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 (VP 10) or continuous exposure conditions (VPC2, VPC3 and VPC4). Orders of resistance expressed (ranging from 3- to 33-fold based on ICS0 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 IIα 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 IIα expression in the low-level resistant DXR-10 and VP 10 sublines, contrasting with the 2-fold decreases identified in the VPC2, VPC3 and VPC4 sublines. Significantly, however, mRNA levels of two alternately spliced Topo IIβ mRNAs were markedly decreased (2- to 9-fold) in all the drug-selected resistant sublincs. No mutations in consensus ATP-binding sequences or in the DNA-binding region of Topo Ma were detected by single strand conformations I polymorphism analysis. Significant Pgp over express ion 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 μM) 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 predominate mechanism underlying low level (< 10-fold) resistance. © 1994 Wiley-Liss, Inc.