A novel
monoclonal antibody has been developed that reacts strongly with human
prostatic cancer, especially
tumors of high grade. This antibody (7E11C-5) is currently in Phase 3 trials as an imaging agent for metastatic disease. We have cloned the gene that encodes the
antigen that is recognized by the 7E11C-5
monoclonal antibody and have designated this unique
protein prostate-specific membrane (
PSM) antigen.
PSM antigen is a putative class II transmembranous
glycoprotein exhibiting a molecular size of Mr 94,000. Functionally, class II
membrane proteins 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
monoclonal antibody. We observed substantial
carboxypeptidase activity as
folate hydrolase associated with
PSM antigen. The purpose of our study was to demonstrate that human prostatic
carcinoma cells expressing
PSM antigen exhibit
folate hydrolase activity using
methotrexate triglutamate (
MTXGlu3) and
pteroylpentaglutamate (PteGlu5) as substrates. Isolated membrane fractions from four human
prostate cancer cell lines (LNCaP, PC-3, TSU-Prl, and Duke-145) were examined for
folate hydrolase activity using capillary electrophoresis. After timed incubations at various pH ranges and in the presence and absence of
thiol reagents, separation of pteroyl(
glutamate)n derivatives was achieved with an
electrolyte of
sodium borate and SDS, while absorbance was monitored at 300 nm. The results demonstrate clearly that LNCaP cells, which highly express PSM, hydrolyze gamma-glutamyl linkages of
MTXGlu3. The membrane-bound
enzyme is an
exopeptidase, because it progressively liberates
glutamates from
MTXGlu3 and PteGlu5 with accumulation of MTX and PteGlu1, respectively. The semipurified
enzyme has a broad activity 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 glutathione,
homocysteine, and
p-hydroxymercuribenzoate (0.05-0.5 mm) but was inhibited weakly by DTT (>/=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 comparable
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
monoclonal antibody and demonstrated
folate hydrolase activities and optimum pH activity profiles identical to those of LNCaP cells. The membrane-bound
enzymes from both LNCaP- and PC-3-transfected cells also have a capacity to hydrolyze an alpha-linked glutamyl moiety from N-acetyl-alpha-
aspartylglutamate. We have identified that
PSM antigen is a pteroyl 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 future therapeutic strategies in the treatment of
prostate cancer that utilize
folate antagonists need to consider this mechanism of resistance.