Effect of the serine protease inhibitor N-tosyl-l-phenylalanine-chloromethyl ketone (TPCK) on MCF-7 mammary tumour cells growth and differentiation.

Previous studies from this and other laboratories indicated that the oestrogen-regulated heat shock protein HSP27 is involved in the control of MCF-7 cells growth and differentiation, as it also appears to be in other cell types, including osteoblasts and HL-60 cells. In the latter instance, induction of differentiation is associated with the downregulation of myeloblastin, a serine protease now identified as proteinase 3 (hence its designation as PR3/Mbn), mirrored by an increase in the cellular content of the small heat shock protein HSP27, a substrate to this enzyme. Besides, antisense inhibition of PR3/Mbn production sufficed for inducing HL-60 cells monocytic differentiation. This prompted us to examine the hypothesis that a post-translational control on HSP27 levels (and by this on differentiation) by a serine protease might also be operating in human mammary tumour cells. As part of our attempt to evaluate this hypothesis, the present work consisted of testing the effects of a treatment of MCF-7 cells with the serine protease inhibitor N-tosyl-L-phenylalanine-chloromethyl ketone (TPCK). Our data show that this resulted in a four-fold increase in HSP27 content, associated with a 2.5-fold decrease in growth rate, the formation of cytoplasmic vesicles and increased secretion of 52 kDa peptides, identified by Western immunoblot as the isoforms of the oestrogen-regulated protein, cathepsin D. TPCK only affected growth in MDAMB-231 cells (in which HSP27 levels are very low and remained below MCF-7 cells basal levels after treatment) and failed to affect L929 cells, in which the hsp27 gene is silent. This provides circumstantial support for the assumption that effects of TPCK on the MCF-7 cells phenotype are linked to the associated increase in HSP27 content. Our recent demonstration that MCF-7 cells do in fact express PR3/Mbn fits with our concept and opens the way to test it directly, using antisense strategy.