Electrophoretic Characterization of Ribosomal Subunits and Proteins in Apoptosis:  Specific Downregulation of S11 in Staurosporine-Treated Human Breast Carcinoma Cells

Abstract

Stimulation of death receptors (Fas on human T-cell leukemia Jurkat cells and tumor necrosis factor receptor-1 on human monoblastic leukemia U937 cells) triggers the specific degradation of 28S ribosomal RNA, and this process may contribute to cell death through the inhibition of protein synthesis. We have developed an analytical method using a polyacrylamide-agarose composite gel to evaluate ribosomal subunits in apoptotic cells (human breast carcinoma MCF-7 cells treated with staurosporine and human 293T cells irradiated with ultraviolet light were used in addition to the two apoptosis systems described above). No alterations were detected by this method, suggesting that apoptosis, including the process of ribosomal RNA degradation, does not cause fragmentation or extensive conformational changes in the ribosome. We also examined the status of 21 different ribosomal proteins in apoptotic cells by immunoblotting with polyclonal antibodies. S11 was specifically downregulated in apoptotic MCF-7 cells and in other apoptotic breast carcinoma cells. Previous studies have shown that S11 is heterogeneously expressed in cancer cells. Taken together, it appears that particular intracellular environments regulate the expression of S11 protein. However, the mechanism by which this process is modulated is as yet unknown. Furthermore, we have demonstrated that our composite gel electrophoresis system can efficiently detect ubiquitination of ribosomal subunits.

Structural Change of Ribosomes during Apoptosis: Degradation and Externalization of Ribosomal Proteins in Doxorubicin-Treated Jurkat Cells

“…It is not likely that these proteins are cleaved directly by caspases since they contain no amino acid sequences that are targeted by caspases. These results are distinct from those previously obtained in the analysis with human breast carcinoma cells, in which the amount of Sll specifically decreased during apoptosis (16). Changes in the amount of ribosomal proteins in apoptotic cells are thus likely to be cell-type specific.…”

“…In some experiments, the specificity of the antibody binding was examined by pre-incubating the first antibodies for 1 h with excess amounts of the corresponding antigen peptides prior to the reaction with the membrane. To analyze the formation of the ribosome complex, we employed a newly developed method (16). Ribosome-enriched fractions were electrophoretically separated on a composite gel consisting of polyacrylamide (2%) and agarose (0.7%), and ribosome subunits were detected by Western blotting as described above.…”

“…These findings indicate that ribosomal proteins and the ribosome complex are structurally modified in apoptotic cells, and suggest that some ribosomal proteins have extra-ribosomal functions. However, the previous studies dealt with only a limited number of ribosomal proteins, and changes in the overall structure of the ribosome in apoptotic cells have not been intensively investigated-Nadano et al recently examined structural changes of ribosome subunits and proteins using anti-ribosomal protein antibodies and found that the amount of Sll decreases in human tumor-derived cells undergoing staurosporine-induced apoptosis (16). This change seemed to occur in a cell type-specific manner it was evident in four different cell lines derived from human breast carcinoma, but not in cells from other types of tumors (26).…”

Differential Expression of Ribosomal Proteins in Human Normal and Neoplastic Colorectum

“…Recently, S11 was shown to be specifically downregulated in apoptotic breast carcinoma MCF-7 cells induced by staurosporine (Nadano et al 2001). Therefore, the present result suggests that S11 ribosomal protein expressed in the colorectal cancer cells may inhibit apoptosis of cancer cells.…”

Active Multienzyme Assemblies for Long-Chain Olefinic Hydrocarbon Biosynthesis

“…2A, tag arrangement 4) were purified as described above. Native gels were made as described by Nadano et al (26) and contained 1.9% polyacrylamide and 0.75% agarose in 40 mM Tris-acetate buffer at pH 8.4. Protein was mixed with loading buffer (15% sucrose in Tris-acetate) and run at 100 V. Gels were transferred to a nitrocellulose membrane and washed thoroughly with 200 mM NaCl, 20 mM NaPO 4 (pH 7.4), and 0.1% Tween 20 as a blocking agent.…”