Visual Universitätsmedizin Mainz

Dr. Teodora Nikolova

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DNA repair and the sensitivity of tumour cells to alkylating agents

Alkylating genotoxins have long been used in the treatment of malignant tumours due to their strong cytotoxic effect on rapidly dividing cells. Methylating and chloroethylating agents induce primary and secondary DNA damage which can trigger programmed death (apoptosis and necrosis) in the affected cells in case of non-repair. In this process, the DNA adduct O6-methylguanine, which is induced by methylating agents, and the O6-Chlorethylguanine (with chloroethylnitrosaureae) step forward as critical primary DNA lesions. These adducts are repaired by transfer of the alkyl group to the repair protein in tumour cells that express the repair enzyme MGMT. The lesions in cells due to loss of MGMT function lead, however, to the formation of secondary DNA damage (for instance, DNA cross-linking strands after treatment with chloroethyl nitrosoureas). Additionally, these are converted into DNA double-strand breaks which can, in turn, trigger apoptosis or necrosis. There are two mechanisms to repair DSBs in human cells: the homologous recombination and the non-homologous-end-joining. During the formation of DSBs, repair protein complexes occur inside the cell nucleus. These immunocytochemical complexes are detected as discrete intranuclear foci.

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a. Intranuclear foci from DSB-marker γH2AX and phospho-53BP1induced by lomustine in glioblastoma cells.

b. Glioblastoma cells with double-marked mitochondria with AIF-antibodies and MitoTracker Orange.

c. DNA fiber spreads from replicating glioblastoma cells marked with chloro-deoxyuridine (CldU) and iodo-deoxyuridine (IdU).

d. Apoptic glioblastoma cell marked with Annexin V in the cell membrane and with γH2AX-Foci induced by nimustine in the nucleus.

Focal points of the current scientific work of our group are topics that deal with issues of DNA double-strand break repair and sensitivity of tumor cells to alkylating genotoxins. Due to their DNA damaging effects and killing activity in cancer cells, methylating agents such as temozolomide (TMZ) and chloroethylating agents such as the nitrosoureas (CNUs) CCNU (lomustine) and ACNU (Nimustin) are first-choice drugs for the chemotherapy of brain tumors and brain metastases of various origins. Currently, Dr. Nikolova leads a project funded by the German Research Foundation (DFG) with the topic: "Preclinical studies with inhibitors of DNA double-strand break repair for the improved chemotherapy of malignant brain tumors". The aim of the project group is to determine the role of DNA DSB repair for glioblastoma sensitivity to the alkylating chemotherapeutic agents. Specific HR inhibitors (RAD51 inhibitors) and PARP inhibitors are used for the modulation of DSB repair and/or sensitivity to TMZ and CNUs. In addition, we also investigate the cytotoxic effects of co-administered combinatorial treatments with RAD51 and PARP inhibitors. Since PARP participates in the backup NHEJ mechanisms for the repair of DSB in the absence of functional HR, HR-defective cells, and especially those with BRCA2 mutations, show hypersensitivity to PARP inhibitors = PARPi. This chemosensitization of BRCA2 mutant cells resembles ultimately the phenomenon of "synthetic lethality," a term that describes the lethal effect of the simultaneous disruption of two DNA repair pathways through mutations. Synthetic lethality is of clinical interest as it allows genetically-based stratification of patients into effective therapies. Therefore, we are currently investigating whether combinations of PARPi with newly developed RAD51 inhibitors can induce "synthetic lethality" -like effects in glioblastoma cells in vitro and in vivo.

 

Masters and PhD students are both invited to work on parts of the project and to apply for positions within the scope of their work.

1. Nikolova T, Kiweler N, Krämer OH. Interstrand Crosslink Repair as a Target for HDAC Inhibition. Trends Pharmacol Sci. 2017;38(9):822-836. Review.

 

2. Nikolova T, Roos WP, Krämer OH, Strik HM, Kaina B. Chloroethylating nitrosoureas in cancer therapy: DNA damage, repair and cell death signaling. Biochim Biophys Acta. 2017;1868(1):29-39. Review.

 

3. Berte N, Piée-Staffa A, Piecha N, Wang M, Borgmann K, Kaina B, Nikolova T* (last author). Targeting Homologous Recombination by Pharmacological Inhibitors Enhances the Killing Response of Glioblastoma Cells Treated with Alkylating Drugs. Mol Cancer Ther. 2016;15(11):2665-2678.

 

4. Ensminger M, Iloff L, Ebel C, Nikolova T, Kaina B, Lӧbrich M. DNA breaks and chromosomal aberrations arise when replication meets base excision repair. J Cell Biol. 2014 7;206(1):29-43.

 

5. Nikolova T, Dvorak M, Jung F, Adam I, Krämer E, Gerhold-Ay A, Kaina B. The γH2AX assay for genotoxic and nongenotoxic agents: comparison of H2AX phosphorylation with cell death response. Toxicol Sci. 2014;140(1):103-17.

 

6. Knizhnik AV, Roos WP, Nikolova T, Quiros S, Tomaszowski KH, Christmann M, Kaina B. Survival and death strategies in glioma cells: autophagy, senescence and apoptosis triggered by a single type of temozolomide-induced DNA damage. PLoS One. 2013;8(1):e55665.

 

7. Nikolova T*, Hennekes F*, Bhatti A., Kaina B. (2012) Chloroethylnitrosourea-induced cell death and genotoxicity: cell cycle dependence and the role of DNA double-strand breaks, HR and NHEJ. Cell Cycle. 11(14):2606-2619.

 

8. Nikolova T., M. Emsinger, M. Loebrich and B. Kaina. (2010) Homologous recombination protects mammalian cells from replication-associated DNA double-strand breaks arising in response to methyl methanesulfonate. DNA Repair (Amst) 9(10):1050 - 1063.