Differential DNA repair pathway choice in cancer cells after proton- and photon-irradiation.

BACKGROUND AND PURPOSE: Non-homologous end-joining (NHEJ) and homologous recombination (HR) contribute to the repair of irradiation-induced DNA double-strand breaks (DSBs). We investigated the impact of the two major DSB repair machineries for cellular survival of human tumor cells in response to proton- and photon-irradiation. MATERIALS AND METHODS: DNA damage repair and cell survival were analyzed in wildtype, HR- and NHEJ-repair-compromised and pharmacologically DNA-PKcs-inhibited human tumor cells in response to clinically relevant, low-linear energy transfer proton- and 200-keV photon-irradiation. RESULTS: Pharmacological inhibition of DNA-PKcs strongly radiosensitized lung adenocarcinoma and glioblastoma cells to photon- but to a much lower extent to proton-irradiation. Enhanced radiosensitization correlated with strongly delayed repair kinetics with elevated amounts of γH2AX foci after photon-irradiation. Interestingly, we observed reduced phosphorylation of DNA-PKcs at Ser-2056 and Thr-2609 clusters after proton-irradiation compared to photon-irradiation. In contrast, A549 cells depleted of the RAD51 recombinase were markedly hypersensitive to proton-irradiation in comparison with control cells. Likewise, human BRCA2-deficient ovarian carcinoma cells were hypersensitive toward proton- in comparison with photon-irradiation. CONCLUSION: A differential DNA damage response with enhanced susceptibility of HR-deficient tumor cells to proton-irradiation and increased sensitivity of photon-irradiated tumor cells to NHEJ inhibitors were demonstrated.

DNA damage response and DNA repair - dog as a model?

BACKGROUND: Companion animals like dogs frequently develop tumors with age and similarly to human malignancies, display interpatient tumoral heterogeneity. Tumors are frequently characterized with regard to their mutation spectra, changes in gene expression or protein levels. Among others, these changes affect proteins involved in the DNA damage response (DDR), which served as a basis for the development of numerous clinically relevant cancer therapies. Even though the effects of different DNA damaging agents, as well as DDR kinetics, have been well characterized in mammalian cells in vitro, very little is so far known about the kinetics of DDR in tumor and normal tissues in vivo. DISCUSSION: Due to (i) the similarities between human and canine genomes, (ii) the course of spontaneous tumor development, as well as (iii) common exposure to environmental agents, canine tumors are potentially an excellent model to study DDR in vivo. This is further supported by the fact that dogs show approximately the same rate of tumor development with age as humans. Though similarities between human and dog osteosarcoma, as well as mammary tumors have been well established, only few studies using canine tumor samples addressed the importance of affected DDR pathways in tumor progression, thus leaving many questions unanswered. SUMMARY: Studies in humans showed that misregulated DDR pathways play an important role during tumor development, as well as in treatment response. Since dogs are proposed to be a good tumor model in many aspects of cancer research, we herein critically investigate the current knowledge of canine DDR and discuss (i) its future potential for studies on the in vivo level, as well as (ii) its possible translation to veterinary and human medicine.

Deficiency in homologous recombination renders Mammalian cells more sensitive to proton versus photon irradiation.

PURPOSE: To investigate the impact of the 2 major DNA repair machineries on cellular survival in response to irradiation with the 2 types of ionizing radiation. METHODS AND MATERIALS: The DNA repair and cell survival endpoints in wild-type, homologous recombination (HR)-deficient, and nonhomologous end-joining-deficient cells were analyzed after irradiation with clinically relevant, low-linear energy transfer (LET) protons and 200-keV photons. RESULTS: All cell lines were more sensitive to proton irradiation compared with photon irradiation, despite no differences in the induction of DNA breaks. Interestingly, HR-deficient cells and wild-type cells with small interfering RNA-down-regulated Rad51 were markedly hypersensitive to proton irradiation, resulting in an increased relative biological effectiveness in comparison with the relative biological effectiveness determined in wild-type cells. In contrast, lack of nonhomologous end-joining did not result in hypersensitivity toward proton irradiation. Repair kinetics of DNA damage in wild-type cells were equal after both types of irradiation, although proton irradiation resulted in more lethal chromosomal aberrations. Finally, repair kinetics in HR-deficient cells were significantly delayed after proton irradiation, with elevated amounts of residual γH2AX foci after irradiation. CONCLUSION: Our data indicate a differential quality of DNA damage by proton versus photon irradiation, with a specific requirement for homologous recombination for DNA repair and enhanced cell survival. This has potential relevance for clinical stratification of patients carrying mutations in the DNA damage response pathways.

Microtubule stabilising agents and ionising radiation: multiple exploitable mechanisms for combined treatment.

Combined radiochemotherapy treatment modalities are in use for many indications and therefore of high interest. Even though a combined modality in clinical use is often driven by pragmatic aspects, mechanistic preclinical-based concepts of interaction are of importance in order to translate and implement an optimal combination and scheduling of two modalities into the clinics. The use of microtubule stabilising agents is a promising strategy for anti-cancer therapy as a part of combined treatment modality with ionising radiation. Traditionally, microtubule targeting agents are classified as cytotoxic chemotherapeutics and are mostly used in a maximally tolerated dose regimen. Apart from direct cytotoxicity and similar to mechanisms of molecular targeting agents, microtubule stabilising agents interfere with multiple cellular processes, which can be exploited as part of combined treatment modalities. Recent preclinical investigations on the combination of ionising radiation and microtubule stabilising agents reveal new mechanistic interactions on the cellular and tumour level and elucidate the supra-additive tumour response observed particularly in vivo. The major focus on the mechanism of interaction was primarily based on radiosensitisation due to cell cycle arrest in the most radiosensitive G2/M-phase of the cell cycle. However, other mechanisms of interaction such as reoxygenation and direct as well as indirect endothelial damage have also been identified. In this review we summarise and allocate additive and synergistic effects induced by the combined treatment of clinically relevant microtubule stabilising agents and ionising radiation along a described radiobiological framework encompassing distinct mechanisms relevant for exploiting the combination of drugs and ionising radiation.

Silencing of human DNA polymerase λ causes replication stress and is synthetically lethal with an impaired S phase checkpoint.

Human DNA polymerase (pol) λ functions in base excision repair and non-homologous end joining. We have previously shown that DNA pol λ is involved in accurate bypass of the two frequent oxidative lesions, 7,8-dihydro-8-oxoguanine and 1,2-dihydro-2-oxoadenine during the S phase. However, nothing is known so far about the relationship of DNA pol λ with the S phase DNA damage response checkpoint. Here, we show that a knockdown of DNA pol λ, but not of its close homologue DNA pol ß, results in replication fork stress and activates the S phase checkpoint, slowing S phase progression in different human cancer cell lines. We furthermore show that DNA pol λ protects cells from oxidative DNA damage and also functions in rescuing stalled replication forks. Its absence becomes lethal for a cell when a functional checkpoint is missing, suggesting a DNA synthesis deficiency. Our results provide the first evidence, to our knowledge, that DNA pol λ is required for cell cycle progression and is functionally connected to the S phase DNA damage response machinery in cancer cells.