Multiple mechanisms of resistance in a series of human testicular teratoma cell lines selected for increasing resistance to etoposide.

Mechanisms of resistance to VP-16 were monitored in a series of sublines of the human testicular teratoma cell line (SuSa) derived following exposure either to fractionated X-irradiation (DXR-10) or to VP-16 using pulsed 24-hr exposures (VP10) or continuous exposure conditions (VPC2, VPC3 and VPC4). Orders of resistance expressed (ranging from 3- to 33-fold based on IC50 values derived from colony forming assays) were comparable with those likely to be encountered clinically. All of these resistant sublines showed some cross-resistance to VCR, and the 3 drug-selected sublines tested also proved cross-resistant to ADR. Resistance was not associated with modified 3H-VP-16 accumulation. However, decreased VP-16-induced SSBs were detectable in all the resistant sublines and a strong positive correlation was noted between the extent of SSB formation and VP-16 resistance by linear regression analysis. Topo II alpha protein content, as judged by Western blotting, was significantly decreased only in the sublines derived by continuous exposure to VP-16, but this was not progressive with increasing levels of resistance expressed. RNase protection assays also showed no significant differences in Topo II alpha expression in the low-level resistant DXR-10 and VP10 sublines, contrasting with the 2-fold decreases identified in the VPC2, VPC3 and VPC4 sublines. Significantly, however, mRNA levels of two alternately spliced Topo II beta mRNAs were markedly decreased (2- to 9-fold) in all the drug-selected resistant sublines. No mutations in consensus ATP-binding sequences or in the DNA-binding region of Topo II alpha were detected by single strand conformational polymorphism analysis. Significant Pgp overexpression was only identified in the most highly resistant sublines VPC3 and VPC4, which both showed 4-fold cross-resistance to VCR. Decreased 3H-VCR accumulation and partial reversal of resistance by VPM (6.6 microM) addition was also identified, consistent with a functional Pgp being overexpressed in these sublines. Modifications of Topo II expression therefore appear to precede Pgp overexpression in this series of sequentially derived VP-16 resistant sublines and to represent the predominant mechanism underlying low level (< 10-fold) resistance.