Abstract. We have studied the posttranslational modifications of the 52-kD protein, an estrogenl~gulated autocrine mitogen secreted by several human breast cancer cells in culture (Westley, B., and H. Rochefort, 1980, Cell, 20:353-362). The secreted 52-kD protein was found to be phosphorylated mostly (94%) on high-mannose N-linked oligosaccharide chains, and mannose-6-phosphate signals were identified. The phosphate signal was totally removed by alkaline phosphatase hydrolysis. The secreted 52-kD protein was partly taken up by MCF7 cells via mannose-6-phosphate receptors and processed into 48-and 34-kD protein moieties as with lysosomal hydrolases. By electron microscopy, immunoperoxidase staining revealed most of the reactive proteins in lysosomes. After complete purification by immunoaffinity chromatography, we identified both the secreted 52-kD protein and its processed cellular forms as aspartic and acidic proteinases specifically inhibited by pepstatin. The 52-kD protease is secreted in breast cancer cells under its inactive proenzyme form, which can be autoactivated at acidic pH with a slight decrease of molecular mass. The enzyme of breast cancer cells, when compared with cathepsin D(s) of normal tissue, was found to be similar in molecular weight, enzymatic activities (inhibitors, substrates, specific activities), and immunoreactivity. However, the 52-kD protein and its cellular processed forms of breast cancer cells were totally sensitive to endo-13-N-acetylglucosaminidase H (Endo H), whereas several cellular cathepsin D(s) of normal tissue were partially Endo H-resistant. This difference, in addition to others concerning tissue distribution, mitogenic activity and hormonal regulation, strongly suggests that the 52-kD cathepsin D-like enzyme of breast cancer cells is different from previously described cathepsin D(s). The 52-kD estrogen-induced lysosomal proteinase may have important functions in facilitating the mammary cancer cells to proliferate, migrate, and metastasize.
Rat-liver nucleoli (10-15 micrograms DNA) were digested with either 0.6 or 3 units of DNase I for various times (up to 1 h). RNA synthesis was then measured in the absence or presence of 3 units of Escherichia coli RNA polymerase. It was found that the nucleolar chromatin supporting the endogenous engaged RNA polymerase I transcription was completely destroyed in 3 min with either concentration of DNase I. The nucleolar chromatin template transcribed by E. coli RNA polymerase retained 50% of its original capacity even 60 min after 3 units of DNase I digestion. When hybridization experiments were conducted, it was found that the DNAs derived from both levels of DNase-I-digested nucleoli were incapable of forming hybrids with the labelled nucleolar RNA synthesized by the engaged RNA polymerase I from the untreated nucleoli. Since the engaged RNA polymerase I transcribes only the physiologically active genes of the nucleolar chromatin, and the RNA transcripts represent active gene product, these data suggest that DNase I digestion has completely destroyed the active genes of the nucleolar chromatin, and E. coli RNA polymerase is able to transcribe the inactive nucleolar chromatin template.
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