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Purpose: Hyperthermia (40-44 °C) effectively sensitises tumours to radiotherapy by locally altering tumour biology. One of the effects of heat at the cellular level is inhibition of DNA repair by homologous recombination via degradation of the BRCA2-protein. This suggests that hyperthermia can expand the group of patients that benefit from PARP-inhibitors, a drug exploiting homologous recombination deficiency. Here, we explore whether the molecular mechanisms that cause heat-mediated degradation of BRCA2 are conserved in cell lines from various origins and, most importantly, whether, BRCA2 protein levels can be attenuated by heat in freshly biopted human tumours. Experimental design: Cells from four established cell lines and from freshly biopsied material of cervical (15), head- and neck (9) or bladder tumours (27) were heated to 42 °C for 60 min ex vivo. In vivo hyperthermia was studied by taking two biopsies of the same breast or cervical tumour: one before and one after treatment. BRCA2 protein levels were measured by immunoblotting. Results: We found decreased BRCA2-levels after hyperthermia in all established cell lines and in 91% of all tumours treated ex vivo. For tumours treated with hyperthermia in vivo, technical issues and intra-tumour heterogeneity prevented obtaining interpretable results. Conclusions: This study demonstrates that heat-mediated degradation of BRCA2 occurs in tumour material directly derived from patients. Although BRCA2-degradation may not be a practical biomarker for heat deposition in situ, it does suggest that application of hyperthermia could be an effective method to expand the patient group that could benefit from PARP-inhibitors. [ABSTRACT FROM AUTHOR]

It has long been established that hyperthermia increases the therapeutic benefit of radiation and chemotherapy in cancer treatment. During the last few years there have been substantial technical improvements in the sources used to apply and measure heat, which greatly increases enthusiasm for the clinical use of hyperthermia. These advances are converging with a better understanding of the physiological and molecular effects of hyperthermia. Therefore, we are now at a juncture where the parameters that will influence the efficacy of hyperthermia in cancer treatment can be optimised in a more systematic and rational manner. In addition, the novel insights in hyperthermia’s many biological effects on tumour cells will ultimately result in new treatment regimes. For example, the molecular effects of hyperthermia on the essential cellular process of DNA repair suggest novel combination therapies, with DNA damage response targeting drugs that should now be clinically explored. Here, we provide an overview of recent studies on the various macroscopic and microscopic biological effects of hyperthermia. We indicate the significance of these effects on current treatments and suggest how they will help design novel future treatments. [ABSTRACT FROM AUTHOR]

Regional hyperthermia is described as a targeted therapy and the definitions of six hallmarks of hyperthermia are proposed, representing the pleiotropic effect of this therapeutic modality to counteract tumour growth and progression. We recommend the considerations of these hallmarks in the design of clinical trials involving regional hyperthermia as targeted therapy. Randomised clinical studies using loco-regional hyperthermia as an adjuvant to radiotherapy or to chemotherapy for locally advanced tumours demonstrate the benefit of the combination compared to either of the standard treatments alone for tumour response, disease control, and patient survival outcome. These impressive results were obtained from proof-of-concept trials for superficial or deep-seated malignancies in unselected patients. None of these trials was designed as tailored approaches for the treatment of specified targets or to select potentially more sensitive subpopulations of patients using eligibility criteria. Based upon clinical examples of targeted chemotherapy, some guidelines are described for the successful development of targeted therapeutic combinations. We also retrospectively analyse the stepwise process of generating an ongoing new clinical trial using hyperthermia as targeted therapy to evade DNA repair in combination with a DNA damaging anticancer agent to implement this new vision. [ABSTRACT FROM AUTHOR]

Local hyperthermia is an effective treatment modality to augment radio- and chemotherapy-based anti-cancer treatments. Although the effect of hyperthermia is pleotropic, recent experiments revealed that homologous recombination, a pathway of DNA repair, is directly inhibited by hyperthermia. The hyperthermia-induced DNA repair deficiency is enhanced by inhibitors of the cellular heat-shock response. Taken together, these results provide the rationale for the development of novel anti-cancer therapies that combine hyperthermia-induced homologous recombination deficiency with the systemic administration of drugs that specifically affect the viability of homologous recombination deficient cells and/or inhibit the heat-shock response, to locally sensitise cancer cells to DNA damaging agents. [ABSTRACT FROM AUTHOR]

Interstrand cross-links (ICLs) prevent DNA strand separation and, therefore, transcription and replication, making them extremely cytotoxic. The precise mechanism by which ICLs are removed from mammalian genomes largely remains elusive. Genetic evidence implicates ATR, the Fanconi anemia proteins, proteins required for homologous recombination, translesion synthesis, and at least two endonucleases, MUS81-EME1 and XPF-ERCC1. ICLs cause replication-dependent DNA double-strand breaks (DSBs), and MUS81-EME1 facilitates DSB formation. The subsequent repair of these DSBs occurs via homologous recombination after the ICL is unhooked by XPF-ERCC1. Here, we examined the effect of the loss of either nuclease on FANCD2 monoubiquitination to determine if the nucleolytic processing of ICLs is required for the activation of the Fanconi anemia pathway. FANCD2 was monoubiquitinated in Mus81-/-, Ercc1-/-, and XPF-deficient human, mouse, and hamster cells exposed to cross-linking agents. However, the monoubiquitinated form of FANCD2 persisted longer in XPF-ERCC1-deficient cells than in wild-type cells. Moreover, the levels of chromatin-bound FANCD2 were dramatically reduced and the number of ICL-induced FANCD2 foci significantly lower in XPF-ERCC1-deficient cells. These data demonstrate that the unhooking of an ICL by XPF-ERCC1 is necessary for the stable localization of FANCD2 to the chromatin and subsequent homologous recombination-mediated DSB repair. [ABSTRACT FROM AUTHOR]

