Background: Cisplatin based chemotherapy cures over 80% of metastatic testicular germ cell tumours (TGCT). In contrast, almost all other solid cancers in adults are incurable once they have spread beyond the primary site. Cell lines derived from TGCTs are hypersensitive to cisplatin reflecting the clinical response. Earlier findings suggested that a reduced repair capacity might contribute to the cisplatin hypersensitivity of testis tumour cells (TTC), but the critical DNA damage has not been defined. This study was aimed at investigating the formation and repair of intrastrand and interstrand crosslinks (ICLs) induced by cisplatin in TTC and their contribution to TTC hypersensitivity. Results: We observed that repair of intrastrand crosslinks is similar in cisplatin sensitive TTC and resistant bladder cancer cells, whereas repair of ICLs was significantly reduced in TTC. γH2AX formation, which serves as a marker of DNA breaks formed in response to ICLs, persisted in cisplatin-treated TTC and correlated with sustained phosphorylation of Chk2 and enhanced PARP-1 cleavage. Expression of the nucleotide excision repair factor ERCC1-XPF, which is implicated in the processing of ICLs, is reduced in TTC. To analyse the causal role of ERCC1-XPF for ICL repair and cisplatin sensitivity, we over-expressed ERCC1-XPF in TTC by transient transfection. Over-expression increased ICL repair and rendered TTC more resistant to cisplatin, which suggests that ERCC1-XPF is rate-limiting for repair of ICLs resulting in the observed cisplatin hypersensitivity of TTC. Conclusion: Our data indicate for the first time that the exceptional sensitivity of TTC and, therefore, very likely the curability of TGCT rests on their limited ICL repair due to low level of expression of ERCC1-XPF.

The pronounced neurotoxicity of the potent antitumor drug cisplatin frequently results in the onset of peripheral polyneuropathy (PNP), which is assumed to be initially triggered by platination products in the nuclear DNA of affected tissues. To further elucidate the molecular mechanisms, we analyzed in a mouse model the formation and processing of the main cisplatin-induced DNA adduct (guanine–guanine intrastrand cross-link) in distinct neuronal cell types by adduct-specific monoclonal antibodies. Comparison of the adduct kinetics in cisplatin-injected mice either proficient or deficient for nucleotide excision repair (NER) functions revealed the essential role of this DNA repair pathway in protecting differentiated cells of the nervous system from excessive formation of such lesions. Hence, chronic exposure to cisplatin resulted in an accelerated accumulation of unrepaired intrastrand cross-links in neuronal cells of mice with dysfunctional NER. The augmented adduct levels in dorsal root ganglion (DRG) cells of those animals coincided with an earlier onset of PNP-like functional disturbance of their sensory nervous system. Independently from the respective repair phenotype, the amount of persisting DNA cross-links in DRG neurons at a given cumulative dose was significantly correlated to the degree of sensory impairment as measured by electroneurography. Collectively, these findings suggest a new model for the processing of cisplatin adducts in primary neuronal cells and accentuate the crucial role of effectual DNA repair capacity in the target cells for the individual risk of therapy-induced PNP.

While platinum-based chemotherapeutic agents are widely used to treat various solid tumors, the acquired platinum resistance is a major impediment in their successful treatment. Since enhanced DNA repair capacity is a major factor in conferring cisplatin resistance, targeting of DNA repair pathways is an effective stratagem for overcoming cisplatin resistance. This study was designed to delineate the role of nucleotide excision repair (NER), the principal mechanism for the removal of cisplatin-induced DNA intrastrand crosslinks, in cisplatin resistance and reveal the impact of DNA repair interference on cisplatin sensitivity in human ovarian cancer cells. We assessed the inherent NER efficiency of multiple matched pairs of cisplatin-sensitive and -resistant ovarian cancer cell lines and their expression of NER-related factors at mRNA and protein levels. Our results showed that only the cisplatin-resistant ovarian cancer cell line PEO4 possessed an increased NER capacity compared to its inherently NER-inefficient parental line PEO1. Several other cisplatin-resistant cell lines, including CP70, CDDP and 2008C13, exhibited a normal and parental cell-comparable NER capacity for removing cisplatin-induced DNA intrastrand cross-links (Pt-GG). Concomitant gene expression analysis revealed discordance in mRNA and protein levels of NER factors in various ovarian cancer cell lines and NER proteins level were unrelated to the cisplatin sensitivity of these cell lines. Although knockdown of NER factors was able to compromise the NER efficiency, it only caused a minimal effect on cisplatin sensitivity. On the contrary, downregulation of BRCA2, a critical protein for homologous recombination repair (HRR), significantly enhanced the efficacy of cisplatin in killing ovarian cancer cell line PEO4. Our studies indicate that the level of NER factors in ovarian cancer cell lines is neither a determinant of their NER capacity nor of the sensitivity to cisplatin, and suggest that manipulation of the HRR but not the NER factor expression provides an effective strategy for sensitizing cisplatin-resistant tumors to platinating agents.

