Aufgrund seiner infausten Prognose und des häufigen Auftretens nimmt das GBM unter den Hirntumoren eine besondere Rolle ein. Viele intrazelluläre Signalwege und Tumormarker sind bereits gut erforscht und verstanden. Hierzu gehört auch der epigenetisch determinierte Methylierungsgrad des MGMT-Genpromotors. Die Bestimmung des MGMT-Status gehört bei allen Patienten mittlerweile zur Standarddiagnostik, um den Effekt der Radiochemotherapie auf den Tumor zu prognostizieren. Ist der MGMT-Genpromotor unmethyliert, haben alkylierende Substanzen wie TMZ nur einen geringen Effekt auf die Tumorzellen. Solche Patienten profitieren kaum von der Standardtherapie nach dem Stupp-Schema. Es sind jedoch Fälle aufgetreten, bei denen sich der Methylierungsgrad des MGMT-Genpromotors im Behandlungsverlauf der Patienten verändert hat. Aufgrund dessen untersuchte ich in meiner Arbeit, ob man Änderungen im MGMT-Genmethylierungsstatus und in der MGMT-Genexpression auf mRNA-und Proteinebene unter Nachahmung der Standardtherapie experimentell auslösen kann. Mit den verwendeten Versuchsansätzen konnte ich in der Zellkultur keine Veränderungen feststellen. Lediglich auf mRNA-Ebene konnte nach 5 Tagen fraktionierter Bestrahlung bei der methylierten Zelllinie U87 eine leichte Steigerung der MGMT-mRNA-Expression verzeichnet werden. Diese Expressionssteigerung stand allerdings nicht im Zusammenhang mit einer Änderung des MGMT-Methylierungsstatus und spiegelte sich auch nicht auf Proteinebene wider. Dieses Ergebnis lässt weitere Forschungen in die Richtung der therapieinduzierten Änderungen am MGMT-Genpromotor sinnvoll erscheinen, um letztendlich die Therapie am Patienten effektiver und individueller zu gestalten und das mediane Überleben sowie dieLebensqualität unter der Behandlung vor allem für Patienten mit unmethyliertem MGMT-Genpromotor zu verbessern. / Due to its inaccurate prognosis and frequent occurrence, GBM plays a special role among brain tumors. Many intracellular signaling pathways and tumor markers are already well understood and understood. This includes the epigenetically determined degree of methylation of the MGMT gene promoter. Determination of MGMT status has become standard diagnostic practice in all patients to predict the effect of chemoradiotherapy on the tumor. If the MGMT gene promoter is unmethylated, alkylating agents such as TMZ have little effect on the tumor cells. Such patients hardly benefit from the standard therapy according to the Stupp-Scheme. However, cases have occurred in which the degree of methylation of the MGMT gene promoter has changed in the course of treatment of patients. As a result, I investigated in my work whether it is possible to experimentally trigger changes in MGMT gene methylation status and in MGMT gene expression at mRNA and protein level, following the standard therapy. With the experimental approaches I was unable to detect any changes in the cell culture. Only at the mRNA level could a slight increase in MGMT mRNA expression be recorded after 5 days of fractionated irradiation in the methylated cell line U87. However, this expression increase was not associated with a change in MGMT methylation status and was not reflected at the protein level. This result suggests further research into the therapy-induced changes in the MGMT gene promoter to ultimately make patient therapy more effective and individualized, and to improve median survival and quality of life under treatment, especially for patients with unmethylated MGMT gene promoter.

