PMC

Chromosomal alterations in RT-112-LTT by aCGH. Chromosomal alterations of RT-112-LTT hybridized against its parental cell line as detected for each chromosome by aCGH.

Karyotype changes in LTTs. Karyotype of (a) RT-112, (b) RT-112-LTT, (c) J82, and (d) J82-LTT as revealed by GTG-banding analysis.

Common chromosomal alterations across all LTT sublines as detected by aCGH. 100% denotes change in all cell lines.

Allele frequency of mutations found in LTTs. Heat map indicating allele frequencies in four LTT sublines from low (green) to high (red).

Exemplary chromosomal changes in LTTs. (a) The region 17q21.32 around the HOXB gene cluster was gained in all four LTTs as detected by aCGH. (b) The region 19p13.33 containing the KEAP1 gene was lost in RT-112-LTT at five different time points (T1–T5) according to aCGH.

Characterization of genetic alterations in LTT lines. (a) Correlation between the total number of nucleotide variants and maintenance cisplatin concentration in RT-112-LTT, J82-LTT, 253J-LTT, and T-24-LTT. (b) Total number of exchanges for the six possible types of substitutions for each LTT subline. (c) Total number of exchanges for the twelve types of substitutions for each LTT subline; color codes as in (b). Lighter corresponding color shades denote substitutions on the nontranscribed strand.

Mutation profiles of LTTs. Mutational spectra of (a) RT-112-LTT, (b) J82-LTT, (c) 253J-LTT, and (d) T-24-LTT relative to their parental UCCs are displayed according to the 96 substitution classification defined by the substitution class and sequence context of the mutated base by Alexandrov et al. (2014). Note that the number of mutations in J82-LTT is too low to allow the generation of a representative profile. (e) Common variants found in RT-112-LTT, J82-LTT, 253J-LTT, and T-24-LTT for all variants (left Venn diagram) and exonic nonsynonymous variants (right Venn diagram).

Protein domain structure of genes commonly mutated in LTTs. Overview of the domain structures of (a) ATP7B, (b) NRXN2 (Neurexin-2), (c) FAM205A, and (d) HECW1 (NEDL1). Mutations found in the TCGA data set as annotated from cBioportal and mutations found in LTTs were included. All mutations in the LTTs are missense mutations except for one FAM205A mutation in RT-112 (Table ). (e) ATP7B mRNA expression was measured by qRT-PCR in parental UCCs and LTTs after treatment with siRNA against ATP7B or a non-targeting control. SDHA mRNA was used as a reference. (f) Relative cell viability after 48 h cisplatin treatment as measured by CellTiterGlo assay in parental UCCs and LTTs transfected with siRNA targeting ATP7B or a non-targeting negative control siRNA. Values represent the mean ± SD of biological quadruplicates, *p < 0.05 compared to non-targeting control. HECT: Homologous to the E6-AP Carboxyl Terminus; HECWN: HECTW1 N-terminal domain; H: Helical bundle; C2: C2 domain; WW: WW domain; HMA: Heavy metal-associated domain.