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Updated: Jul 24, 2025
Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis
Published on: May 12, 2019
Characterization of Native COMPASS Complex in Urothelial Carcinoma Cells by Size Exclusion Chromatography
Christoph Peter1, Wolfgang A Schulz2, Patcharawalai Whongsiri3
1Institute of Molecular Medicine I, Medical Faculty, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
Abstract:
The human COMPASS complexes regulate gene expression during development and cell differentiation. Three distinct subunits, KMT2C, KMT2D, and KDM6A (also known as UTX), are frequently mutated in urothelial carcinoma, possibly disrupting the formation of functional COMPASS complexes. Here, we describe methods to evaluate the formation of these large native protein complexes in urothelial carcinoma (UC) cell lines harboring different mutations in KMT2C/D. To this end COMPASS complexes were purified from nuclear extracts by size exclusion chromatography (SEC) using a Sepharose 6 column. SEC fractions were then separated by 3-8% Tris-acetate gradient polyacrylamide gel and the COMPASS complex subunits KMT2C, UTX, WDR5, and RBBP5 were detected by immunoblotting. In this fashion, the formation of a COMPASS complex could be observed in UC cells with wild-type but not in cells with mutant KMT2C and KMTD.
Insights
The study investigated COMPASS complexes in urothelial carcinoma (UC). Functional COMPASS complexes were detected in UC cells with wild-type KMT2C/D, but not in those with mutations, suggesting disrupted complex formation in cancer.
Area of Science:
- Molecular Biology
- Epigenetics
- Cancer Biology
Background:
- COMPASS complexes regulate gene expression crucial for development and differentiation.
- Mutations in KMT2C, KMT2D, and KDM6A (UTX) are common in urothelial carcinoma (UC).
- These mutations may impair the formation of functional COMPASS complexes in UC.
Purpose of the Study:
- To develop and apply methods for evaluating COMPASS complex formation in UC cell lines.
- To assess the impact of KMT2C/D mutations on COMPASS complex assembly.
Main Methods:
- Purification of COMPASS complexes from nuclear extracts using size exclusion chromatography (SEC) on a Sepharose 6 column.
- Separation of SEC fractions using gradient polyacrylamide gel electrophoresis (PAGE).
- Detection of COMPASS subunits (KMT2C, UTX, WDR5, RBBP5) via immunoblotting.
Main Results:
- Established a method to analyze large native protein complex formation in UC cells.
- Demonstrated the presence of COMPASS complexes in UC cells with wild-type KMT2C/D.
- Showed the absence of detectable COMPASS complex formation in UC cells with mutant KMT2C and KMTD.
Conclusions:
- The described method effectively evaluates COMPASS complex formation in UC.
- Mutations in KMT2C/D disrupt the assembly of functional COMPASS complexes in urothelial carcinoma.
- This finding has implications for understanding UC pathogenesis and potential therapeutic strategies.
