One of the major mechanisms responsible for multidrug resistance in cancer chemotherapy is overexpression of some members of the ATP-binding cassette (ABC) transporter superfamily, including ABCB1, ABCC1 and ABCG2, resulting in decreased intracellular levels of drugs [reviewed in (58)]. and 7-BIA deletions as determined by the Nexus software algorithm. To quantify genomic breakage, log2 ratio differences larger than 0.3 were used to Rabbit Polyclonal to CDKA2 discriminate putative DNA breakage points. We identified those breakage points at the edges of segments of copy number gains and losses as well as points of abrupt copy number changes called within larger aberrations. The precision of this type of measurement is determined by the resolution of the array. The smallest aberration we were able to detect with confidence on the 180k platform was 100 kb in length. In addition, the abnormality needed to be present in at least 10-20% of the cells in order to be detected. To obtain a global genomic insight into the development of acquired CPT resistance and to identify putative candidate genes involved in this process, we took advantage of the CPT-K5 cell line, which exhibits highly stable CPT resistance following prolonged CPT-selective growth of its parental RPMI-8402 cell line (11,24,34). For the purposes of the present study, we wanted to confirm the cellular and biochemical characteristics of these cell lines using current methods. Firstly, immunophenotyping of the parental cell line RPMI-8402 confirmed that it is indeed a T-ALL derived cell line (Table I). The CPT-K5 cell line has not previously been characterized by immunophenotyping but this analysis confirmed that CPT-K5 retains the predominant T-ALL immunophenotype (Table I). Secondly, at the genetic level it was confirmed that the RPMI-8402 harbors two T-ALL specific chromosomal aberrations: i) the common deletion at chromosome region 1p32; and ii) the rare t(11;14)(p15;q11)/(11) and Kjeldsen (24), and establish that the CPT-K5 cell line is a T-ALL derived 7-BIA cell line with unique characteristics. due to reduced gene dosage; ii) mutations that render the enzyme drug-resistant; iii) reduced intracellular active drug content by decreased drug influx or increased drug 7-BIA efflux; iv) resistance to apoptosis; and v) efficient repair of TOP1-cc by the cell. A first line of defense for a cell against CPT-induced damage is down-regulation of its TOP1 activity, thereby reducing TOP1-cc (55,56). Our subtractive oligo-based aCGH analysis revealed that the gene dosage of the locus was diminished by a factor of approximately 7-BIA 1.7, which was confirmed by FISH (Figure 5). The loss of gene copy numbers correlated with a reduced level of cellular TOP1 protein in CPT-K5 cells (Figure 2). Other studies have shown that a copy number change of the gene correlates with the cellular amount of enzyme and seems to be a common mechanism contributing to CPT resistance (56). By CGH analysis of the CPT-resistant cell lines HT-29/CPT, A549/CPT and st-4/CPT, a reduced DNA copy number of TOP1 was shown together with a reduced relative expression of TOP1 (44). In our subtractive oligo-based aCGH analysis, we further screened for copy number changes of other type I topoisomerases (and and (also known as and exhibited large losses, and loci of TOP3B and TOP2B were gained, while and displayed no copy number change between the CPT-K5 and RPMI-8402 cells (Table IV). Open in a separate window Figure 5 Fluorescence in situ hybridization (FISH) validation of copy number changes at specific genomic regions important for the camptothecin resistance in CPT-K5 cells. Left hand-side panel: Genomic profiles of chromosome 20 (upper panel), chromosome 4 (middle panel) and chromosome 14 (lower panel) together with their respective ideograms beneath. Magnified genomic profile views of corresponding altered chromosomal regions are given below chromosome 20, chromosome 4, and chromosome 14 indicating deleted region at 20q12 containing topoisomerase 1 (TOP1) gene, highly amplified region at 4q22.1 containing ATP-binding cassette sub-family G member 2 (ABCG2) gene and amplified region at 14q31.3q32.11 containing the tyrosyl-DNA phosphodiesterase 1 (TDP1) gene, respectively. Blue and red bars around the ideograms indicate regions of gains and losses, respectively. Regions with high gains and losses are indicated by double bars in their respective color. A yellow bar in the copy number profile of chromosome 14 indicates.