Senile plaque and paired helical filament (PHF) formation are characteristic of Alzheimer's disease, but the mechanisms leading to these lesions still remain unclear. To understand them better, we have performed different immunolabellings of amyloid protein and PHF. We describe a very specific immunodetection of PHF with AD2, a monoclonal antibody directed against a hyperphosphorylated epitope of PHF-tau, and use double immunolabelling to show that PHF and plaque amyloid are discretely labelled by different antibodies. We also discuss different mechanisms of PHF maturation.

We have studied the intracellular trafficking of cathepsin D in different colon carcinoma cell populations: the HT-29 cell line, composed of >95% undifferentiated cells; 2 subpopulations derived from this cell line, containing cells committed to differentiation into mucin-secreting cells (HT-29 MTX) or enterocyte-like cells (HT-29 G-) after confluence; and the Caco-2 cell line, which spontaneously differentiates into enterocyte-like cells after confluence. Post-confluent undifferentiated HT-29 cells and differentiated enterocyte-like HT-29 G- and Caco-2 cells secrete significant levels of cathepsin D in culture medium, in contrast to post-confluent differentiated mucin-secreting HT-29 MTX cells, which secrete this enzyme at a very low level. The intracellular content and the mRNA level of cathepsin D increase after confluence in the different cell types, particularly in Caco-2 cells, which intensify the secretion of cathepsin D along with the differentiation process post-confluence. Membrane-associated mature cathepsin D was detected in HT-29 cells but not in Caco-2 cells. In the different types of cell, pro-cathepsin D associates with the membrane concomitantly to its binding to an Mr 72,000 protein. Membrane association persists after dissociation of the complex in HT-29 cells but not in Caco-2 cells. In the mucin-secreting HT-29 MTX cells, cathepsin D was immunolocalised to the membrane of mucin vacuoles localised under the brush border. Our results show that cathepsin D can be regulated differently in colon carcinoma cells, and this finding might have specific functional implications for each cell type.

Int. J. Cancer: 68,479-484 (1996) 0 1996 Wilcy-Liss, Inc. In human skin fibroblasts, cathepsin D is synthesised as a glycosylated precursor form of M, 53,000 with 2 asparaginelinked oligosaccharide side chains in the rough endoplasmic reticulum (Hasilik and Von Figura, 1981; Gieselman et al., 1983). A portion of these N-linked oligosaccharides may become phosphorylated or converted into complex oligosaccharides in the Golgi apparatus (Hasilik and Von Figura, 1981; Giesclman et al., 1983). Phosphomannosyl residucs are lysosoma1 targcting signals which will bind to the mcmbrancassociated mannose-6-phosphate specific receptors (MPRs) in the trans-Golgi network (Dahms et al., 1989). During transport to lysosomes, the precursor form is successively converted into an intermediate M, 47,000 form by cleavage of the pro-peptide in a pre-lysosomal compartment and then into the mature M, 31,000 form found in lysosomes (Gieselman et al., 1983). REGULATION OF CATHEPSIN D DEPENDENT ON THE PHENOTYPE OF COLON CARCINOMA CELLSSylvianeThe oestrogen-stimulated over-cxprcssion of cathepsin D in breast cancer cells has becn suggested to induce a saturation of the mannose-6-phosphatc/IGF-II receptor that Icd to the abnormal secretion of the pro-enzyme (Rochefort et al., 1990). Subsequently, intracellular targeting of cathepsin D was also shown to occur in a mannose-6-phosphate indcpendent manner in HepG2 cells (Rijnboutt et al., 1991a, b ) and breast cancer cells (Capony et al., 1994). In the MPR indcpendent transport in HepG2 cells, cathepsin D also becomes transiently membrane-associated in the Golgi apparatus through binding to an M, 72,000 pro-saposin form (Zhu and Conner, 1994). After delivery to lysosomes and subsequent proteolytic processing, the complex is dissociated and both proteins are released from the membrane. In addition, pro-cathepsin D associates to an M, 68,000 pro-saposin to form a soluble complex.By Western blotting, we have previously shown that the HT-29 colon carcinoma cell line, which contains >95% undiffcrentiatcd HT-29 cclls, secretes pro-cathepsin D in the culture medium, whcreas a subpopulation derived from this cell line, which contains differentiated mucin-secreting HT-29 cells, appears unable to secrete the pro-enzyme. Furthermore, using immunocytochcmistry, we have found that cathepsin D is localised, like mucins, in the apical compartment of the cells, under the brush border (Huct et al., 1994).These observations Icd us to examine the synthesis and intracellular trafficking of cathepsin D in the HT-29 cell line, composed of > 95% undifferentiated cells, and in different types of differentiated colon carcinoma cells: differentiated mucin-secreting HT-29 goblet cells, obtained by a stepwise adaptation to the anti-cancer drug methotrcxatc ( M) (Ixsumeur et al., 1990); differentiated cnterocyte-like HT-29 cells, selected by hexose deprivation (Lcsumeur et al., 1991); and Caco-2 cells, which spontaneously display structural and functional differentiation characteristics of mature enterocytcs at late c...