Transactivation of the metallothionein promoter in cisplatin-resistant cancer cells: a specific gene therapy strategy.

Abstract

Cisplatin (cis-diamminedichloroplatinum) is one of the most active agents against a broad range of malignancies, including ovarian cancer. Cisplatin resistance appears to be associated with several molecular alterations, including overexpression of metallothionein, a metal-binding protein. In the present study, we attempted to take advantage of metallothionein overexpression to overcome cisplatin resistance. Using a virus-free system (liposomes), we sought to express the suicide gene, thymidine kinase (TK), driven by the promoter of the human metallothionein IIa (hMTIIa) gene using the pMT-TK plasmid. We used cisplatin-resistant human ovarian carcinoma cells as a model. We first analyzed metallothionein expression using a ribonuclease protection assay. In comparison to parental cells, the cisplatin-resistant cells were found to have increased expression of metallothionein messenger RNA (mRNA). Metallothionein overexpression in these cells was not associated with an increased copy number of the hMTIIa gene or with different transfection efficiencies. Furthermore, we showed by reverse transcription-polymerase chain reaction analysis that transfection of the pMT-TK plasmid results in a 56-fold higher expression of thymidine kinase mRNA in cisplatin-resistant cells compared with parental cells, consistent with increased metallothionein promoter-mediated transactivation in the cisplatin-resistant cells. Transfection of resistant cells with pMT-TK or a control plasmid (pCD3-TK) resulted in a marked sensitization to ganciclovir, with a 50% cell growth-inhibitory concentration (IC(50)) of 20 microg/mL and 9 microg/mL, respectively. Transfections of the cisplatin-sensitive cells resulted in no sensitization to ganciclovir with pMT-TK (IC(50) 200 microg/mL) and a high sensitization with pCD3-TK (IC(50) = 6 microg/mL). These studies suggest that pMT-TK gene therapy may provide an alternative treatment for cisplatin-refractory ovarian tumors.