Yes / Topoisomerase inhibitors are in common use as chemotherapeutic agents although they can display reduced efficacy in chemotherapy-resistant tumours, which have inactivated DNA damage response (DDR) genes, such as ATM and TP53. Here, we characterise the cellular response to the dual-acting agent, Alchemix (ALX), which is a modified anthraquinone that functions as a topoisomerase inhibitor as well as an alkylating agent. We show that ALX induces a robust DDR at nano-molar concentrations and this is mediated primarily through ATR- and DNA-PK- but not ATM-dependent pathways, despite DNA double strand breaks being generated after prolonged exposure to the drug. Interestingly, exposure of epithelial tumour cell lines to ALX in vitro resulted in potent activation of the G2/M checkpoint, which after a prolonged arrest, was bypassed allowing cells to progress into mitosis where they ultimately died by mitotic catastrophe. We also observed effective killing of lymphoid tumour cell lines in vitro following exposure to ALX, although, in contrast, this tended to occur via activation of a p53-independent apoptotic pathway. Lastly, we validate the effectiveness of ALX as a chemotherapeutic agent in vivo by demonstrating its ability to cause a significant reduction in tumour cell growth, irrespective of TP53 status, using a mouse leukaemia xenograft model. Taken together, these data demonstrate that ALX, through its dual action as an alkylating agent and topoisomerase inhibitor, represents a novel anti-cancer agent that could be potentially used clinically to treat refractory or relapsed tumours, particularly those harbouring mutations in DDR genes.

Das humane Zytomegalievirus, ist ein ubiquitäres Pathogen, welches akute und persistierende Infektionen verursacht. Bei immunsupprimierten Patienten kann das Virus zu schweren Erkrankungen, wie Hepatitis, Pneumonie und bei kongenitaler Infektion außerdem zu Schädigungen des ZNS führen. HCMV blockiert die Zellproliferation durch einen Arrest am G1/S-Übergang des Zellzyklus, andererseits wird aber gleichzeitig die Expression S-Phase spezifischer Gene aktiviert. Teilweise lässt sich dies durch eine Virus vermittelte spezifische Inhibition der zellulären DNA-Repliaktion sowie durch eine massive Deregulation Zyklin-assozzierter Kinasen erklären. Zellkulturexperimente deuten darauf hin, dass die Zellzyklusalterationen wichtige Voraussetzungen für eine erfolgreiche Virusreplikation darstellen. Es ist hingegen nicht bekannt, welche Relevanz sie für die Virusvermehrung in vivo und das pathologische Erscheinungsbild im erkrankten Organismus besitzen. Diese Frage kann nur in einem Tiermodell sinnvoll angegangen werden. Aufgrund der Wirtsspezifität der Zytomegalieviren, ist man dabei auf die Verwendung der jeweiligen artspezifischen CMV angewiesen. Murines CMV (MCMV) und Ratten-CMV (RCMV) sind dabei die bislang bestuntersuchtesten Systeme. Das Anliegen dieser Arbeit war es zu prüfen, inwieweit die für HCMV beschriebenen Zellzyklusregulationen in MCMV und RCMV auf Zellkulturbasis konserviert sind. Es konnte gezeigt werden, dass sowohl RCMV als auch MCMV einen antiproliferativen Effekt auf infizierte Zellen besitzen und ebenso wie HCMV zu einem Zellzyklusarrest führen. Nager-Zytomegalieviren können Zellen auch in der G2-Phase arretieren und in dieser Zellzyklusphase auch effizient replizieren können. Die Infektion mit Nager-CMV führt außerdem auf breiter Basis zur Veränderung Zyklin-assoziierter Kinaseaktivitäten. Allen Zytomegalieviren ist die Hemmung der zellulären DNA-Synthese am G1/S-Übergang durch die Inhibition des replication licensing, dem Beginn der DNA-Synthese gemein. Durch diese vergleichende Studie wird einerseits deutlich, dass wesentliche funktionelle Schritte der Zellzyklusregulation zwischen den Zytomegalieviren konserviert sind, aber andererseits die zu Grunde liegenden molekularen Mechanismen zum Teil deutlich variieren. / Human Cytomegalovirus (HCMV) is an ubiquitous, species-specific beta-herpesvirus that, like other herpesviruses, can establish lifelong latency following primary infection. HCMV infection becomes virulent only in immunocompromised patients such as premature infants, transplant recipients and AIDS patients where the virus causes severe disease like hepatitis, pneumonitis and retinitis. Congenital infection produces birth defects, most commonly hearing loss. To develop rational-based strategies for prevention and treatment of HCMV infection, it is crucial to understand the interactions between the virus and its host cell that support the establishment and progression of the virus replicative cycle. In general, herpesviruses are known to replicate most efficiently in the absence of cellular DNA synthesis. What is more, they have evolved mechanisms to avoid the cell´s DNA replication phase by blocking cell cycle progression outside S phase. HCMV has been shown to specifically inhibit the onset of cellular DNA synthesis resulting in cells arrested with a G1 DNA content. Towards a better understanding of CMV-mediated cell cycle alterations in vivo, we tested murine and rat CMV (MCMV/RCMV), being common animal models for CMV infection, for their influence on the host cell cycle. It was found that both MCMV and RCMV exhibit a strong anti-proliferative capacity on immortalised and primary embryonic fibroblasts after lytic infection. This results from specific cell cycle blocks in G1 and G2 as demonstrated by flow cytometry analysis. The G1 arrest is at least in part caused by a specific inhibition of cellular DNA synthesis and involves both the formation and activation of the cells’ DNA replication machinery. Interestingly, and in contrast to HCMV, the replicative cycle of rodent CMVs started from G2 as efficiently as from G1. Whilst the cell cycle arrest is accompanied by a broad induction of cyclin-cdk2 and cyclin-cdk1 activity, cyclin D1-cdk4/6 activity is selectively suppressed in MCMV and RCMV infected cells. Thus, given that both rodent and human CMVs are anti-proliferative and arrest cell cycle progression we found a surprising divergence of some of the underlying mechanisms. Therefore, any question put forward to a rodent CMV model involving cell cycle regulation has to be well defined in order to extrapolate meaningful information for the human system.