As a hydrophilic biopolymer, a DNA molecule is surrounded by water molecules in aqueous solution. The charge hopping mechanism indicates the competition between radical cation quenching by water molecules and migration along DNA partially determines the distance and efficiency of charge transport in DNA. Lipid can effectively bind DNA to induce hydrophobic environment around the DNA helix and reduce the water contact with bases in the DNA duplex. Therefore, the effect of water molecules on charge transport can be studied by comparison between nature DNA and DNA-lipid complexes. We synthesized several cationic lipids with various lengths of dialkyl chain (2, 8, 18) and spermine (Sp4+) binding core in this research, which posses strong DNA binding affinity due to their multi-charged spermine head-groups. Among those, C8GlySp4+ and C2GlySp4+ can form stable complex with DNA oligomer in aqueous solution, characterized by time dependent UV and CD spectrometry. C2GlySp4+ showed the similar inhibition on oxidative damage in GG steps as spermine while C8GlySp4+ demonstrated much more significant prohibitive effect at the same concentration. Since all the lipids bear the same binding core, they should afford the similar binding affinity towards DNA duplexes. we attributed the observation to the longer length of dialkyl group in C8GlySp4+, which can more effectively shield the DNA duplex from the water molecules than either spermine or C2GlySp4+. A kinetic model based on phonon-assist polaron hopping mechanism was proposed to rationalize the experimental results. The finding may give insight on the protection of DNA oxidative damage by reducing the access of the water molecule to DNA duplex and may have potential impact on the application of DNA as conducting biopolymer and protection of DNA in biological system.

Die WHO-Klassifikation der Hirntumoren von 2016 ebnete den Weg für molekulare Marker und Therapie-Angriffspunkte. Der Transkriptionsfaktor ATF5 könnte ein solcher sein. Er unterdrückt die Differenzierung von neuronalen Vorläuferzellen und wird in Glioblastomen (GBM) überexprimiert. Daten zur ATF5-Expression in WHO Grad II Gliomen (LGG) und GBM-Rezidiven sind nur spärlich vorhanden. Daher untersuchten wir 79 GBM, 40 LGG und 10 Normalhirnproben auf ihre ATF5-mRNA- und Proteinexpression mit quantitativer Echtzeit-PCR bzw. Immunhistochemie und verglichen sie mit multiplen, retrospektiv erhobenen klinischen Charakteristika der Patienten. ATF5 war in LGG und GBM verglichen zum Normalhirn sowohl auf mRNA-, als auch Proteinebene überexprimiert. Obwohl die ATF5-mRNA-Expression im GBM eine erhebliche Fluktuationsrate zeigte, gab es keine signifikanten Expressionsunterschiede zwischen GBM-Gruppen unterschiedlicher biologischer Wachstumsmuster. ATF5-mRNA korrelierte mit dem Alter der Patienten und invers mit der Ki67-Färbung. Kaplan Meier- und Cox-Regressionsanalysen zeigten eine signifikante Korrelation der ATF5-mRNA-Expression mit dem Überleben nach 12 Monaten sowie dem progressionsfreien Überleben. Die Methylierung des Promotors der O6-Methylguanin-DNA-Methyltransferase (MGMT) ist ein etablierter Marker in der Therapie des GBMs. Sie ist mit dem therapeutischen Ansprechen auf Temozolomid und dem Überleben assoziiert. Uns fielen inzidentell Veränderungen der MGMT-Promotormethylierung auf, woraufhin wir den aktuellen Wissensstand mittels einer ausführlichen Literatur-Metaanalyse zusammenfassten. Dabei fanden wir Veränderungen der MGMT-Promotormethylierung bei 115 der 476 Patienten. Wir schlussfolgern, dass die ATF5-mRNA-Expression als prognostischer Faktor für das Überleben der Patienten dienen könnte. Da seine in vitro-Inhibition zu einem selektiven Zelltod von Gliomzellen führte und wir eine Überexpression in glialen Tumoren nachweisen konnten, zeigt ATF5 Potential