PROSTATE-SPECIFIC MEMBRANE ANTIGEN - A NOVEL FOLATE HYDROLASE IN HUMAN PROSTATIC-CARCINOMA CELLS

A novel monoclonal antibody has been developed that reacts strongly wi th human prostatic cancer, especially tumors of high grade. This antib ody (7E11C-5) is currently in Phase 3 trials as an imaging agent for m etastatic disease. We have cloned the gene that encodes the antigen th at is recognized by the 7E11C-5 monoclonal antibody and have designate d this unique protein prostate-specific membrane (PSM) antigen. PSM an tigen is a putative class II transmembranous glycoprotein exhibiting a molecular size of M(r) 94,000. Functionally, class II membrane protei ns serve as transport or binding proteins or have hydrolytic activity. Preliminary studies have demonstrated binding of pteroylmonoglutamate (folate) to membrane fractions that also cross-reacted with the PSM m onoclonal antibody. We observed substantial carboxypeptidase activity as folate hydrolase associated with PSM antigen. The purpose of our st udy was to demonstrate that human prostatic carcinoma cells expressing PSM antigen exhibit folate hydrolase activity using methotrexate trig lutamate (MTXGlu(3)) and pteroylpentaglutamate (PteGlu(5)) as substrat es. Isolated membrane fractions from four human prostate cancer cell l ines (LNCaP, PC-3, TSU-Pr1, and Duke-145) were examined for folate hyd rolase activity using capillary electrophoresis. After timed incubatio ns at various pH ranges and in the presence and absence of thiol reage nts, separation of pteroyl(glutamate)(n) derivatives was achieved with an electrolyte of sodium berate and SDS, while absorbance was monitor ed at 300 nm. The results demonstrate clearly that LNCaP cells, which highly express PSM, hydrolyze gamma-glutamyl linkages of MTXGlu(3). Th e membrane-bound enzyme is an exopeptidase, because it progressively l iberates glutamates from MTXGlu(3) and PteGlu(5) with accumulation of MTX and PteGlu(1), respectively. The semipurified enzyme has a broad a ctivity from pH 2.5 to 9.5 and exhibits activity maxima at pH 5 and 8. Enzymatic activity is maintained in the presence of reduced glutathio ne, homocysteine, and p-hydroxymercuribenzoate (0.05-0.5 mM) but was i nhibited weakly by DTT (greater than or equal to 0.2 mM). By contrast to LNCaP cell membranes, membranes isolated from other human prostate adenocarcinoma cells (PC-3, Duke-145, and TSU-Pr1) did not exhibit com parable hydrolase activity, nor did they react with 7E11-C5 monoclonal antibody. After transfection of PC-3 cells with a full-length 2.65-kb PSM cDNA subcloned into a pREP7 eukaryotic expression vector, non-PSM antigen-expressing PC-3 cells developed immunoreactivity to 7E11-C5 m onoclonal antibody and demonstrated folate hydrolase activities and op timum pH activity profiles identical to those of LNCaP cells. The memb rane-bound enzymes from both LNCaP- and PC-3-transfected cells also ha ve a capacity to hydrolyze an alpha-linked glutamyl moiety from N-acet yl-alpha-aspartylglutamate. We have identified that PSM antigen is a p teroyl poly-gamma-glutamyl carboxypeptidase (folate hydrolase) and is expressed strongly in human prostate cancer. Cancer cells that express this enzyme are resistant to methotrexate therapy. Those developing f uture therapeutic strategies in the treatment of prostate cancer that utilize folate antagonists need to consider this mechanism of resistan ce.