Alkylating agents are a diverse family of compounds whose toxic, mutagenic and carcinogenic effects in living organisms are due to their ability to damage DNA. Humans are exposed to these agents through lifestyle, diet, occupation and some forms of chemotherapy, but they are also formed endogenously. To protect against these adverse effects, a variety of DNA repair process have evolved. Among these is O6-methylguanine-DNA methyltransferase (MGMT), a damage reversal protein that repairs O6-alkylguanine (O6-alkG) adducts by capturing the alkyl group onto a cysteine residue within the protein in an autoinactivating mechanism. Human MGMT is known to be present in at least 12 polymorphic variant forms and numerous studies have partially characterised one or a few of these in relation to cancer susceptibility. The main objective in this thesis was to compare the substrate specificity of seven MGMT variant proteins, and also a putative size-variant of the normal MGMT, using short ODNs containing 12 different O6-alkylguanines (O6-alkGs). A variety of analytical methods was used in these studies, including radioisotope-based assays, enzyme-linked immunosorbent assays (ELISA), mass spectrometry (MS) and surface plasmon resonance (SPR). The most potent inactivators of all MGMTs were ODNs containing O6-BzG followed by O6-MeG and O6-CMG. For the other ODNs, the MGMT proteins could be classed in three groups: firstly Wt, and the F84, V143/R178 and F84/V143/R178 variants for which the potencies were O6-PrG > O6-PobG > O6-EtG; secondly R160 and F84/R160 for which O6-PrG > O6-EtG > O6- PobG and finally Q128 for which O6- PobG > O6-PrG > O6-EtG. Alkyl group transfer from six of the ODNs to the active site cysteine of Wt MGMT was demonstrated by MS. The Q128 variant was the most resistant to inactivation by all modified ODNs. The R160 and F84/R160 variants were more resistant than the other variants to inactivation by the O6-CMG and O6-PobG containing ODNs. It was also shown that ZnCl2 supplementation during Wt MGMT expression in E.coli significantly increased MGMT specific activity and decreased sensitivity to inactivation by the ODNs. In addition both 5-methylcytosine adjacent and opposite O6-MeG significantly affected the inactivation of Wt MGMT. Finally, it was confirmed that O6-CMG is, contrary to previous reports, a substrate for MGMT, and the inefficient repair of O6-CMG by the E.coli Ogt alkyltransferase protein was shown to be the basis of the error. Studies with human cells suggest that MGMT also repairs O6-CMG in vivo. The results suggest that MGMT variants repair different O6-alkGs at different rates and hence the consequence of alkylating agents exposure will depend not only on the nature of the exposure but also an individual’s own specific MGMT variant.

No description available.

DNR pažaidų atitaisymas ir apoptozė - dvi pagrindinės grandys, palaikančios žmogaus genomo vientisumą. Sutrikus šiems procesams, ląstelė išgyvena, nepaisant susikaupusių DNR pažaidų ir sudaromas pagrindas tolesnei transformacijai. Tyrimu įvertinome DNR pažaidų atitaisymo funkcijoje dalyvaujančių (hMLH1, MGMT) ir su apoptoze susijusių (DAPK-1, CASP8) genų epigenetinio reguliavimo - metilinimo aspektus pakopiniame skrandžio onkogenezės procese. Šio mokslinio tyrimo metu pirmą kartą buvo nustatytas skirtingas hMLH1 geno metilinimo dažnis atskirose skrandžio anatominėse dalyse atrofiniu pangastritu sergančiųjų audinyje. Įvertinta, kad hMLH1 geno metilinimas sergančiųjų skrandžio vėžiu aplinkiniame nenavikiniame audinyje sietinas su pacientų amžiumi. Išgyvenamumo analizės rezultatai parodė, kad MGMT geno metilinimas agresyvios skrandžio vėžio histologinės formos atveju yra geresnės prognozės rodiklis. Tyrimo metu nustatėme mokslinėje periodikoje neaprašytų tirtųjų genų metilinimo derinių sąsajų su klinikiniais, morfologiniais ir prognoziniais onkologinės ligos ypatumais. / DNA repair and apoptosis are two main pathways supporting the integrity of human genome. After the disturbance of these processes the cell survives, despite the accumulation of DNA lesions, and in this way a basis for a subsequent transformation is formed. In our research we evaluated the epigenetic regulation - methylation - aspects of genes participating in DNA repair function (hMLH1 and MGMT) and also of apoptosis-related genes (DAPK-1, CASP8) in relation to a stepwise gastric oncogenesis process. During this investigation a different hMLH1 gene methylation observation frequency in tissues obtained from separate anatomical parts of the stomach in atrophic pangastritis patients was determined for the first time. It was estimated, that hMLH1 gene methylation in tumor-surrounding non-cancerous tissue in gastric cancer patients could be associated with patient age. Results of survival analysis indicated that MGMT gene methylation is an indicator of better prognosis in case of diffuse form of gastric cancer. During the study we determined some additional associations (not described in previous publications) between methylation combinations of analyzed genes and clinical, morphological and prognostic features of oncological illness.