als ubiquitäres Therapieziel in Gliomen. Zum aktuellen Zeitpunkt ergibt sich keine klare Indikation, den klinischen Standard der MGMT-Teststrategie zu verändern. Trotzdem könnte eine erneute Testung der MGMT-Promotormethylierung für zukünftige Therapieentscheidungen sinnvoll sein und wir regen an, dass dieses Thema in klinischen Studien weiter untersucht wird. / The 2016 WHO classification for brain tumors signaled a major paradigm shift and paves the way for molecular markers and molecular targets. One such target could be the transcription factor ATF5. It suppresses differentiation of neuroprogenitor cells and is overexpressed in glioblastoma (GBM). Data on ATF5 expression in glioma of WHO grade II (LGG) are scarce and lacking on recurrent GBM. Therefore, we examined 79 GBM, 40 LGG and 10 normal brain (NB) specimens for their ATF5-mRNA and protein expression by quantitative real-time PCR and immunohistochemistry, respectively, and compared it to multiple retrospectively obtained clinical characteristics of the patients. ATF5-mRNA was overexpressed in LGG and GBM compared to NB on mRNA and protein level. Although ATF5-mRNA expression in GBM showed a considerable fluctuation range, GBM groups of varying biological behavior were not significantly different. ATF5-mRNA correlated with the patients' age and inversely with Ki67-staining. Kaplan-Meier analysis and Cox regression indicated that ATF5-mRNA expression was significantly associated with survival after 12 months and progression-free survival. Methylation of the O6-Methylguanin DNA methyltransferase (MGMT) promoter is a well-established strong prognostic factor in the therapy of GBM. It is associated with an improved response to chemotherapy with temozolomide and longer overall survival. We made the incidental finding of patients with changing MGMT promoter methylation during the clinical course, which prompted us to further investigate this topic. Indeed, a meta-analysis of the literature revealed changes in MGMT promoter methylation in 115 of 476 patients. To conclude, ATF5-mRNA expression could be identified as an additional, though not independent factor correlating with patients‘ survival. Since its inhibition might lead to the selective death of glioma cells, it might serve as a potential ubiquitous therapeutic target in astrocytic tumors. Clinical implications of the observed changes in MGMT promoter methylation are still ambiguous and do not yet support a change in clinical practice. However, retesting MGMT methylation might be useful for future treatment decisions and we encourage clinical studies to address this topic.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

A produção de radicais de carbono \"in vivo\" durante a biotransformação da hidrazina foi demonstrada por ressonância para magnética eletrônica, utilizando o método do captador de spin. Eritrócitos de rato também oxidaram a hidrazina, formando radicais de carbono e nitrogênio, além de espécies reativas de oxigênio. Todas estas espécies, possivelmente formadas \"in vivo\", são potencialmente causadoras de dano a macromoléculas. Podem, por exemplo, iniciar reações secundárias formando radicais de componentes celulares, como ocorreu com a hemoglobina que foi oxidada a radicais tiil-hemoglobina em eritrócitos tratados com hídrazina. Radicais de carbono formados durante a biotransformação da hidrazina em animais expostos provêm necessariamente de substâncias endógenas e podem ser direta ou indiretamente responsáveis pela modificação ( alquilação ) de bases no DNA \"in vivo\". A hidrazona do formaldeído é descrita na literatura como um intermediário da alquilação induzida por hidrazina \"in vivo\". Células L 1210, catalase ou oxihemoglobina de rato foram capazes de formar radicais de carbono durante a oxidação da hidrazona do