Proteomic analysis of T24T derived bladder cancer cell lines using differential in-gel electrophoresis and LC-MS/MS
Authors: Gunjan Malik, Saurabh Gupta, Michael Ward, Shamina Mitchell, Krista White, Richard Drake, Dan Theodorescu, O. John Semmes, Jose Diaz
Journal: Cancer Research (2007)

2719  Bladder Cancer (BCa) is the fifth most common cancer in the United States with an estimated 61,420 new cases in 2006. The muscle-invasive form of the disease accounts for 30% of BCa patients and is associated with poor prognosis. The relative 5-year survival rate for metastatic BCa is 16% as opposed to 95% for an early stage, noninvasive cancer.  Investigation of molecular changes associated with BCa progression has been restricted due to poor availability of human metastatic BCa cell lines in a suitable animal model. The development of T24T cells (Gildea JJ et al., 2000) as a highly tumorigenic variant of the widely used T24 TCC cell line has led to the establishment of a model system for studying BCa progression. T24T was subsequently utilized for the derivation of progressively more efficiently metastatic human BCa cell lines specific to distant organs like lung (FL) and liver (SLT).  Using 2-D DIGE and LC-MS/MS, we analyzed T24T and its highly metastatic derivatives. Whole cell protein extracts were labeled with fluorescent cyanine dyes and separated by 2-D gel electrophoresis. DeCyder2.0 software analysis generated up to 2844 protein spots on the master gel with 9.7% of the ‘filtered’ spots differentially expressed between the two cell lines (>2.5 fold). 18 protein spots with significant differential expression between the two cell lines were ‘picked’ from the gel and identified by LC-MS/MS. 15 proteins were up-regulated and 3 were down-regulated in the metastatic SLT4 cell line. A notable finding was the up-regulation of a ~76 kDa protein in SLT4 identified as bone morphogenetic protein endothelial cell precursor-derived regulator (BMPER). BMPER, an analog of CV-2, is known to inhibit BMP2/4 function. BMPs are TGF-beta family morphogens involved in bone and cartilage formation. They have recently been shown to regulate a large range of biological functions in various cell types. In recent studies, BMPs and their antagonists have also been linked to tumor growth and development for their involvement in cellular processes such as apoptosis, cell proliferation, cell differentiation and angiogenesis.  We are currently assessing the expression levels of various BMPs and BMP-related proteins in several T24T derived BCa cell lines. Initial Western results using antibodies against BMP2/4 and BMP receptors displayed decreased levels of BMP2/4 in SLT4. We are currently developing antibodies against rBMPER to validate the DIGE results. Since BMPER is a secreted protein, we will also analyze its levels in culture supernatants. The potential role of BMPER as a biomarker for BCa metastasis is also under investigation.  Assessing the genetic and proteomic changes associated with the degree of metastatic potential in T24T derived tumor cell lines could help understand the mechanism of BCa invasion and metastasis, lead to the discovery of prognostic biomarkers for BCa and define molecular targets for BCa therapy.