No description available.

Yes / Temozolomide (TMZ) is the standard first line treatment for malignant glioma, reaching “blockbuster” status in 2010, yet it remains the only drug in its class. The main constraints on the clinical effectiveness of TMZ therapy are its requirement for active DNA mismatch repair (MMR) proteins for activity, and inherent resistance through O6-methyl guanine-DNA methyl transferase (MGMT) activity. Moreover, acquired resistance, due to MMR mutation, results in aggressive TMZ-resistant tumour regrowth following good initial responses. Much of the attraction in TMZ as a drug lies in its PK/PD properties: it is acid stable and has 100% oral bioavailability; it also has excellent distribution properties, crosses the blood-brain barrier, and there is direct evidence of tumour localisation. This review seeks to unravel some of the mysteries of the imidazotetrazine class of compounds to which TMZ belongs. In addition to an overview of different synthetic strategies, we explore the somewhat unusual chemical reactivity of the imidazotetrazines, probing their mechanisms of reaction, examining which attributes are required for an active drug molecule and reviewing the use of this combined knowledge towards the development of new and improved anti-cancer agents.

Yes / Glioblastoma multiforme (GBM) is a grade IV brain tumor characterized by a heterogeneous population of cells that are highly infiltrative, angiogenic and resistant to chemotherapy. The current standard of care, comprised of surgical resection followed by radiation and the chemotherapeutic agent temozolomide, only provides patients with a 12–14 month survival period post-diagnosis. Long-term survival for GBM patients remains uncommon as cells with intrinsic or acquired resistance to treatment repopulate the tumor. In this review we will describe the mechanisms of resistance, and how they may be overcome to improve the survival of GBM patients by implementing novel chemotherapy drugs, new drug combinations and new approaches relating to DNA damage, angiogenesis and autophagy.

Les gliomes malins de l’enfant (GME), de pronostic sombre, se distinguent des gliomes malins de l’adulte (GMA) sur le plan biologique mais aussi clinique, avec des taux de réponse au témozolomide (chimiothérapie alkylante de référence chez l’adulte) moindres. L’efficacité du temozolomide est réduite par l’action de l’enzyme de réparation de l’ADN, l’O6-methylguanine-DNA-methyltransferase (MGMT), dont l’expression est fréquemment inhibée par méthylation du promoteur de son gène dans les GMA. En première partie, la mise au point d’une nouvelle technique de PCR spécifique de méthylation a montré une fréquence plus faible dans les GME (15%) vs les GMA (45%, p<0,001). En deuxième partie, l’hypoxie intra-tumorale et la dérégulation en amont de l’axe mTOR-HIF-1α, connus pour être impliqués dans la chimio-résistance, ont été étudiés dans les GME et ciblés par l’association rapamycin-irinotecan dans une étude in vitro, pour laquelle des lignées dérivées de GME ont été développées. / Pediatric malignant glioma (PMGs), are associated with a very dismal prognosis. They are distinct from their adult counterparts (AMGs), biologically but also clinically, with a lower response to temozolomide (the current reference alkylating chemotherapy) compared to AMGs. Temozolomide efficacy is reduced by the activity of the DNA repair enzyme, O6-methylguanine-DNA-methyltransferase (MGMT), whose expression is frequently silenced by promoter methylation. First, the development of a new methylation-specific PCR showed a lower frequency of MGMT methylation in PMGs (15%) vs AMGs (45%, p<0,001). Secondly, intra-tumor hypoxia and the upstream deregulation of mTOR-HIF-1α axis, well-known to be involved in chemo-resistance and the up-regulation of MGMT expression, were studied in a PMG cohort. The targeting of this axis was then studied in vitro using a therapy combining rapamycin and irinotecan. For this, pediatric patient-derived malignant glioma cell lines were developed.

