Abstract
The DNA-damaging compound cisplatin is broadly employed for cancer chemotherapy. The mutagenic effects of cisplatin on cancer cell genomes are poorly studied and might even contribute to drug resistance. We have therefore analyzed mutations and chromosomal alterations in four cisplatin-resistant bladder cancer cell lines (LTTs) by whole-exome-sequencing and array-CGH. 720–7479 genes in the LTTs contained point mutations, with a characteristic mutational signature. Only 53 genes were mutated in all LTTs, including the presumed cisplatin exporter ATP7B. Chromosomal alterations were characterized by segmented deletions and gains leading to severely altered karyotypes. The few chromosomal changes shared among LTTs included gains involving the anti-apoptotic BCL2L1 gene and losses involving the NRF2 regulator KEAP1. Overall, the extent of genomic changes paralleled cisplatin treatment concentrations. In conclusion, bladder cancer cell lines selected for cisplatin-resistance contain abundant and characteristic drug-induced genomic changes. Cisplatin treatment may therefore generate novel tumor genomes during patient treatment.
Highlights
Following activation in the cell by aquation, cisplatin forms monoadducts with guanine bases in DNA which react with adjacent purine bases to form intrastrand crosslinks, namely about 65% cis-Pt(NH3)2-d(GpG), 25% cis-Pt(NH3)2-d(ApG) 1,2-intrastrand adducts and 5–10% 1,3-intrastrand adducts[10,11,12]
Www.nature.com/scientificreports but increased in the respective cisplatin-resistant variants to 10 to 210 μM, yielding resistance indices of 5–17 fold[7]. They are maintained with regular cisplatin treatment, which would be lethal for the parental cells, but from which the Long-term cisplatin-treated sublines (LTTs) recover within a few days
To which extent does cisplatin induce point mutations in bladder cancer cell lines? Second, do these mutations conform to the signature proposed by recent studies? Third, is there evidence for induction of chromosomal alterations by cisplatin? Fourth, are mutations and chromosomal alterations gained during initial selection or accrued gradually? Fifth, is it possible to identify genomic changes accounting for the observed mechanisms of resistance in the LTTs?
Summary
Following activation in the cell by aquation, cisplatin forms monoadducts with guanine bases in DNA which react with adjacent purine bases to form intrastrand crosslinks, namely about 65% cis-Pt(NH3)2-d(GpG), 25% cis-Pt(NH3)2-d(ApG) 1,2-intrastrand adducts and 5–10% 1,3-intrastrand adducts[10,11,12]. Many mechanisms are involved in the resistance of cancer cells to cisplatin treatment, including changes in transport and metabolism of the drug, altered DNA repair and checkpoint signaling as well as evasion of apoptosis[9,14]. These mechanisms have been studied in depth in many cancer types, especially in cell line models. We report on the genomic differences between the parental RT-112, J82, 253J, and T-24 cell lines and their cisplatin-resistant LTT variants, as determined by whole exome sequencing (WES), array comparative genomic hybridization (aCGH) and karyotyping Based on this data, we addressed several questions. To which extent does cisplatin induce point mutations in bladder cancer cell lines? Second, do these mutations conform to the signature proposed by recent studies? Third, is there evidence for induction of chromosomal alterations by cisplatin? Fourth, are mutations and chromosomal alterations gained during initial selection or accrued gradually? Fifth, is it possible to identify genomic changes accounting for the observed mechanisms of resistance in the LTTs?
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