DNA clearing from noncovalently bound agents in mammalian cells as a new mechanism of drug resistance

Earlier. we have described the process of active dissociation or "DNA clearing" from noncovalently bound agents in living mammalian cells. The vital fluorescent bisbenzimidazole dye Hoechst 33342, which binds tightly but not covalently to DNA in the minor groove, was used for studying interactions of noncovalently binding agents with DNA. Multiple drug resistance (MDR) in tumour cells is related to the expression of transport proteins that alter cellular drug transport and distribution. Three different groups of genes (mdr, MRP, and LRP) and their products are implicated in MDR (review, Krishan A., Fitz C.M., Andritsch I.//Cytometry. 1997. V. 29. P. 279-285). To obtain new cell lines characterised by enhanced process of active dissociation of noncovalently bound agent from DNA or "DNA clearing", we selected rodent cell lines that are hyperresistant to Hoechst 33342, The cell lines selected from AA8 were named AA8Hoe-R-1-AA8Hoe-R-10, and the cell lines selected from L cells were called LHoe-R-1-LHoe-R-10. The most resistant of them, AA8Hoe-R-6 and AA8Hoe-R-7, were able to grow in the presence of 80 mu g/ml of Hoechst 33342 in the cell culture medium. All mutants were analysed with flow cytometric technique and were divided into two different groups. We conclude that the drug resistance of the first group of cell lines was due to the changes in transport proteins. The second group of the resistant cell lines was characterised by an enchanced dissociation of the bisbenzimidazole dye - DNA complex, As we believe, the enchanced level "DNA clearing" was caused by the amplification of some genes because the gradual increase of Hoechst resistance in the same cell line resulted from the increase in the ability to remove the dye from DNA. These lines were shown to be also resistant to netropsin.