Capony F, Braulke T, Rougeot C, Roux S, Montcourrier P, Rochefort H
Institut National de la Santé et de la Recherche Médicale, Unité Hormones et Cancer (U 148), Montpellier, France.
Exp Cell Res. 1994 Nov;215(1):154-63. doi: 10.1006/excr.1994.1327.
The secretion of pro-cathepsin D (pro-cath-D) in some human metastatic breast cancer cells (MCF7, MDA/MB231), contrary to normal mammary cells, is not increased by ammonium chloride treatment, indicating a mannose-6-phosphate-independent sorting to lysosomes. By studying a variety of cell lines and lysosomal enzymes, we show that secretion of newly synthesized pro-cath-D was not mediated by the 46-kDa mannose-6-phosphate receptor (MPR) and that its resistance to NH4Cl for secretion was specific to cath-D and not to other lysosomal enzymes. This resistance appeared to be correlated with the basal hypersecretion of pro-cath-D, but not with its overexpression. By contrast, pro-cath-D secretion was increased by NH4Cl in fibroblasts and nontumoral epithelial mammary cells, suggesting a specificity for cancer cells. Immunofluorescence staining showed that pro-cath-D, but neither cathepsin B nor beta-hexosaminidase, accumulated in intracytoplasmic vesicles of cells treated with ammonium chloride. In pulse--chase experiments and by subcellular fractionation on Percoll gradient, cath-D was found to be sorted into dense lysosomes whether cells were treated or not by NH4Cl. Treatment of cells with NH4Cl, however, inhibited processing and maturation of pro-cath-D, which was also observed in light vesicles in the absence of NH4Cl. Part of pro-cath-D, but not processed enzyme, was also found to be membrane associated in saponin-permeabilized cells. We conclude that in breast cancer cells, the MPR-independent pathway of pro-cath-D to lysosome is predominant compared to normal cells and other lysosomal enzymes. This alternative pathway should therefore be considered, in addition to MPR, to explain pro-cath-D sorting and activation in breast cancer cells.
与正常乳腺细胞相反，氯化铵处理不会增加某些人转移性乳腺癌细胞（MCF7、MDA/MB231）中组织蛋白酶D原（pro-cath-D）的分泌，这表明其向溶酶体的分选不依赖于甘露糖-6-磷酸。通过研究多种细胞系和溶酶体酶，我们发现新合成的pro-cath-D的分泌不是由46 kDa的甘露糖-6-磷酸受体（MPR）介导的，并且其分泌对氯化铵的抗性是组织蛋白酶D特有的，而不是其他溶酶体酶。这种抗性似乎与pro-cath-D的基础高分泌相关，但与其过表达无关。相比之下，氯化铵可增加成纤维细胞和非肿瘤性乳腺上皮细胞中pro-cath-D的分泌，这表明癌细胞具有特异性。免疫荧光染色显示，氯化铵处理的细胞的胞质内囊泡中积累了pro-cath-D，但没有组织蛋白酶B或β-己糖胺酶。在脉冲追踪实验和通过Percoll梯度进行亚细胞分级分离中，发现无论细胞是否用氯化铵处理，组织蛋白酶D都会被分选到致密溶酶体中。然而，氯化铵处理会抑制pro-cath-D的加工和成熟，在没有氯化铵的情况下，轻囊泡中也观察到了这种情况。在皂素通透的细胞中，还发现部分pro-cath-D而非加工后的酶与膜相关。我们得出结论，在乳腺癌细胞中，与正常细胞和其他溶酶体酶相比，pro-cath-D向溶酶体的不依赖MPR的途径占主导地位。因此，除了MPR之外，还应考虑这种替代途径来解释乳腺癌细胞中pro-cath-D的分选和激活。