formaldeído. A oxidação da hidrazona do formaldeído pela catalase foi estudada \"in vítro\" e os radicais de carbono formados, identificados como radicais metila. A base modificada C8 -metil-guanina foi formada em animais expostos, como demonstrado por cromatografia líquida de alta eficiência associada à detecção eletroquímica, sugerindo que ocorreu alquilação do DNA por radicais metila durante a biotransformação da hidrazina \"in vivo\". / The production of carbon-centered radicais during hydrazine biotransformation \"in vivo\" was demonstrated by electron paramagnetic resonance ( EPR ) spin trapping technique. Rat red blood cells also oxidized hydrazine, forming carbon and nitrogen centered radicais, besides oxygen reactive speties. Ali these species, possibly formed \"in vivo\", are potentially harmful to macromolecules. For example, they can initiate secondary reactions in which the radicais from cell components are formed, as it occurred with hemoglobin, forming thiyl-hemoglobin radicais in the red blood cells treated with hydrazine. Carbon-centered radicais produced during the biotransformation of hydrazine in exposed animais must be derived from endogenous sources and may be directly or indirectly responsible for the modificaton ( alkylation ) of DNA bases \"in vivo\". The formaldehyde hydrazone is reported in the literature as an intermediate of hydrazine-induced alkylation \"in vivo\". L1210 cells, catalase and rat hemoglobin were able to produce carbon-centered radicais during the oxidation of the formaldehyde hydrazone. The oxidation of formaldehyde hydrazone by catalase was studied \"in vitro\" and the generated carbon-centered radicais were identified as methyl radicais. The modified base C8 -methylguanine was formed in exposed animais, as demonstrated by high performance liquid chromatography with electrochemical detection, suggesting that DNA alkylation by methyl radicais occurred during hydrazine biotransformation \"in vivo.\"

Pancreatic endocrine tumors and gastrointestinal stromal tumors are rare. Evidence regarding prognostic factors, and in the former also treatment, is scarce. We evaluated the survival and prognostic factors in a consecutive series of 324 patients with pancreatic endocrine tumors treated at a single institution. Radical surgery, WHO classification, TNM stage, age and Ki67 ≥2% emerged as independent prognostic factors. Having a non-functioning tumor was not an independent prognostic marker, and neither was hereditary tumor disease. We present the first evaluation of the newly proposed TNM staging system for these patients. A separate analysis of well-differentiated neuroendocrine carcinomas is reported, suggesting tumor size ≥5cm and Ki67 ≥2% as negative prognostic markers in this group. The first 36 patients with advanced neuroendocrine tumors treated with temozolomide at our clinic were evaluated. The median time to progression was seven months. Fourteen percent showed partial regression and 53% stabilization of disease. Side effects were generally mild. Investigation of O6-methylguanine DNA methyltransferase revealed a low expression in a subset of tumors. Four out of five patients responding to treatment had tumors with low expression. Concomitant expression of the orexigen ghrelin and its receptor in pancreatic endocrine tumors is demonstrated. No significant difference in mean plasma ghrelin between patients and controls were found, but elevated plasma ghrelin was seen in five patients. We provide the first report of expression of ghrelin and its receptor in gastrointestinal stromal tumors. Concomitant expression was frequent, indicating the presence of an autocrine loop. The tumors also expressed the neuroendocrine marker synaptic vesicle protein 2. Together, these findings are suggestive of neuroendocrine features.

