Proteome-level display by 2-dimensional chromatography of extracellular matrix-dependent modulation of the phenotype of bladder cancer cells

Robert E Hurst,Anil Singh,Kimberly D Kyker,Hiroyuki Matsumoto,Mikhail G Dozmorov,Nobuaki Takemori,Ricardo Saban,Michael H Simonian,Edna Betgovargez

doi:10.1186/1477-5956-4-13

Abstract

BackgroundThe extracellular matrix can have a profound effect upon the phenotype of cancer cells. Previous work has shown that growth of bladder cancer cells on a matrix derived from normal basement membrane suppresses many malignant features that are displayed when the cells are grown on a matrix that has been modified by malignant tumors. This work was undertaken to investigate proteome-level changes as determined by a new commercially available proteome display involving 2-dimensional chromatography for bladder cancer cells grown on different extracellular matrix preparations that modulate the expression of the malignant phenotype.ResultsDepending on the matrix, between 1300 and 2000 distinct peaks were detected by two-dimensional chromatographic fractionation of 2.1 – 4.4 mg of total cellular protein. The fractions eluting from the reversed-phase fractionation were suitable for mass spectrometric identification following only lyophilization and trypsin digestion and achieved approximately 10-fold higher sensitivity than was obtained with gel-based separations. Abundant proteins that were unique to cells grown on one of the matrices were identified by mass spectrometry. Following concentration, peaks of 0.03 AU provided unambiguous identification of protein components when 10% of the sample was analyzed, whereas peaks of 0.05 AU was approximately the lower limit of detection when the entire sample was separated on a gel and in-gel digestion was used. Although some fractions were homogeneous, others were not, and up to 3 proteins per fraction were identified. Strong evidence for post-translational modification of the unique proteins was noted. All 13 of the unique proteins from cells grown on Matrigel were related to MYC pathway.ConclusionThe system provides a viable alternative to 2-dimensional gel electrophoresis for proteomic display of biological systems. The findings suggest the importance of MYC to the malignant phenotype of bladder cancer cells.

Highlights

The extracellular matrix can have a profound effect upon the phenotype of cancer cells
In earlier reports from our laboratory we have developed a model for investigating the effect of extracellular matrix (ECM) on cancer cells by growing them on ECM derived from normal tissue, from malignant tissue, and on plastic in conventional tissue culture where ECM effects are essentially absent [8,9]
We describe the application of 2-dimensional chromatographic proteomics to describing the effect of ECM on the phenotype of bladder cancer cells, including the mass spectrometric identification of some of the major differences in the proteomes of cells grown on Matrigel, SISgel and plastic

Summary

Introduction

The extracellular matrix can have a profound effect upon the phenotype of cancer cells. On SISgel, a gel-forming ECM product derived from normal porcine small intestine submucosa (SIS), the same bladder cancer cell lines display a much more normalized phenotype in which invasion is suppressed and the lowest grade cell line forms a multi-layered structure reminiscent of normal bladder epithelium. The mechanism for this effect is not known and may reflect protein-level changes not reflected in the transcriptome

Methods

Results

Conclusion