Identification of genetic changes associated with drug resistance by reverse in situ hybridization.

The molecular cytogenetic techniques of comparative genomic hybridization (CGH) and reverse in situ hybridization (REVISH) allow the entire genomes of tumours to be screened for genetic changes without the requirement for specific probes or markers. In order to define the ability of REVISH to detect and map regions of amplification associated with drug resistance, we investigated a panel of cell lines selected for resistance to doxorubicin and intrinsic sensitivity to topoisomerase II-inhibitory drugs. We have defined a modified REVISH protocol, which involves double hybridizations with genomic DNA from the test cell lines and chromosome-specific whole chromosome paints to identify the chromosomes to which the amplicons localize. Sites of amplification are then mapped by fractional length measurements (Flpter), using published genome databases. Our findings show that amplification of the topoisomerase II alpha gene is readily detected and mapped, as is amplification of the MDR and MRP loci. Interestingly, REVISH detected a new amplicon in the doxorubicin-resistant lung cancer cell line, GLC4-ADR, which mapped to chromosome 1q. REVISH is therefore ideally suited to characterize genetic changes specific for drug resistance within a background of genetic anomalies associated with tumour progression.