCR, the enzyme activity, and flow cytometry. Kinetic parameters Km and Vmax of PSMA enzymatic activity for the synthetic substrate (PABGγG) were determined to be 393 nM and 220 pmol min(-1) mg protein(-1) , respectively. The inhibitor core 1 and fluorescent inhibitor 2 were found to be potent reversible inhibitors (IC50 = 13.2 and 1.6 nM, respectively) of PSMA expressed on the Leo cell line. Fluorescent labeling of Leo cells demonstrated that the fluorescent PSMA inhibitor 2 can be used for the detection of PSMA-positive canine prostate tumor cells. Expression of PSMA on Ace-1 was low and not detectable by flow cytometry. CONCLUSIONS: The results described herein have demonstrated that PSMA is expressed on canine prostate tumor cells and exhibits similar enzymatic characteristics as human PSMA. The findings show that the small molecule enzyme inhibitors currently being studied for use in diagnosis and therapy of human prostate cancer can also be extended to include canine prostate cancer. Importantly, the findings demonstrate that the potential of the inhibitors for use in diagnosis and therapy can be evaluated in an immunocompetent animal model that naturally develops prostate cancer before use in humans.

Revisiting the Chesapeake Bay phytoplankton index of biotic integrity.

In 2006, a phytoplankton index of biotic integrity (PIBI) was published for Chesapeake Bay Lacouture et al. (Estuaries 29(4):598-616, 2006). The PIBI was developed from data collected during the first 18 years (1985-2002) of the Chesapeake Bay Program long-term phytoplankton and water quality monitoring programs. Combinations of up to nine phytoplankton metrics were selected to characterize bay habitat health according to plankton community condition in spring and summer seasons across four salinity zones. The independent data available at the time for index validation was not sufficient to test the PIBI because they lacked critical index parameters (pheophytin and dissolved organic carbon) and reference samples for some seasons and salinity zones. An additional 8 years of monitoring data (2003-2010) are now available to validate the original index, reassess index performance and re-examine long-term trends in PIBI conditions in the Bay. The PIBI remains sensitive to changes in nutrient and light conditions. Evaluation of the PIBI results over the entire 1985-2010 time period shows no discernible trends in the overall health of Bay habitat based on phytoplankton community conditions. This lack of overall PIBI trend appears to be a combined response to declines in water clarity and improvements in dissolved inorganic nitrogen and dissolved phosphorus conditions in the bay.

Prolonged androgen deprivation leads to downregulation of androgen receptor and prostate-specific membrane antigen in prostate cancer cells.

Emergence of androgen-independent cancer cells during androgen deprivation therapy presents a significant challenge to successful treatment outcomes in prostate cancer. Elucidating the role of androgen deprivation in the transition from an androgen-dependent to an androgen-independent state may enable the development of more effective therapeutic strategies against prostate cancer. Herein, we describe an in vitro model for assessing the effects of continuous androgen-deprivation on prostate cancer cells (LNCaP) with respect to the expression of two prostate-specific markers: the androgen receptor (AR) and prostate-specific membrane antigen (PSMA). Compared with androgen-containing normal growth medium, androgen-deprived medium apparently induced the concomitant downregulation of AR and PSMA over time. Decreased protein levels were confirmed by fluorescence imaging, western blotting and enzymatic activity studies. In contrast to the current understanding of AR and PSMA in prostate cancer progression, our data demonstrated that androgen-deprivation induced a decrease in AR and PSMA levels in androgen-sensitive LNCaP cells, which may be associated with the development of more aggressive disease-state following androgen deprivation therapy.