Three immunoassays based on monoclonal antibodies specific for prostate specific antigen (PSA), alpha-1-antichymotrypsin (ACT), and the PSA-ACT complex.

BACKGROUND: Prostate specific antigen (PSA) has been widely used as a biomarker for the screening and diagnosis of prostate cancer. PSA in serum predominantly exists as a complex with alpha-1-antichymotrypsin (ACT), and measurement of free PSA and the PSA-ACT complex may improve the utility of the serum PSA assay for differential diagnosis of prostate cancer and non-malignant prostate diseases, such as benign prostatic hyperplasia (BPH). METHODS: Monoclonal antibodies (MAbs) against PSA, ACT, and the PSA-ACT complex were produced by immunizing mice with an incubated mixture of PSA and ACT, and characterized by Western blot analyses and several enzyme-linked immunosorbant assay (ELISA) methods. RESULTS: The MAbs produced in this study are capable of distinguishing the PSA-ACT complex from free PSA and ACT. Four MAbs have been selected and utilized to construct three ELISA systems for the separate measurements of free PSA, the PSA-ACT complex, and total PSA. CONCLUSIONS: The three PSA assay systems developed in this study can specifically measure free PSA, total PSA, and the PSA-ACT complex with equal molar sensitivity. It is expected that these PSA assay systems could be useful in the diagnosis of prostate cancer.

Cross-talk between calpain and caspase proteolytic systems during neuronal apoptosis.

Cross-talk between calpain and caspase proteolytic systems has complicated efforts to determine their distinct roles in apoptotic cell death. This study examined the effect of overexpressing calpastatin, the specific endogenous calpain inhibitor, on the activity of the two proteolytic systems following an apoptotic stimulus. Human SH-SY5Y neuroblastoma cells were stably transfected with full-length human calpastatin cDNA resulting in 20-fold overexpression based on Western blot and 5-fold greater calpain inhibitory activity in cell extracts. Wild type and calpastatin overexpressing (CST1) cells were neuronally differentiated and apoptosis-induced with staurosporine (0.1-1.0 microm). Calpastatin overexpression decreased calpain activation, increased caspase-3-like activity, and accelerated the appearance of apoptotic nuclear morphology. Following 0.1-0.2 microm staurosporine, plasma membrane integrity based on calcein-acetoxymethyl fluorescence was significantly greater at 24 h in differentiated CST1 compared with differentiated wild type cells. However, this protective effect was lost at higher staurosporine doses (0.5-1.0 microm), which resulted in pronounced caspase-mediated degradation of the overexpressed calpastatin. These results suggest a dual role for calpains during neuronal apoptosis. In the early execution phase, calpain down-regulates caspase-3-like activity and slows progression of apoptotic nuclear morphology. Subsequent calpain activity, facilitated by caspase-mediated degradation of calpastatin, contributes to plasma membrane disruption and secondary necrosis.

Comparison of calpain and caspase activities in the adult rat brain after transient forebrain ischemia.

The role of calpain and caspase family proteases in postischemic neuronal death remains controversial. This study compared the timing, location, and relative activity of calpains and caspases in the adult rat brain following 10 min of transient forebrain ischemia. Western blots of cortical, striatal, and hippocampal homogenates demonstrated a alpha-spectrin cleavage pattern indicative of predominant calpain activity, which peaked between 24 and 48 h after reperfusion. However, immunohistochemical evidence of both caspase 3 activation and caspase-mediated substrate cleavage was detected as early as 1 h and as late as 7 days after reperfusion in circumscribed neuronal populations. Simultaneous or sequential caspase and calpain activation was also observed suggesting the potential for interaction of these protease systems. The complex spatiotemporal pattern of calpain and caspase activity observed in this study provides important insights for the development and evaluation of therapeutic strategies to reduce protease-mediated injury following global brain ischemia.