Background: Using differential display (DD), we discovered a new membe
r of the serine protease family of protein-cleaving enzymes, named pro
tease M. The gene is most closely related by sequence to the kallikrei
ns, to prostate-specific antigen (PSA), and to trypsin. The diagnostic
use of PSA in prostate cancer suggested that a related molecule might
be a predictor for breast or ovarian cancer. This, in turn, led to st
udies designed to characterize the protein and to screen for its expre
ssion in cancer. Materials and Methods: The isolation of protease M by
DD, the cloning and sequencing of the cDNA, and the comparison of the
predicted protein structure with related proteins are described, as a
re methods to produce recombinant proteins and polyclonal antibody pre
parations. Protease M expression was examined in mammary, prostate, an
d ovarian cancer, as well as normal cells and tissues. Stable transfec
tants expressing the protease M gene were produced in mammary carcinom
a cells. Results: Protease M was localized by fluorescent in situ hybr
idization analysis to chromosome 19q13.3, in a region to which other k
allikreins and PSA also map. The gene is expressed in the primary mamm
ary carcinoma lines tested but not in the corresponding cell lines of
metastatic origin. It is strongly expressed in ovarian cancer;tissues
and cell lines. The enzyme activity could not be established, because
of difficulties in producing sufficient recombinant protein, a common
problem with proteases. Transfectants were selected that overexpress t
he mRNA, but the protein levels remained very low. Conclusions: Protea
se M expression (mRNA) may be a useful marker in the detection of prim
ary mammary carcinomas, as well as primary ovarian cancers. Other medi
cal applications are also likely, based on sequence relatedness to try
psin and PSA.