Increased phosphorylation of DNA topoisomerase II in etoposide resistant mutants of human glioma cell line.

The efficacy of the epipodophyllotoxins VP-16 and VM-26 is limited by the occurrence of drug resistance in the tumor cell population. Cellular insensitivity to drugs that stabilize the cleavable complex is frequently expressed as multidrug resistance (MDR). In some cell lines, overexpression of MDR-1/P-glycoprotein or the multidrug resistance associated protein (MRP) has been demonstrated and implicated as the mechanism of resistance. Typically, these cells have reduced drug accumulation, secondary to increased drug efflux. In other cell lines, an atypical MDR phenotype has been identified, with the predominant mechanism of resistance shown to be qualitative and/or quantitative changes in the levels and activity of topoisomerase II. For VP-16, increased expression of MDR-1 or MRP and alterations in topoisomerase II have been shown to confer tolerance. To further understand resistance to VP-16, T98G-VP(1000) was initially isolated as a single clone from parental cell, T98G, by exposure to VP-16. Subsequently, a population of cells from this subline was exposed to three-fold higher drug concentration allowing stable sublines to be established at higher extracellular drug concentration. Characterization of the resistant sublines demonstrates the adaptation that occurs with advancing drug concentrations during in vitro selections. Reduced topoisomerase II mRNA and protein levels were observed in the initial isolate. This reduction was accompanied by a decrease in topoisomerase II activity and cellular growth rate and was associated with 47-fold resistance to topoisomerase II poisons. With advancing resistance, MRP expression increased, with increased VP-16 efflux and reduced accumulation. This adaptation allowed for partial restoration of topoisomerase II activity secondary to increased expression and hyperphosphorylation, with a resultant increase in growth rate. In this cell line, hyperphosphorylation coincided with increased casein kinase II mRNA protein levels, without increased PKC protein levels, suggesting a role for this kinase in the acquired hyperphosphorylation. In this cell line, hyperphosphorylation mediated the increased activity despite a fall in topoisomerase II protein levels secondary to an acquired 615 bp deletion in one topoisomerase II allele, which resulted in reduced protein levels. In this subline, high levels of resistance were attained as a result of synergism between the reduced topoisomerase II levels and MRP overexpression. These studies demonstrate how cellular adaptation to increasing drug pressure occurs and how more than one mechanism can contribute to the resistant phenotype when increasing selecting pressure is applied. Reduced expression of topoisomerase II is sufficient to confer substantial resistance early in the selection process, with synergy from additional mechanisms helping to confer high levels of resistance.