The chloroethylating anticancer drug ACNU induces FRA1 that is involved in drug resistance of glioma cells

AbstractFRA1 belongs, together with c-Fos and FosB, to the family of Fos proteins that form with members of the ATF and Jun family the transcription factor AP-1 (activator protein 1). Previously we showed that c-Fos protects mouse embryonic fibroblasts against the cytotoxic effects of ultraviolet (UV) light by induction of the endonuclease XPF, leading to enhanced nucleotide excision repair (NER) activity. Here, we analyzed the regulation of FRA1 in glioma cells treated with the anticancer drug nimustine (ACNU) and its role in ACNU-induced toxicity. We show that FRA1 is upregulated in glioblastoma cells following ACNU on mRNA and protein levels. Knockdown of FRA1 by either siRNA or shRNA clearly sensitized glioma cells towards ACNU-induced cell death. Despite decreased AP-1 binding activity upon FRA1 knockdown, this effect is independent on regulation of the AP-1 target genes fasL, ercc1 and xpf. In addition, FRA1 knockdown does not affect DNA repair capacity. However, lack of FRA1 attenuated the ACNU-induced phosphorylation of CHK1 and led to a reduced arrest of cells in G2/M and, thereby, presumably leads to enhanced cell death in the subsequent cell cycle

Delayed c-Fos activation in human cells triggers XPF induction and an adaptive response to UVC-induced DNA damage and cytotoxicity

The oncoprotein c-Fos has been commonly found differently expressed in cancer cells. Our previous work showed that mouse cells lacking the immediate-early gene c-fos are hypersensitive to ultraviolet (UVC) light. Here, we demonstrate that in human diploid fibroblasts UV-triggered induction of c-Fos protein is a delayed and long-lasting event. Sustained upregulation of c-Fos goes along with transcriptional stimulation of the NER gene xpf, which harbors an AP-1 binding site in the promoter. Data gained on c-Fos knockdown and c-Fos overexpressing human cells provide evidence that c-Fos/AP-1 stimulates upregulation of XPF, thereby increasing the cellular repair capacity protecting from UVC-induced DNA damage. When these cells are pre-exposed to a low non-toxic UVC dose and challenged with a subsequent high dose of UVC irradiation, they show accelerated repair of UVC-induced DNA adducts and reduced cell kill. The data indicate a protective role of c-Fos induction by triggering an adaptive response pathway

Rolle der FOS-Proteine und des MAPK-Signallings in der Regulation der zellulären Antwort auf genotoxischen Stress