The DNA polymerase processivity factor proliferating cell nuclear antigen (PCNA) is central to both DNA replication and repair. The ring-shaped homotrimeric PCNA encircles and slides along double-stranded DNA, acting as a ‘sliding clamp’ that localizes proteins to DNA. We determined the behavior of green fluorescent protein-tagged human PCNA (GFP-hPCNA) in living cells to analyze its different engagements in DNA replication and repair. Photobleaching and tracking of replication foci revealed a dynamic equilibrium between two kinetic pools of PCNA, i.e., bound to replication foci and as a free mobile fraction. To simultaneously monitor PCNA action in DNA replication and repair, we locally inflicted UV-induced DNA damage. A surprisingly longer residence time of PCNA at damaged areas than at replication foci was observed. Using DNA repair mutants, we showed that the initial recruitment of PCNA to damaged sites was dependent on nucleotide excision repair. Local accumulation of PCNA at damaged regions was observed during all cell cycle stages but temporarily disappeared during early S phase. The reappearance of PCNA accumulation in discrete foci at later stages of S phase likely reflects engagements of PCNA in distinct genome maintenance processes dealing with stalled replication forks, such as translesion synthesis (TLS). Using a ubiquitination mutant of GFP-hPCNA that is unable to participate in TLS, we noticed a significantly shorter residence time in damaged areas. Our results show that changes in the position of PCNA result from de novo assembly of freely mobile replication factors in the nucleoplasmic pool and indicate different binding affinities for PCNA in DNA replication and repair. [ABSTRACT FROM AUTHOR]

The BRCT domain is a highly conserved module found in many proteins that participate in DNA damage checkpoint regulation, DNA repair, and cell cycle control. Here we describe the cloning, characterization, and targeted mutagenesis of Brctx, a novel gene with a BRCT motif. Brctx was found to be expressed ubiquitously in adult tissues and during development, with the highest levels found in testis. Brctx-deficient mice develop normally, show no pathological abnormalities, and are fertile. BRCTx binds to the C terminus of hRAD18 in yeast two-hybrid and immunoprecipitation assays and colocalizes with this protein in the nucleus. Despite this, Brctx-deficient murine embryonic fibroblasts (MEFs) do not show overt sensitivity to DNA-damaging agents. MEFs from Brctx-deficient embryos grow at a similar rate to wild-type MEF CD4/CD8 expressions, and the cell cycle parameters of thymocytes from wild-type and Brctx knockout animals are indistinguishable. Intriguingly, the BRCT domain of BRCTx is responsible for mediating its localization to the nucleus and centrosome in interphase cells. We conclude that, although highly conserved, Brctx is not essential for the above-mentioned processes and may be redundant. [ABSTRACT FROM AUTHOR]

Interstrand cross-links (ICLs) are an extremely toxic class of DNA damage incurred during normal metabolism or cancer chemotherapy. ICLs covalently tether both strands of duplex DNA, preventing the strand unwinding that is essential for polymerase access. The mechanism of ICL repair in mammalian cells is poorly understood. However, genetic data implicate the Ercc1-Xpf endonuclease and proteins required for homologous recombination-mediated double-strand break (DSB) repair. To examine the role of Ercc1-Xpf in ICL repair, we monitored the phosphorylation of histone variant H2AX (γ-H2AX). The phosphoprotein accumulates at DSBs, forming foci that can be detected by immunostaining. Treatment of wild-type cells with mitomycin C (MMC) induced γ-H2AX foci and increased the amount of DSBs detected by pulsed-field gel electrophoresis. Surprisingly, γ-H2AX foci were also induced in Ercc1-/- cells by MMC treatment. Thus, DSBs occur after cross-link damage via an Ercc1-independent mechanism. Instead, ICL-induced DSB formation required cell cycle progression into S phase, suggesting that DSBs are an intermediate of ICL repair that form during DNA replication. In Ercc1-/- cells, MMC-induced γ-H2AX foci persisted at least 48 h longer than in wild-type cells, demonstrating that Ercc1 is required for the resolution of cross-link-induced DSBs. MMC triggered sister chromatid exchanges in wild-type cells but chromatid fusions in Ercc1-/- and Xpf mutant cells, indicating that in their absence, repair of DSBs is prevented. Collectively, these data support a role for Ercc1-Xpf in processing ICL-induced DSBs so that these cytotoxic intermediates can be repaired by homologous recombination. [ABSTRACT FROM AUTHOR]