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Development of an in-vitro model system to investigate the mechanism of muscle protein catabolism induced by proteolysis-inducing factor.

Gomes-Marcondes MC, Smith HJ, Cooper JC, Tisdale MJ - Br. J. Cancer (2002)

Bottom Line: In myoblasts this followed a bell-shaped dose-response curve with maximal effects at a proteolysis-inducing factor concentration between 2 and 4 nM, while in myotubes increased protein degradation was seen at all concentrations of proteolysis-inducing factor up to 10 nM, again with a maximum of 4 nM proteolysis-inducing factor.There was also an increased expression of the 19S regulatory complex as well as the ubiquitin-conjugating enzyme (E2(14k)), and in myotubes a decrease in myosin expression was seen with increasing concentrations of proteolysis-inducing factor.These results show that proteolysis-inducing factor co-ordinately upregulates both ubiquitin conjugation and proteasome activity in both myoblasts and myotubes and may play an important role in the muscle wasting seen in cancer cachexia.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of Campinas, UNICAMP, SP, Brazil 13083-970. M.J.Tisdale@aston.ac.uk

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(A) Western blot of soluble extracts of C2C12 myoblasts detected with MCP231, a murine monoclonal antibody to the proteasome α-subunits after treatment of cells with PBS (A) or 1 (B), 2 (C), 4 (D), 10.5 (E) or 17 nM PIF (F) alone or with cycloheximide (1 μM) (G) or with cycloheximide (1 μM) plus 1 (H), 2 (I), 4 (J), 10.5 (K) or 17 nM PIF (L). (B) Western blot analysis of soluble extracts of C2C12 myoblasts either untreated (A) or treated with 0.4 (B), 1 (C), 2 (D), 4 (E) or 10 nM PIF (F) and detected with mouse anti-human MSSI antibody. (C) Western blot analysis of soluble extracts of C2C12 myoblasts either untreated (A) or treated with 0.4 (B), 1 (C), 2 (D), 4 (E) or 10 nM PIF (F) and detected with E214k rabbit polyclonal antisera.
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fig2: (A) Western blot of soluble extracts of C2C12 myoblasts detected with MCP231, a murine monoclonal antibody to the proteasome α-subunits after treatment of cells with PBS (A) or 1 (B), 2 (C), 4 (D), 10.5 (E) or 17 nM PIF (F) alone or with cycloheximide (1 μM) (G) or with cycloheximide (1 μM) plus 1 (H), 2 (I), 4 (J), 10.5 (K) or 17 nM PIF (L). (B) Western blot analysis of soluble extracts of C2C12 myoblasts either untreated (A) or treated with 0.4 (B), 1 (C), 2 (D), 4 (E) or 10 nM PIF (F) and detected with mouse anti-human MSSI antibody. (C) Western blot analysis of soluble extracts of C2C12 myoblasts either untreated (A) or treated with 0.4 (B), 1 (C), 2 (D), 4 (E) or 10 nM PIF (F) and detected with E214k rabbit polyclonal antisera.

Mentions: To determine whether PIF-induced protein catabolism was mediated through the ATP-ubiquitin-dependent pathway proteasome functional activity was determined by measuring ‘chymotrypsin-like’ enzyme activity, the major proteolytic activity of the β-subunits. Using the fluorogenic substrate succinyl LLVY-MCA an increase in enzyme activity was detectable at concentrations of PIF between 2 and 4 nM (Figure 1B). The effect on protein expression of proteasome subunits in the presence of PIF was determined by immunoblotting. Cellular supernatants of PIF-treated cells were Western blotted using MCP 231 antibody, a murine monoclonal to the 20S proteasome, which reacts with the six different α-type subunits. Three bands were detected at approximate molecular weight of 29 000, 32 000 and 35 000 (Figure 2AFigure 2


Development of an in-vitro model system to investigate the mechanism of muscle protein catabolism induced by proteolysis-inducing factor.

Gomes-Marcondes MC, Smith HJ, Cooper JC, Tisdale MJ - Br. J. Cancer (2002)

(A) Western blot of soluble extracts of C2C12 myoblasts detected with MCP231, a murine monoclonal antibody to the proteasome α-subunits after treatment of cells with PBS (A) or 1 (B), 2 (C), 4 (D), 10.5 (E) or 17 nM PIF (F) alone or with cycloheximide (1 μM) (G) or with cycloheximide (1 μM) plus 1 (H), 2 (I), 4 (J), 10.5 (K) or 17 nM PIF (L). (B) Western blot analysis of soluble extracts of C2C12 myoblasts either untreated (A) or treated with 0.4 (B), 1 (C), 2 (D), 4 (E) or 10 nM PIF (F) and detected with mouse anti-human MSSI antibody. (C) Western blot analysis of soluble extracts of C2C12 myoblasts either untreated (A) or treated with 0.4 (B), 1 (C), 2 (D), 4 (E) or 10 nM PIF (F) and detected with E214k rabbit polyclonal antisera.
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Related In: Results  -  Collection

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fig2: (A) Western blot of soluble extracts of C2C12 myoblasts detected with MCP231, a murine monoclonal antibody to the proteasome α-subunits after treatment of cells with PBS (A) or 1 (B), 2 (C), 4 (D), 10.5 (E) or 17 nM PIF (F) alone or with cycloheximide (1 μM) (G) or with cycloheximide (1 μM) plus 1 (H), 2 (I), 4 (J), 10.5 (K) or 17 nM PIF (L). (B) Western blot analysis of soluble extracts of C2C12 myoblasts either untreated (A) or treated with 0.4 (B), 1 (C), 2 (D), 4 (E) or 10 nM PIF (F) and detected with mouse anti-human MSSI antibody. (C) Western blot analysis of soluble extracts of C2C12 myoblasts either untreated (A) or treated with 0.4 (B), 1 (C), 2 (D), 4 (E) or 10 nM PIF (F) and detected with E214k rabbit polyclonal antisera.
Mentions: To determine whether PIF-induced protein catabolism was mediated through the ATP-ubiquitin-dependent pathway proteasome functional activity was determined by measuring ‘chymotrypsin-like’ enzyme activity, the major proteolytic activity of the β-subunits. Using the fluorogenic substrate succinyl LLVY-MCA an increase in enzyme activity was detectable at concentrations of PIF between 2 and 4 nM (Figure 1B). The effect on protein expression of proteasome subunits in the presence of PIF was determined by immunoblotting. Cellular supernatants of PIF-treated cells were Western blotted using MCP 231 antibody, a murine monoclonal to the 20S proteasome, which reacts with the six different α-type subunits. Three bands were detected at approximate molecular weight of 29 000, 32 000 and 35 000 (Figure 2AFigure 2

Bottom Line: In myoblasts this followed a bell-shaped dose-response curve with maximal effects at a proteolysis-inducing factor concentration between 2 and 4 nM, while in myotubes increased protein degradation was seen at all concentrations of proteolysis-inducing factor up to 10 nM, again with a maximum of 4 nM proteolysis-inducing factor.There was also an increased expression of the 19S regulatory complex as well as the ubiquitin-conjugating enzyme (E2(14k)), and in myotubes a decrease in myosin expression was seen with increasing concentrations of proteolysis-inducing factor.These results show that proteolysis-inducing factor co-ordinately upregulates both ubiquitin conjugation and proteasome activity in both myoblasts and myotubes and may play an important role in the muscle wasting seen in cancer cachexia.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of Campinas, UNICAMP, SP, Brazil 13083-970. M.J.Tisdale@aston.ac.uk

Show MeSH
Related in: MedlinePlus