| Metallothionein Isoform 3 as a Potential Biomarker for Human Bladder Cancer Mary Ann Sens,1 Seema Somji,2 Donald L. Lamm,2 Scott H. Garrett,2 Frank Slovinsky,1 John H. Todd,1 and Donald A. Sens2 1Robert C. Byrd Health Sciences Center, Department of Pathology, 2Department of Urology, West Virginia University, Morgantown, West Virginia, USA Abstract
The goal of the present study was to determine if the expression of metallothionein isoform 3 (MT-3) might serve as a biomarker for human bladder cancer. To accomplish this goal, we defined the localization and expression of MT-3 protein and mRNA using fresh and archival biopsy specimens obtained from patients undergoing differential diagnosis for a variety of bladder disorders. We used immunohistochemistry, immunoblot, and RT-PCR analysis to define the localization and expression of MT-3 protein and mRNA. Immunohistochemical analysis disclosed no immunoreactivity for MT-3 in normal bladder cells. The absence of MT-3 expression in the normal bladder was further confirmed by demonstrating that MT-3 mRNA could not be detected using reverse transcriptase-polymerase chain reaction (RT-PCR) or MT-3 protein using immunoblot. Immunohistochemistry also disclosed no immunoreactivity for MT-3 in archival biopsy specimens from patients with interstitial cystitis and related disorders. Immunohistochemical analysis demonstrated that MT-3 was expressed in carcinoma in situ (CIS) , high-grade bladder cancer, low-grade bladder cancer, and dysplastic lesions. MT-3 immunostaining was intense in both CIS and high-grade bladder cancer, and low to moderate in low-grade bladder cancer and dysplastic lesions. We determined MT-3 mRNA expression in a subset of these bladder cancer specimens ; expression was elevated as compared to that of the housekeeping gene, ß-actin. The cDNA from the RT-PCR reaction primed for MT-3 contained a FokI restriction site, a site unique for MT-3 as compared to other MT family members. In conclusion, this study demonstrates that MT-3 is up-regulated in human bladder cancer and that this up-regulation increases with increasing tumor grade. The finding that MT-3 expression is minimal in normal bladder suggests that MT-3 might be developed into an effective biomarker for bladder cancer. Key words: biomarker, bladder, bladder cancer, carcinoma in situ, cystitis, immunohistochemistry, inflammation, interstitial cystitis, metallothionein, RT-PCR, transitional cell carcinoma, urothelium. Environ Health Perspect 108:413-418 (2000) . [Online 17 March 2000] http://ehpnet1.niehs.nih.gov/docs/2000/108p413-418sens/ abstract.html Address correspondence to D.A. Sens, Department of Urology, West Virginia University, PO Box 9251, Morgantown, WV 26506-9251 USA. Telephone: (304) 293-3212. Fax: (304) 293-6249. E-mail: dsens@wvu.edu Received 31 August 1999 ; accepted 12 November 1999.
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