Although DNA repair processes have been shown to considerably modulate the cytotoxic effects of alkylating agents, little information is available on the role of these mechanisms in chemotherapy-induced myelosuppression. Therefore, we have analyzed in detail the DNA repair capacity of primary human hematopoietic cells from cord blood (CB) or bone marrow (BM) by 2 functional assays, the immunocytologic assay (ICA) and single-cell gel electrophoresis (comet assay). Besides substantial interindividual differences, we consistently observed significantly lower repair capacity of CD34⁺ cells in comparison to CD34⁻, CD19⁺, or CD33⁺ cells of the same donor. After exposure to the alkylating agent ethylnitrosourea (EtNU), the comet assay displayed on average twice as many DNA single-strand breaks (SSBs) in CD34⁺ cells and a tripled half-life of these lesions in comparison to corresponding CD34⁻ cells. Similarly, reduced SSB repair activity in CD34⁺ cells was detected following melphalan or cisplatin application. When specific antibodies were used to monitor DNA reaction products of these drugs, adduct levels were significantly higher and lesions persisted longer in the CD34⁺ fraction. To assess the contribution of individual pathways to overall DNA repair, modulators blocking defined steps in repair processes were coapplied with alkylating drugs. Similar “modulation pattern” in corresponding CD34⁺ and CD34⁻ cell fractions indicated a generalized reduction in DNA repair capacity of CD34⁺ cells, rather than deficiencies in a specific pathway. Because CD34⁺ cells also displayed higher frequencies of apoptosis in response to melphalan or cisplatin, these findings may help to explain the myelosuppression after exposure to alkylating agents.

Therapeutic efficacy of cisplatin-based treatment of late stage urothelial carcinoma (UC) is limited by chemoresistance. To elucidate underlying mechanisms and to develop new approaches for overcoming resistance, we generated long-term cisplatin treated (LTT) UC cell lines, characterised their cisplatin response, and determined the expression of molecules involved in cisplatin transport and detoxification, DNA repair, and apoptosis. Inhibitors of metallothioneins and Survivin were applied to investigate their ability to sensitise towards cisplatin. Cell growth, proliferation, and clonogenicity were examined after cisplatin treatment by MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, EdU (5-ethynyl-2’-deoxyuridine) incorporation assay, and Giemsa staining, respectively. Cell cycle distribution and apoptosis were quantified by flow cytometry. mRNA and protein expressions were measured by real-time quantitative (qRT)-PCR, western blot, or immunofluorescence staining. LTTs recovered rapidly from cisplatin stress compared to parental cells. In LTTs, to various extents, cisplatin exporters and metallothioneins were induced, cisplatin adduct levels and DNA damage were decreased, whereas expression of DNA repair factors and specific anti-apoptotic factors was elevated. Pharmacological inhibition of Survivin, but not of metallothioneins, sensitised LTTs to cisplatin, in an additive manner. LTTs minimise cisplatin-induced DNA damage and evade apoptosis by increased expression of anti-apoptotic factors. The observed diversity among the four LTTs highlights the complexity of cisplatin resistance mechanisms even within one tumour entity, explaining heterogeneity in patient responses to chemotherapy.

To improve the treatment of pancreatic ductal adenocarcinoma (PDAC), a promising strategy consists of personalized chemotherapy based on gene expression profiles. Investigating a panel of PDAC-derived human cell lines, we found that their sensitivities towards cisplatin fall in two distinct classes. The platinum-sensitive class is characterized by the expression of GATA6, miRNA-200a, and miRNA-200b, which might be developable as predictive biomarkers. In the case of resistant PDAC cells, we identified a synergism of cisplatin with HSP90 inhibitors. Mechanistic explanations of this synergy include the degradation of Fanconi anemia pathway factors upon HSP90 inhibition. Treatment with the drug combination resulted in increased DNA damage and chromosome fragmentation, as we have reported previously for ovarian cancer cells. On top of this, HSP90 inhibition also enhanced the accumulation of DNA-bound platinum. We next investigated an orthotopic syngeneic animal model consisting of tumors arising from KPC cells (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre, C57/BL6 genetic background). Here again, when treating established tumors, the combination of cisplatin with the HSP90 inhibitor onalespib was highly effective and almost completely prevented further tumor growth. We propose that the combination of platinum drugs and HSP90 inhibitors might be worth testing in the clinics for the treatment of cisplatin-resistant PDACs.