Die mittlere Überlebenszeit nach Erkennung eines Glioblastoms ohne Behandlung liegt bei 3 Monaten und kann durch die Behandlung mit Temozolomid (TMZ) auf etwa 15 Monate gesteigert werden. Neben TMZ sind die chlorethylierenden Nitrosoharnstoffe die meistversprechendsten und am häufigsten eingesetzten Chemotherapeutika in der Gliomtherapie. Hier liegt die mittlere Überlebenszeit bei 17,3 Monaten. Um die Therapie des Glioblastoms noch effektiver zu gestalten und Resistenzen zu begegnen, werden unterschiedlichste Ansätze untersucht. Eine zentrale Rolle spielen hierbei das activator protein 1 (AP-1) und die mitogen aktivierten Proteinkinasen (MAPK), deren Funktion in bisherigen Arbeiten noch unzureichend beleuchtet wurde.rnBesonders mit der Rolle des AP-1-bildenden Proteins FRA-1 in der Therapie des Glioblastoms haben sich bisher nur wenige Arbeiten beschäftigt, weshalb im ersten Teil der vorliegenden Arbeit dessen Funktion in der Regulation der Chemosensitivität gegenüber dem chlorethylierenden Agenz ACNU genauer untersucht wurde. Es konnte gezeigt werden, dass die FRA 1-Expression durch Behandlung mit ACNU induziert wird. Die Induktion erfolgte über die beiden MAPKs ERK1/2 und p38K. JNK hatte keinen Einfluss auf die Induktion. Durch die Herunterregulation der FRA-1-Expression mit Hilfe von siRNA und eines shRNA exprimierenden Plasmids kam es zu einer signifikanten Sensitivierung gegenüber ACNU. Dabei konnte gezeigt werden, dass die Herunterregulation der FRA-1-Expression in einer verminderten AP 1-Bildung, bedingt durch eine reduzierte Menge an FRA-1 im AP-1-Komplex resultiert. Die Sensitivierung gegenüber ACNU ist weder durch eine Veränderung in der DNA-Reparatur, noch in der Modulation der FAS-Ligand- bzw. FAS-Rezeptor-Expression bedingt. Auch die hier untersuchten BCL 2-Familienmitglieder wiesen keine Unterschiede in der Expression durch Modulation der FRA 1-Expression auf. Allerdings kam es durch die verminderte FRA-1-Expression zu einer Reduktion der Zellzahl in der G2/M-Phase nach Behandlung mit ACNU. Diese ging einher mit einer reduzierten Menge an phosphoryliertem und unphosphoryliertem CHK1, weshalb davon auszugehen ist, dass FRA 1 nach ACNU-Behandlung in Gliomzellen vor der Apoptose schützt, indem es modulierend auf die Zellzykluskontrolle einwirkt.rnIm zweiten Teil dieser Arbeit wurde die Regulation der apoptotischen Antwort nach Behandlung mit ACNU und TMZ genauer beleuchtet, wobei ein spezielles Augen¬merk auf AP 1 und die MAPKs gelegt wurde. Hier konnte gezeigt werden, dass die Apoptose nach Behandlung mit ACNU bzw. TMZ sowohl durch Spaltung von Pro-Caspase 8, als auch Pro-Caspase 9 eingeleitet wird. Dabei akkumulierte in beiden Fällen p53 vermehrt im Zellkern. Eine Inhibierung der transkriptionellen Aktivität von p53 führte nach ACNU-Behandlung zu einer Sensitivierung der Zellen, nach TMZ-Behandlung kam es zu einem leichten Anstieg in der Vitälität. Der FAS-Rezeptor wurde nach ACNU- und nach TMZ-Behandlung aktiviert und auch die DNA-Reparaturproteine DDB2 und XPC wurden in beiden Fällen vermehrt exprimiert. Für die MAPKs JNK und ERK1/2 konnte gezeigt werden, dass diese pro-apoptotisch wirken. Die AP-1-Bildung nach ACNU-Behandlung erfolgte bereits nach 24 h und war von langer Dauer, wohingegen nach TMZ-Behandlung nur eine transiente AP 1-Bildung zu relativ späten Zeitpunkten detektiert werden konnte. Ebenso konnte für das AP-1-Zielgen FAS-Ligand nach ACNU-Behandlung eine relativ schnelle, lang anhaltende Aktivierung detektiert werden, wohingegen nach TMZ-Behandlung zu einem späten Zeitpunkt ein kurzer Anstieg im Signal zu verzeichnen war. In späteren Experimenten konnte gezeigt werden, dass das BCL-2-Familienmitglied BIM eine zentrale Rolle in der Regulation des intrinsischen Apoptosesignalweges nach Behandlung mit ACNU und TMZ spielt. Die hier entstanden Ergebnisse tragen entscheidend zum Verständnis der durch diese beiden Agenzien gesteuerten, apoptotischen Signalwege bei und bieten eine fundierte Grundlage für weitere Untersuchungen.rnAfter detection of glioblastoma, the average survival time without treatment is 3 months. This can be prolonged to 15 months by treatment with temozolomide (TMZ). Besides TMZ the chlorethylating nitrosocompounds are one of the most promising and most often applied chemotherapeutic drugs in glioma therapy. Here, the average survival time amounts to 17.3 months. With the aim to improve glioblastoma therapy and to counter resistance, diverse approaches are currently under investigation. Though activator protein (AP-1) and the mitogen activated protein kinases (MAPKs) play a central role, so far, their function has been insufficiently examined.rnEspecially the role of the AP-1-forming protein FRA-1 in glioblastoma therapy has only been considered in a few papers, why the first part of the present thesis deals with the question of its function in the regulation of chemosensitivity towards the chlorethylator ACNU. It could be shown that FRA-1-expression is induced by ACNU treatment. The induction occurred via the MAPKs ERK1/2 and p38K, whereas JNK had no impact on FRA-1 expression. RNA interference-mediated knockdown of FRA-1-expression caused a decrease in AP-1 formation and a significant sensitization towards ACNU. It was additionally shown that the knock down of FRA-1-expression causes a decrease in AP-1-formation, due to a reduced amount of FRA-1 in the AP-1-complex. The sensitization towards ACNU was not caused by a decrease in DNA repair or by a modulation of FAS ligand or FAS receptor expression. Also, members of the BCL-2 family did not exhibit any differences in expression by modulation of FRA-1-expression. However, the reduced FRA-1-expression resulted in a decrease of the amount of cells in G2/M-phase after ACNU treatment. This was accompanied by a reduced amount of phosphorylated and unphosphorylated CHK1. These observations lead to the assumption that in glioma cells after ACNU treatment, FRA-1 protects from apoptosis by modulating cell cycle control. rnIn the second part of the present thesis, the regulation of the apoptotic response after treatment with ACNU and TMZ was examined. Special attention was given to AP-1 and the MAPKs. Here it was shown that apoptosis after ACNU or TMZ treatment was induced by cleavage of pro-caspase 8 and pro-caspase 9. In both cases, p53 accumulated in the nucleus. Inhibition of transcriptional activity of p53 led to a sensitization to ACNU, after TMZ treatment, a small increase in vitality was detected. FAS receptor expression was increased after ACNU and TMZ treatment, as well as the expression of the DNA repair proteins DDB2 and XPC. For the MAPKs, JNK and ERK1/2 could be shown to promote apoptosis. AP-1-formation after ACNU-treatment was already observed after 24 h and long-lasting, whereas after TMZ-treatment only transient AP-1-formation, at rather late time points, could be detected. Similarly, after ACNU-treatment, the AP-1-target gene FAS ligand showed fast and long-lasting activation, whereas after TMZ-treatment, a short increase in the signal at one, rather late time point was detected. In subsequent experiments, it could be shown that BIM, a member of the BCL-2 family, plays a central role in the regulation of the intrinsic apoptotic pathway after treatment with ACNU and TMZ. The data from the present work is crucially important for the better understanding of the apoptotic pathways triggered by ACNU and TMZ and offers a substantiated basis for further research.r

