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 MTprotein following exposure to CdCl₂ or NaAsO₂ at lethal and sublethal levels. Acute exposure was modeled by treating confluent cultures with 100 μM NaAsO₂ or 53.4 μM CdCl₂ for 4 h followed by a 48-h recovery period. Extended exposure was modeled by treating confluent cells with 1, 4, and 8 μM As³⁺ or 1, 5, and 9 μM 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/μg 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 Cd ²⁺ or As²⁺ for a 16-d time period.