The aim of the study was to identify molecular markers that can determine progression of low grade glioma. This was done using various approaches such as IDH1 and IDH2 mutation analysis, MGMT methylation analysis, copy number analysis using array comparative genomic hybridisation and identification of differentially expressed miRNAs using miRNA microarray analysis. IDH1 mutation was present at a frequency of 71% in low grade glioma and was identified as an independent marker for improved OS in a multivariate analysis, which confirms the previous findings in low grade glioma studies. IDH1 mutation was associated with MGMT promoter methylation when partially methylated tumours were grouped with methylated tumours. Grade II and grade III tumour comparison analysis revealed 14 novel significant miRNAs with differential expression. A miRNA signature was shown for histological subtypes, oligoastrocytoma and anaplastic oligoastrocytoma, following the miRNA expression analysis in grade II and grade III tumors based on histology. Oligoastrocytoma presented a more similar profile to oligodendroglioma, but anaplastic oligoastrocytoma was more similar to anaplastic astrocytoma. Five novel miRNAs were identified in grade III tumours, when comparing IDH1 mutant and IDH1 wild type tumours. Analysis of paired samples of primary/recurrent tumours revealed that additional genomic changes may promote tumour progression. For each of the pair, the two samples were genomically different and in each case, the reccurent tumours had more copy number aberrations than the corresponding primary tumours. Cell cultures derived from the tumour biopsies were not representative of the low grade glioma in vivo, which was evident from the differences identified in the miRNA expression and copy number changes in the paired samples. IDH1 mutation present in tumour biopsies was not maintained in their respective cell cultures. These findings give an insight into the molecular mechanisms involved in the tumourigenesis of low grade glioma and also tumour progression.

Primary brain tumours, and especially the most common form malignant gliomas, usually display a pronounced resistance to other treatment modalities when surgery fails to cure. Growth factors, such as EGF and its receptor, frequently amplified and overexpressed in malignant gliomas, and factors associated with multidrug resistance have been suggested to at least partially explain the poor outcome. The aim of this thesis was to characterise factors in primary brain tumours associated with the development of resistance with focus on the epidermal growth factor receptor (ErbB) family, and multidrug resistance (MDR). Influences of irradiation on the expression and activity of P-glycoprotein (Pgp) in malignant gliomas was evaluated. The effects showed that irradiation increased the efflux activity of Pgp in rat brain vascular endothelial cells, but not in glioma cells. In the intracranial BT4C glioma model, Pgp was detected in the capillary endothelium in the tumour tissue but not in glioma cells. Expression of several factors coupled to MDR (Pgp, MRP1, LRP, and MGMT) in primary brain tumours were analysed and correlated to clinical data. In gliomas, Pgp and MRP1 were predominantly observed in capillary endothelium and in scattered tumour cells, whereas LRP occurred only in tumour cells. In meningiomas, expression of the analysed markers was demonstrated in the capillary endothelium, with a higher expression of Pgp and MRP1 in transitional compared to meningothelial meningiomas. A pronounced expression of MGMT was found independently of the histopathological grade or tumour type. Survival analysis indicated a shorter overall survival for patients suffering from low-grade gliomas with high expression of Pgp. To explore the importance of the epidermal growth factor receptor (EGFR), expression levels of the family members (EGFR, ErbB2-4) were analysed and their relations to various clinical parameters were evaluated in gliomas and meningiomas. In gliomas, the highest EGFR expression was observed in high-grade tumours, while ErbB4 expression was most pronounced in low-grade tumours. In meningiomas, expression of EGFR, ErbB2, and ErbB4 was observed in the majority of the tumours. An intriguing observation in low-grade gliomas was a significantly decreased overall survival for patients with high EGFR protein expression. The effects of different time schedules for administration of the selective EGFR inhibitor ZD1839 in relation to irradiation of glioma cells were analysed. The analyses showed a heterogeneity in the cytotoxic effects of ZD1839 between cell lines, and it was obvious that some of the cell lines showed sensitivity to ZD1839 despite no or low expression of EGFR. The study also demonstrated the importance of timing of ZD1839 administration when this agent is combined with irradiation. In conclusion, in order to enhance the efficacy of radiotherapy by various drugs in malignant gliomas it may be essential to inhibit drug efflux activity in endothelial cells and to deliver drugs in an optimal timing in relation to radiotherapy. The heterogeneity in expression of drug resistance markers, as well as the ErbB family reflects the complexity in classification of primary brain tumours, and indicates that subgroups of patients with low-grade gliomas expressing Pgp and EGFR might benefit from more aggressive and individualised treatment.