Metallothionein isoform 1 and 2 gene expression in a human urothelial cell line (UROtsa) exposed to CdCl2 and NaAsO2

Studies have shown that metallothionein (MT) is overexpressed in some human bladder cancers and that overexpression can predict treatment response to neoadjuvant cisplatin, methotrexate, and vinblastine chemotherapy. in the present study the UROtsa cell line, a model of normal human urothelium, was used to determine the expression of the human MT-1 and MT-2 genes and MT protein following exposure to CdCl2 or NaAsO2 at lethal and sublethal levels. Acute exposure was modeled by treating confluent cultures with 700 PM NaAsO2 or 53.4 muM CdCl2 for 4 h followed by a 48-h recovery period. Extended exposure was modeled by treating confluent cells with 1, 4, and 8 muM As3+ or 1, 5, and 9 muM Cd2+, for 16 d, with the highest concentrations producing cell lethality. The expression of MT mRNAs and protein were determined by reverse-transcription polymerase chain reaction (RT-PCR) and immunoblot analysis. Cell viability was determined by the MTT assay. It was shown that acute exposure to either As3+ or Cd2+ increased the levels of mRNAs for the MT-1E, MT-1X, and MT-2A genes, whereas extended exposure only increased these mRNAs following exposure to Cd2+. It was shown that both acute and extended exposure to either As3+ or Cd2+ increased the levels of MT protein, reaching a maximal value of 8 ng MT protein/mug total protein for acute exposure to Cd2+. This is in contrast to previous studies using cultured human proximal tubule cells, where similar extended treatment with Cd2+ resulted in over 20-fold higher MT protein levels. These studies demonstrate that human urothelial cells accumulate only modest,amounts of MT protein when exposed to either Cd2+ or As2+ for a 16-d time period.