2008 Annual Meeting Abstracts

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Defects in Muscarinic Receptor Cell Signaling in a Bladder Urothelial Cancer Cell Line
Brian T. Tully, Jared R. Berkowitz, Yan Sun*, Mingkai Li*, Toby C. Chai
University of Maryland School of Medicine, Baltimore, MD

Introduction: Bladder urothelial cells (BUC) have been shown to be involved in bladder signaling. BUC can release and respond to various putative neurotransmitters. Based on data from keratinocytes, another epithelial cell type, non-neuronal acetylcholine is critical to keratinocyte growth and differentiation. This study explores the muscarinic receptor signaling in a bladder cancer cell line J82.
Methods:J82 cells were purchased from ATCC and normal human BUC were cultured from cystoscopic biopsies. Using PCR, cells were analyzed for the presence of m1-m5 transcripts. Cells were stimulated with carbachol (100 µM), a cholinergic agonist. Changes in intracellular calcium [Ca2+]i levels were measured using fura-2 ratiometric microfluorimetry. m3 plasmid transfection of J82 cells was performed.
Results:J82 cells expressed neither m2 nor m3 transcripts whereas normal BUC expressed both. Consistently, all J82 cells failed to respond to carbachol (0/64 cells). 47% of normal BUC (8/17 cells) responded to carbachol. m3-transfected cells had a positive m3 band on PCR. m3-transfected J82 cells responded to carbachol, but at a rate of 8% (17/212 cells).
Conclusions:This is the first description of muscarinic signaling in bladder cancer cells. Cancer cells expressed neither m2 nor m3 transcripts. Normal BUC expressed both these transcripts. J82 cells had no response to carbachol as measured by changes in [Ca2+]i whereas a large proportion of normal BUC responded. We could partially reverse the defect in J82 muscarinic signaling phenotype by transfecting these cancer cells with the m3 plasmid. Muscarinic signaling on urothelial cell growth and differentiation is unknown and will be explored further.

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