Breaking photoswitch activation depth limit using ionising radiation stimuli adapted to clinical application.

Electromagnetic radiation-triggered therapeutic effect has attracted a great interest over the last 50 years. However, translation to clinical applications of photoactive molecular systems developed to date is dramatically limited, mainly because their activation requires excitation by low-energy photons from the ultraviolet to near infra-red range, preventing any activation deeper than few millimetres under the skin. Herein we conceive a strategy for photosensitive-system activation potentially adapted to biological tissues without any restriction in depth. High-energy stimuli, such as those employed for radiotherapy, are used to carry energy while molecular activation is provided by local energy conversion. This concept is applied to azobenzene, one of the most established photoswitches, to build a radioswitch. The radiation-responsive molecular system developed is used to trigger cytotoxic effect on cancer cells upon gamma-ray irradiation. This breakthrough activation concept is expected to expand the scope of applications of photosensitive systems and paves the way towards the development of original therapeutic approaches.

Effect of X-ray minibeam radiation therapy on clonogenic survival of glioma cells.

The goal is to compare, in vitro, the efficiency of minibeam radiotherapy (MBRT) and standard RT in inducing clonogenic cell death in glioma cell lines. With this aim, we report on the first in vitro study performed in an X-ray Small Animal Radiation Research Platform (SARRP) modified for minibeam irradiations. F98 rat and U87 human glioma cells were irradiated with either an array of minibeams (MB) or with conventional homogeneous beams (broad beam, BB). A specially designed multislit collimator was used to generate the minibeams with a with of a center-to-center distance of 1465 (±10) µm, and a PVDR value of 12.4 (±2.3) measured at 1 cm depth in a water phantom. Cells were either replated for clonogenic assay directly (immediate plating, IP) or 24 h after irradiation (delayed plating, DP) to assess the effect of potentially lethal damage repair (PLDR) on cell survival. Our hypothesis is that with MBRT, a similar level of clonogenic cell death can be reached compared to standard RT, when using equal mean radiation doses. To prove this, we performed dose escalations to determine the minimum integrated dose needed to reach a similar level of clonogenic cell death for both treatments. We show that this minimum dose can vary per cell line: in F98 cells a dose of 19 Gy was needed to obtain similar levels of clonogenic survival, whereas in U87 cells there was still a slightly increased survival with MB compared to BB 19 Gy treatment. The results suggest also an impairment of DNA damage repair in F98 cells as there is no difference in clonogenic cell survival between immediately and delayed plated cells for each dose and irradiation mode. For U87 cells, a small IP-DP effect was observed in the case of BB irradiation up to a dose of 17 Gy. However, at 19 Gy BB, as well as for the complete dose range of MB irradiation, U87 cells did not show a difference in clonogenic survival between IP and DP. We therefore speculate that MBRT might influence PLDR. The current results show that X-ray MBRT is a promising method for treatment of gliomas: future preclinical and clinical studies should aim at reaching a minimum radiation (valley) dose for effective eradication of gliomas with increased sparing of normal tissues compared to standard RT.

Preclinical study of the DNA repair inhibitor Dbait in combination with chemotherapy in colorectal cancer.

BACKGROUND: Dbait molecules are a new class of DNA repair inhibitors triggering false DNA damage signaling in cancer cells. Dbait has already been shown to be effective in combination with radiotherapy. The aim of this study was to assess the adjuvant impact of Dbait on chemotherapy in vitro and in mouse models of colorectal cancer. METHODS: We assessed DNA repair efficiency over time, in vitro, in human colon adenocarcinoma HT-29 (wild-type KRAS) and HCT-116 (mutated KRAS) cell lines treated with Dbait in combination with 5-fluorouracil and/or camptothecin. Genetically engineered mice spontaneously developing colorectal tumors in the intestines were selected for the evaluation of treatment efficacy. RESULTS: Dbait delayed the repair of DNA damage induced by chemotherapy in vitro. In APC (+/1638N) mutant mice, the combination of Dbait and chemotherapy decreased tumor size more effectively than chemotherapy alone (median size: 3.6 vs. 10.85 mm(2), P < 0.05). In APC (+/1638N)/KRAS ( V12G ) mutant mice, animals treated with a combination of Dbait and chemotherapy survived significantly longer than animals treated by chemotherapy alone (median survival: 210 vs. 194 days, P < 0.05). A quarter of all the animals treated by chemotherapy alone died as rapidly as untreated animals, whereas the first death was delayed by 29 days by the addition of Dbait. No increase in toxicity due to Dbait was observed in either mouse model. CONCLUSIONS: The use of Dbait to inhibit DNA repair may be an effective additional treatment for increasing the efficacy of chemotherapy in colon or rectal cancer, independently of KRAS status.