Effectiveness of Patient Education and Cognitive Behavioural Treatment as a Non-Pharmacological Intervention for Migraine in Adults – a Systematic Review

To evaluate the content of patient education and cognitive behavioural therapy and its effectiveness in the prevention of migraine for adults, a systematic search was conducted in the databases MEDLINE, EMBASE, PsycINFO and CINAHL. RCTs published in the past 10 years in German or English, reporting on any form of patient education or cognitive behavioural therapy for adult migraineurs, were included. Two reviewers independently searched and evaluated search results. The methodological quality of selected studies was assessed using the Cochrane risk of bias tool 2.0 independently by two reviewers. Data on content and effectiveness were extracted in a predesigned table. Across 1059 records, 14 studies were eligible, including 2266 participants (82,7% females). Contents of patient education included explanations on the link between thoughts and feelings, information on lifestyle influences on headaches, relaxation techniques and stress management, advice on diet and the benefit of physical activity. In addition, patient education focussed on the pathogenesis and diagnostic criteria of migraine, triggering factors and effective acute or prophylactic drug use. Outcome measures included headache frequency, medication intake, function and disability, as well as psychosocial status. Education reduced migraine frequency and improved quality of life. Effect sizes were small, and contents were heterogeneous. Educational and behavioural approaches can positively influence headache frequency. However, the content, duration and frequency and education formats varied widely. The level of evidence for education and cognitive behavioural therapy as a non-pharmacological intervention for migraine is still low. The protocol for the systematic review was registered in the database PROSPERO (PROSPERO 2019 CRD42019134463) prior to the data collection.197

Potential analysis of multidisciplinary biopsychosocial rehabilitation for patients with chronic low back pain / Potentialanalyse der multidisziplinären biopsychosozialen Rehabilitation für Patienten/-innen mit chronischen Rückenschmerzen

The prevalence of low back pain ranges from 74 to 85%, almost everybody in Germany suffers from low back pain once in his or her life. The recurrence rate within twelve months is 62%, which often leads to inability to work and reduction in earning capacity. The rate of chronic manifestation of low back pain is 5%. Subsequent loss of mobility and functional disability lowers quality of life and participation in private life and at work