A produção de radicais de carbono \"in vivo\" durante a biotransformação da hidrazina foi demonstrada por ressonância para magnética eletrônica, utilizando o método do captador de spin. Eritrócitos de rato também oxidaram a hidrazina, formando radicais de carbono e nitrogênio, além de espécies reativas de oxigênio. Todas estas espécies, possivelmente formadas \"in vivo\", são potencialmente causadoras de dano a macromoléculas. Podem, por exemplo, iniciar reações secundárias formando radicais de componentes celulares, como ocorreu com a hemoglobina que foi oxidada a radicais tiil-hemoglobina em eritrócitos tratados com hídrazina. Radicais de carbono formados durante a biotransformação da hidrazina em animais expostos provêm necessariamente de substâncias endógenas e podem ser direta ou indiretamente responsáveis pela modificação ( alquilação ) de bases no DNA \"in vivo\". A hidrazona do formaldeído é descrita na literatura como um intermediário da alquilação induzida por hidrazina \"in vivo\". Células L 1210, catalase ou oxihemoglobina de rato foram capazes de formar radicais de carbono durante a oxidação da hidrazona do formaldeído. A oxidação da hidrazona do formaldeído pela catalase foi estudada \"in vítro\" e os radicais de carbono formados, identificados como radicais metila. A base modificada C8 -metil-guanina foi formada em animais expostos, como demonstrado por cromatografia líquida de alta eficiência associada à detecção eletroquímica, sugerindo que ocorreu alquilação do DNA por radicais metila durante a biotransformação da hidrazina \"in vivo\". / The production of carbon-centered radicais during hydrazine biotransformation \"in vivo\" was demonstrated by electron paramagnetic resonance ( EPR ) spin trapping technique. Rat red blood cells also oxidized hydrazine, forming carbon and nitrogen centered radicais, besides oxygen reactive speties. Ali these species, possibly formed \"in vivo\", are potentially harmful to macromolecules. For example, they can initiate secondary reactions in which the radicais from cell components are formed, as it occurred with hemoglobin, forming thiyl-hemoglobin radicais in the red blood cells treated with hydrazine. Carbon-centered radicais produced during the biotransformation of hydrazine in exposed animais must be derived from endogenous sources and may be directly or indirectly responsible for the modificaton ( alkylation ) of DNA bases \"in vivo\". The formaldehyde hydrazone is reported in the literature as an intermediate of hydrazine-induced alkylation \"in vivo\". L1210 cells, catalase and rat hemoglobin were able to produce carbon-centered radicais during the oxidation of the formaldehyde hydrazone. The oxidation of formaldehyde hydrazone by catalase was studied \"in vitro\" and the generated carbon-centered radicais were identified as methyl radicais. The modified base C8 -methylguanine was formed in exposed animais, as demonstrated by high performance liquid chromatography with electrochemical detection, suggesting that DNA alkylation by methyl radicais occurred during hydrazine biotransformation \"in vivo.\"

Yes / The antitumor prodrug temozolomide is compromised by its dependence for activity on DNA mismatch repair (MMR) and the repair of the chemosensitive DNA lesion, O6-methylguanine (O6-MeG), by O6-methylguanine-DNA-methyltransferase (E.C. 2.1.1.63, MGMT). Tumor response is also dependent on wild-type p53. Novel 3-(2-anilinoethyl)-substituted imidazotetrazines are reported that have activity independent of MGMT, MMR, and p53. This is achieved through a switch of mechanism so that bioactivity derives from imidazotetrazine-generated arylaziridinium ions that principally modify guanine-N7 sites on DNA. Mono- and bifunctional analogues are reported, and a quantitative structure-activity relationship (QSAR) study identified the p-tolyl-substituted bifunctional congener as optimized for potency, MGMT-independence, and MMR-independence. NCI60 data show the tumor cell response is distinct from other imidazotetrazines and DNA-guanine-N7 active agents such as nitrogen mustards and cisplatin. The new imidazotetrazine compounds are promising agents for further development, and their improved in vitro activity validates the principles on which they were designed.