Spi-1/PU.1 oncogene accelerates DNA replication fork elongation and promotes genetic instability in the absence of DNA breakage.

The multistage process of cancer formation is driven by the progressive acquisition of somatic mutations. Replication stress creates genomic instability in mammals. Using a well-defined multistep leukemia model driven by Spi-1/PU.1 overexpression in the mouse and Spi-1/PU.1-overexpressing human leukemic cells, we investigated the relationship between DNA replication and cancer progression. Here, using DNA molecular combing and flow cytometry methods, we show that Spi-1 increases the speed of replication by acting specifically on elongation rather than enhancing origin firing. This shortens the S-phase duration. Combining data from Spi-1 knockdown in murine cells with Spi-1 overexpression in human cells, we provide evidence that inappropriate Spi-1 expression is directly responsible for the replication alteration observed. Importantly, the acceleration of replication progression coincides with an increase in the frequency of genomic mutations without inducing DNA breakage. Thus, we propose that the hitherto unsuspected role for spi-1 oncogene in promoting replication elongation and genomic mutation promotes blastic progression during leukemic development.

Small-molecule drugs mimicking DNA damage: a new strategy for sensitizing tumors to radiotherapy.

PURPOSE: Enhanced DNA repair activity is often associated with tumor resistance to radiotherapy. We hypothesized that inhibiting DNA damage repair would sensitize tumors to radiation-induced DNA damage. EXPERIMENTAL DESIGN: A novel strategy for inhibiting DNA repair was tested. We designed small DNA molecules that mimic DNA double-strand breaks (called Dbait) and act by disorganizing damage signaling and DNA repair. We analyzed the effects of Dbait in cultured cells and on xenografted tumors growth and performed preliminary studies of their mechanism(s) of action. RESULTS: The selected Dbait molecules activate H2AX phosphorylation in cell culture and in xenografted tumors. In vitro, this activation correlates with the reduction of Nijmegen breakage syndrome 1 and p53-binding protein 1 repair foci formation after irradiation. Cells are sensitized to irradiation and do not efficiently repair DNA damage. In vivo, Dbait induces regression of radioresistant head and neck squamous cell carcinoma (Hep2) and melanoma (SK28 and LU1205) tumors. The combination of Dbait32Hc treatment and fractionated radiotherapy significantly enhanced the therapeutic effect. Tumor growth control by Dbait molecules depended directly on the dose and was observed with various irradiation protocols. The induction of H2AX phosphorylation in tumors treated with Dbait suggests that it acts in vivo through the induction of "false" DNA damage signaling and repair inhibition. CONCLUSIONS: These data validate the concept of introducing small DNA molecules, which mimic DNA damage, to trigger "false" signaling of DNA damage and impair DNA repair of damaged chromosomes. This new strategy could provide a new method for enhancing radiotherapy efficiency in radioresistant tumors.

Interexperimental and interindividual variations of DNA repair capacities after UV-B and UV-C irradiations of human keratinocytes and fibroblasts.

DNA repair plays a central role in the cellular response to UV. In this work we have studied the response of skin cells (i.e. fibroblasts and keratinocytes) from the same or from different individuals after both ultraviolet-B (UV-B) and ultraviolet-C (UV-C) irradiations using the comet assay to characterize the specific cellular response to UV-induced DNA damage. Cells were irradiated with increasing doses of UV-B or UV-C. To study the UV dose dependency of initial steps of DNA repair, namely recognition and incision at DNA damage level, the comet assay was performed, under alkaline conditions, 60 min after UV irradiation to allow detection of DNA strand breaks. Comparative analysis of tail moment values after UV exposure of cells from the same or from different individuals showed interexperimental and interindividual variations, implying that repeated assays are necessary to characterize the individual DNA repair capacity. With increasing doses of UV in keratinocytes, a plateau was rapidly reached after irradiation, whereas in fibroblasts a linear dose-effect relationship was observed. These interindividual variations associated with cellular specificity in DNA response may be of significance in skin cell and individual susceptibility toward UV-induced carcinogenesis.