SYNTHESIS, MATURATION AND EXTRACELLULAR RELEASE OF PROCATHEPSIN-D AS INFLUENCED BY CELL-PROLIFERATION OR TRANSFORMATION

The relationship between cell growth and intra- and extracellular accu mulation of cathepsin D (CD), a lysosomal endopeptidase involved in ce ll protein breakdown, was examined in cultures of normal and transform ed BALB/c mouse 3T3 fibroblasts grown at various cell densities. In cr owded cultures of normal 3T3 cells (doubling time, Td, 53 hr) intracel lular CD activity was 2-fold higher than in sparse, vapidly-growing (T d, 27 hr) cultures. In uncrowded (Td, 18 hr) and crowded (Td, 32 hr) c ultures of benzo[a]pyrene-transformed cells intracellular CD levels we re one third and two thirds, respectively, of those measured in hyperc onfluent 3T3 cultures. Regardless of cell density, SV-40-virus-transfo rmed cells (Td, 12 hr) contained one third of CD levels found in hyper confluent 3T3 cells. Both transformed cell lines released into the med ium a higher proportion of CD, compared with their untransformed count erpart, yet the amount secreted was not sufficient to account for the reduced ina-acellular level of the enzyme. Serum withdrawal induced a marked increase of both intra- and extracellular levels of CD activity . In both normal and virally or chemically transformed 3T3 cells CD co mprised a precursor (52 kDa) and processed mature polypeptides; the la tter were mostly represented by a 48-kDa peptide, but a minor part was in a double-chain form (31 and 16 kDa respectively). The proportion o f mature enzyme vs. precursor was much higher in confluent, slowly-gro wing cells than in fast-growing cells, whether normal or transformed. In the latter, conversion of mature 48-kDa peptide into the double-cha in form occurred more efficiently. (C) 1995 Wiley-Liss, Inc.