Introduction: Le gène O6-méthylguanine-ADN méthyltransferase (MGMT) code pour une enzyme spécifique réparatrice de l’ADN qui protège les cellules de la toxicité des agents alkylants. Ainsi, l’activité du MGMT est un mécanisme majeur de résistance aux agents alkylants. Il a été démontré qu’une diminution de l’expression du gène MGMT par une hyperméthylation du promoteur résulte en une amélioration de la survie chez les patients avec certains types de tumeurs qui sont traitées avec des agents chimiothérapeuthique alkylants. Objectifs: Déterminer la prévalence de la méthylation du gène MGMT chez des patients avec des cancers épidermoïdes localement avancés de la sphère ORL traités avec chimioradiothérapie et évaluer l’impact de cette méthylation sur la survie. Méthodes: Sur 428 patients consécutifs, traités avec chimioradiothérapie à notre institution et suivis pour un période médiane de 37 mois, 199 spécimens chirurgicaux paraffinés ont été récupérés. L’ADN était extrait et modifié par le traitement au bisulfite. Une réaction en chaîne de la polymérase, spécifique à la méthylation était entreprise pour évaluer l’état de méthylation du promoteur du gène du MGMT. Les résultats de laboratoire étaient corrélés avec la réponse clinique. L’analyse statistique était exécutée à l’aide du test de Fisher pour les données catégoriques et à l’aide des courbes de Kaplan-Meier pour les échecs au traitement. Résultats : Des 199 extraits d’ADN initiaux, 173 (87%) étaient modifiés au bisulfite avec succès. Des ces spécimens modifiés, 71 (41%) ont démontré une hyperméthylation du MGMT. Pour les cas de méthylation et nonméthylation du MGMT, les caractéristiques des patients n’étaient pas significativement différentes. Les taux de réponse étaient 71 et 73% (p=NS) respectivement. Le contrôle locorégional était respectivement 87 et 77% (p=0.26), la survie sans maladie était 80 et 60% (p=0.38), la survie sans métastase à distance était 92 et 78% (p=0.08) et la survie globale était 64 et 62% (p=0.99) à 3 ans. Conclusions : L’état de méthylation du MGMT est fortement prévalent (41%) et semble avoir un possible impact bénéfique sur la survie quand la chimioradiothérapie est administrée aux patients avec des stades avancés de cancers tête et cou. / Background: The O6-methylguanine-DNA methyltransferase (MGMT) gene encodes a specific DNA repair enzyme that protects cells from toxicity of alkylating agents. Thus, MGMT activity is a major mechanism of resistance to alkylating drugs. It has been shown that decreased MGMT gene expression by promoter hypermethylation results in improved survival in patients with certain types of tumors that are treated with alkylating chemotherapeutic agents. Objectives: To determine the prevalence of MGMT methylation in patients with locally advanced Head and Neck Squamous Cell Carcinoma (HNSCC) treated with chemoradiation therapy and to evaluate the impact of this methylation on survival. Methods: Out of 428 consecutive patients treated with chemoradiation therapy at our institution and followed for a median of 37 months, 199 paraffin embedded biopsy or surgical specimens were retrieved. DNA was extracted and subjected to bisulfite treatment. A methylation specific PCR (MSP) was conducted to assess the methylation status of the MGMT gene promoter. Laboratory data was correlated with clinical response. Statistical analysis was performed using Fisher’s test for categorical data and Kaplan-Meier’s curves and logrank statistics for failure times. Results: From the initial 199 DNA extracts, 173 (87%) were successfully modified with bisulfite. Out of these, 71 (41%) demonstrated hypermethylation of MGMT. For MGMT methylated cases and nonmethylated cases, patients characteristics were not significantly different. Response rates were 71 and 73% (p=NS), respectively. Local control rate (LCR) was respectively 87 and 77% (p=0.26), Disease-free survival (DFS) was 80 and 60% (p=0.38), distant metastasis free survival (DMFS) was 92 and 78% (p=0.08) and overall survival (OS) was 64 and 62% (p=0.99) at 3 years respectively. Conclusions: MGMT methylation status is highly prevalent (41%) and seems to have a possible beneficial impact on survival when chemoradiation therapy is given to patients with advanced stage HNSCC.

