Quantification of Biologically Active DNA Alkylation in Temozolomide-Exposed Glioblastoma Cell Lines by Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry: Method Development and Recommendations for Validation.

Quantitative monitoring of biologically active methylations of guanines in samples exposed to temozolomide (TMZ) would be useful in glioblastoma research for preclinical TMZ experiments, for clinical pharmacology questions regarding appropriate exposure, and ultimately for precision oncology. The known biologically active alkylation of DNA induced by TMZ takes place on O6 position of guanines. However, when developing mass spectrometric (MS) assays, the possible signal overlap of O6-methyl-2'-deoxyguanosine (O6-m2dGO) with other methylated 2'-deoxyguanosine species in DNA and methylated guanosines in RNA must be considered. Liquid chromatography-tandem MS (LC-MS/MS) offers the analytical requirements for such assays in terms of specificity and sensitivity, especially when multiple reaction monitoring (MRM) is available. In preclinical research, cancer cell lines are still the gold standard model for in vitro drug screening. Here, we present the development of ultra-performance LC-MRM-MS assays for the quantification of O6-m2dGO in a TMZ-treated glioblastoma cell line. Furthermore, we propose adapted parameters for method validation relevant to the quantification of drug-induced DNA modifications.

Important Requirements for Desorption/Ionization Mass Spectrometric Measurements of Temozolomide-Induced 2'-Deoxyguanosine Methylations in DNA.

In clinical pharmacology, drug quantification is mainly performed from the circulation for pharmacokinetic purposes. Finely monitoring the chemical effect of drugs at their chemical sites of action for pharmacodynamics would have a major impact in several contexts of personalized medicine. Monitoring appropriate drug exposure is particularly challenging for alkylating drugs such as temozolomide (TMZ) because there is no flow equilibrium that would allow reliable conclusions to be drawn about the alkylation of the target site from plasma concentrations. During the treatment of glioblastoma, it appears, therefore, promising to directly monitor the alkylating effect of TMZ rather than plasma exposure, ideally at the site of action. Mass spectrometry (MS) is a method of choice for the quantification of methylated guanines and, more specifically, of O6-methylguanines as a marker of TMZ exposure at the site of action. Depending on the chosen strategy to analyze modified purines and 2'-deoxynucleosides, the analysis of methylated guanines and 2'-deoxyguanosines is prone to important artefacts due to the overlap between masses of (i) guanines from DNA and RNA, and (ii) different methylated species of guanines. Therefore, the specific analysis of O6-methyl-2'deoxyguanosine, which is the product of the TMZ effect, is highly challenging. In this work, we report observations from matrix-assisted laser desorption/ionization (MALDI), and desorption electrospray ionization (DESI) MS analyses. These allow for the construction of a decision tree to initiate studies using desorption/ionization MS for the analysis of 2'-deoxyguanosine methylations induced by TMZ.

Anoctamin 2-chloride channels reduce simple spike activity and mediate inhibition at elevated calcium concentration in cerebellar Purkinje cells.

Modulation of neuronal excitability is a prominent way of shaping the activity of neuronal networks. Recent studies highlight the role of calcium-activated chloride currents in this context, as they can both increase or decrease excitability. The calcium-activated chloride channel Anoctamin 2 (ANO2 alias TMEM16B) has been described in several regions of the mouse brain, including the olivo-cerebellar system. In inferior olivary neurons, ANO2 was proposed to increase excitability by facilitating the generation of high-threshold calcium spikes. An expression of ANO2 in cerebellar Purkinje cells was suggested, but its role in these neurons remains unclear. In the present study, we confirmed the expression of Ano2 mRNA in Purkinje cells and performed electrophysiological recordings to examine the influence of ANO2-chloride channels on the excitability of Purkinje cells by comparing wildtype mice to mice lacking ANO2. Recordings were performed in acute cerebellar slices of adult mice, which provided the possibility to study the role of ANO2 within the cerebellar cortex. Purkinje cells were uncoupled from climbing fiber input to assess specifically the effect of ANO2 channels on Purkinje cell activity. We identified an attenuating effect of ANO2-mediated chloride currents on the instantaneous simple spike activity both during strong current injections and during current injections close to the simple spike threshold. Moreover, we report a reduction of inhibitory currents from GABAergic interneurons upon depolarization, lasting for several seconds. Together with the role of ANO2-chloride channels in inferior olivary neurons, our data extend the evidence for a role of chloride-dependent modulation in the olivo-cerebellar system that might be important for proper cerebellum-dependent motor coordination and learning.