Abstract
Renal cell carcinoma (RCC) displays strong resistance against many chemotherapeutic drugs. Overexpression of P-glycoprotein (Pgp) appears to be part of this resistance. The involvement of another resistance mechanism, involving the decreased activity of DNA topoisomerase II (topoII), remains uncertain. By culturing the human RCC lines RC2 and RC21 in the presence of increasing concentrations of etoposide, we derived the variant sublines RC2E, RC21A and RC21E, that had acquired approximately 30-, 60- and 90-fold resistance to this drug respectively. RC2E, RC21A and RC21E were approximately 50-, 5- and 400-fold cross-resistant to doxorubicin respectively. RC2E and RC21E also showed cross-resistance (approximately 200- and 3500-fold respectively) to vinblastine. Quantitative differences in MDR1 and Pgp expression (elevated in RC2E and RC21E) and topoII alpha (reduced in RC21E and RC21A) were demonstrated using Western blotting and the reverse transcriptase/polymerase chain reaction. Decreased amounts of topoII alpha were reflected in a reduced activity of RC21A and RC21E as measured by unknotting phage P4 DNA. Qualitative changes of the topoII alpha gene, such as point mutations in the motif B/DNBS and DNA-binding regions, or differences in methylation status of the promoter gene of RC21E, were not found. These cell lines represent a model of a solid tumor in which overexpression of Pgp, a combination of increased Pgp and decreased topoII alpha, and a decrease of topoII alpha are represented.
Highlights
30% of patients with renal cell carcinoma (RCC) present with metastases at the time of diagnosis
RC2E derived from RC2, and RC21A and RC21E, both derived from RC21, were used for further studies
To try to account for the decreased expression of topoIIa protein, we examined by single-strand conformational polymorphism (SSCP) analysis two regions of the topoIIa gene where, relatively infrequently, point mutations leading to functional aberrations are known to occur (Danks et al 1993), changing the ATP-binding region (ATP-binding sites: motif B and DNBS) and the DNA-binding region
Summary
30% of patients with renal cell carcinoma (RCC) present with metastases at the time of diagnosis. One of the best known resistance mechanisms implicated in MDR of RCC consists of overexpression of the MDR1 gene and its product P-glycoprotein (Pgp), an energy-dependent multidrug transmembrane transporter that prevents intracellular accumulation of cytotoxic drugs by rapidly extruding them (Kakehi et al 1988). This form of drug resistance (Pgp-MDR) can be circumvented in vitro by chemosensitisers that interact with and block the drug-eux function of Pgp (Kakehi et al 1988). Altered topoIIa activity, which can be caused by quantitative (Fry et al 1991; Gudkov et al 1993; Kim and Beck 1994; Ritke and Yalowich 1993) or qualitative (Danks et al 1993; Hinds et al 1991) changes, can result in reduced numbers of drug-stabilized cleavable complexes and is likely to inuence cellular sensitivity to topoII inhibitors
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