Introduction: Le gène O6-méthylguanine-ADN méthyltransferase (MGMT) code pour une enzyme spécifique réparatrice de l’ADN qui protège les cellules de la toxicité des agents alkylants. Ainsi, l’activité du MGMT est un mécanisme majeur de résistance aux agents alkylants. Il a été démontré qu’une diminution de l’expression du gène MGMT par une hyperméthylation du promoteur résulte en une amélioration de la survie chez les patients avec certains types de tumeurs qui sont traitées avec des agents chimiothérapeuthique alkylants. Objectifs: Déterminer la prévalence de la méthylation du gène MGMT chez des patients avec des cancers épidermoïdes localement avancés de la sphère ORL traités avec chimioradiothérapie et évaluer l’impact de cette méthylation sur la survie. Méthodes: Sur 428 patients consécutifs, traités avec chimioradiothérapie à notre institution et suivis pour un période médiane de 37 mois, 199 spécimens chirurgicaux paraffinés ont été récupérés. L’ADN était extrait et modifié par le traitement au bisulfite. Une réaction en chaîne de la polymérase, spécifique à la méthylation était entreprise pour évaluer l’état de méthylation du promoteur du gène du MGMT. Les résultats de laboratoire étaient corrélés avec la réponse clinique. L’analyse statistique était exécutée à l’aide du test de Fisher pour les données catégoriques et à l’aide des courbes de Kaplan-Meier pour les échecs au traitement. Résultats : Des 199 extraits d’ADN initiaux, 173 (87%) étaient modifiés au bisulfite avec succès. Des ces spécimens modifiés, 71 (41%) ont démontré une hyperméthylation du MGMT. Pour les cas de méthylation et nonméthylation du MGMT, les caractéristiques des patients n’étaient pas significativement différentes. Les taux de réponse étaient 71 et 73% (p=NS) respectivement. Le contrôle locorégional était respectivement 87 et 77% (p=0.26), la survie sans maladie était 80 et 60% (p=0.38), la survie sans métastase à distance était 92 et 78% (p=0.08) et la survie globale était 64 et 62% (p=0.99) à 3 ans. Conclusions : L’état de méthylation du MGMT est fortement prévalent (41%) et semble avoir un possible impact bénéfique sur la survie quand la chimioradiothérapie est administrée aux patients avec des stades avancés de cancers tête et cou. / Background: The O6-methylguanine-DNA methyltransferase (MGMT) gene encodes a specific DNA repair enzyme that protects cells from toxicity of alkylating agents. Thus, MGMT activity is a major mechanism of resistance to alkylating drugs. It has been shown that decreased MGMT gene expression by promoter hypermethylation results in improved survival in patients with certain types of tumors that are treated with alkylating chemotherapeutic agents. Objectives: To determine the prevalence of MGMT methylation in patients with locally advanced Head and Neck Squamous Cell Carcinoma (HNSCC) treated with chemoradiation therapy and to evaluate the impact of this methylation on survival. Methods: Out of 428 consecutive patients treated with chemoradiation therapy at our institution and followed for a median of 37 months, 199 paraffin embedded biopsy or surgical specimens were retrieved. DNA was extracted and subjected to bisulfite treatment. A methylation specific PCR (MSP) was conducted to assess the methylation status of the MGMT gene promoter. Laboratory data was correlated with clinical response. Statistical analysis was performed using Fisher’s test for categorical data and Kaplan-Meier’s curves and logrank statistics for failure times. Results: From the initial 199 DNA extracts, 173 (87%) were successfully modified with bisulfite. Out of these, 71 (41%) demonstrated hypermethylation of MGMT. For MGMT methylated cases and nonmethylated cases, patients characteristics were not significantly different. Response rates were 71 and 73% (p=NS), respectively. Local control rate (LCR) was respectively 87 and 77% (p=0.26), Disease-free survival (DFS) was 80 and 60% (p=0.38), distant metastasis free survival (DMFS) was 92 and 78% (p=0.08) and overall survival (OS) was 64 and 62% (p=0.99) at 3 years respectively. Conclusions: MGMT methylation status is highly prevalent (41%) and seems to have a possible beneficial impact on survival when chemoradiation therapy is given to patients with advanced stage HNSCC.