Hypophosphorylation of topoisomerase IIalpha in etoposide (VP-16)-resistant human carcinoma cell lines associated with carboxy-terminal truncation

Abstract

Topoisomerase IIalpha is a target for many chemotherapeutic agents in clinical use. To define mechanisms of resistance and regions crucial for the function of topoisomerase IIalpha, drug-resistant cell lines have been isolated following exposure to topoisomerase II poisons. Two resistant sublines, T47D-VP and MCF-7-VP, were isolated from human carcinoma cell lines following exposure to 300 or 500 ng / ml etoposide (VP-16). Cytotoxicity studies confirmed resistance to etoposide and other topoisomerase II poisons. KCl-sodium dodecyl sulfate (K-SDS) precipitation assays using intact cells showed reduced DNA-topoisomerase II complex formation following VP-16 or amsacrine (m-AMSA). RNAse protection analysis identified a deletion of 200 base pairs in the topoisomerase IIalpha cDNA of T47D-VP and rising dbl quote, left (low)AA insertion" in the topoisomerase IIalpha cDNA of MCF-7-VP. Reduced topoisomerase IIalpha mRNA and protein levels were observed in both cell lines. It was somewhat surprising to find that nuclear extracts from T47D-VP and MCF-7-VP cells had comparable topoisomerase II activity to that of parental cells. Analysis of the extent of phosphorylation demonstrated that topoisomerase IIalpha from the resistant cells was relatively hypophosphorylated compared to that of parental cells. In these cell lines, hypophosphorylation secondary to loss of a portion of the C-terminal domain of topoisomerase IIalpha mediated the restored activity, despite a fall in topoisomerase IIalpha mRNA and protein, and this resulted in cross resistance to topoisomerase II poisons.