Frédérique Mégnin-Chanet

Control of the G2/M checkpoints after exposure to low doses of ionising radiation: Implications for hyper-radiosensitivity

Two molecularly distinct G2/M cell cycle arrests are induced after exposure to ionising radiation (IR) depending on the cell cycle compartment in which the cells are irradiated. The aims of this study were to determine whether there are threshold doses for their activation and investigate the molecular pathways and possible links between the G2 to M transition and hyper-radiosensitivity (HRS). Two human glioblastoma cell lines (T98G-HRS(+) and U373-HRS(-)) unsynchronized or enriched in G2 were irradiated and flow cytometry with BrdU or histone H3 phosphorylation analysis used to assess cell cycle progression and a clonogenic assay to measure radiation survival. The involvement of ATM, Wee1 and PARP was studied using chemical inhibitors. We found that cells irradiated in either the G1 or S phase of the cell cycle transiently accumulate in G2 in a dose-dependent manner after exposure to doses as low as 0.2Gy. Only Wee1 inhibition reduced this G2 accumulation. A block of the G2 to M transition was found after irradiation in G2 but occurs only above a threshold dose, which is cell line dependent, and requires ATM activity after exposure to doses above 0.5Gy. A failure to activate this early G2/M checkpoint correlates with low dose radiosensitization. These results provide evidence that after exposure to low doses of IR two distinct G2/M checkpoints are activated, each in a dose-dependent manner, with distinct threshold doses and involving different damage signalling pathways and confirm links between the early G2/M checkpoint and hyper-radiosensitivity.

Poly(ADP-ribose) polymerase (PARP-1) is not involved in DNA double-strand break recovery

BackgroundThe cytotoxicity and the rejoining of DNA double-strand breaks induced by γ-rays, H2O2 and neocarzinostatin, were investigated in normal and PARP-1 knockout mouse 3T3 fibroblasts to determine the role of poly(ADP-ribose) polymerase (PARP-1) in DNA double-strand break repair.ResultsPARP-1-/- were considerably more sensitive than PARP-1+/+ 3T3s to induced cell kill by γ-rays and H2O2. However, the two cell lines did not show any significant difference in the susceptibility to neocarzinostatin below 1.5 nM drug. Restoration of PARP-1 expression in PARP-1-/- 3T3s by retroviral transfection of the full PARP-1 cDNA did not induce any change in neocarzinostatin response. Moreover the incidence and the rejoining kinetics of neocarzinostatin-induced DNA double-strand breaks were identical in PARP-1+/+ and PARP-1-/- 3T3s. Poly(ADP-ribose) synthesis following γ-rays and H2O2 was observed in PARP-1-proficient cells only. In contrast neocarzinostatin, even at supra-lethal concentration, was unable to initiate PARP-1 activation yet it induced H2AX histone phosphorylation in both PARP1+/+ and PARP-1-/- 3T3s as efficiently as γ-rays and H2O2.ConclusionsThe results show that PARP-1 is not a major determinant of DNA double-strand break recovery with either strand break rejoining or cell survival as an endpoint. Even though both PARP-1 and ATM activation are major determinants of the cell response to γ-rays and H2O2, data suggest that PARP-1-dependent poly(ADP-ribose) synthesis and ATM-dependent H2AX phosphorylation, are not inter-related in the repair pathway of neocarzinostatin-induced DNA double-strand breaks.

Targeting poly(ADP-ribose) polymerase activity for cancer therapy

Poly(ADP-ribosyl)ation is a ubiquitous protein modification found in mammalian cells that modulates many cellular responses, including DNA repair. The poly(ADP-ribose) polymerase (PARP) family catalyze the formation and addition onto proteins of negatively charged ADP-ribose polymers synthesized from NAD+. The absence of PARP-1 and PARP-2, both of which are activated by DNA damage, results in hypersensitivity to ionizing radiation and alkylating agents. PARP inhibitors that compete with NAD+ at the enzyme’s activity site are effective chemo- and radiopotentiation agents and, in BRCA-deficient tumors, can be used as single-agent therapies acting through the principle of synthetic lethality. Through extensive drug-development programs, third-generation inhibitors have now entered clinical trials and are showing great promise. However, both PARP-1 and PARP-2 are not only involved in DNA repair but also in transcription regulation, chromatin modification, and cellular homeostasis. The impact on these processes of PARP inhibition on long-term therapeutic responses needs to be investigated.

Radiobiological Characterization of Two Therapeutic Proton Beams With Different Initial Energy Spectra Used at the Institut Curie Proton Therapy Center in Orsay

PURPOSE Treatment planning in proton therapy uses a generic value for the relative biological efficiency (RBE) of 1.1 throughout the spread-out Bragg peak (SOBP) generated. In this article, we report on the variation of the RBE with depth in the SOBP of the 76- and 201-MeV proton beams used for treatment at the Institut Curie Proton Therapy Center in Orsay. METHODS AND MATERIALS The RBE (relative to (137)Cs γ-rays) of the two modulated proton beams at three positions in the SOBP was determined in two human tumor cells using as endpoints clonogenic cell survival and the incidence of DNA double-strand breaks (DSBs) as measured by pulse-field gel electrophoresis without and with enzymatic treatment to reveal clustered lesions. RESULTS The RBE for induced cell killing by the 76-MeV beam increased with depth in the SOBP. However for the 201-MeV protons, it was close to that for (137)Cs γ-rays and did not vary significantly. The incidence of DSBs and clustered lesions was higher for protons than for (137)Cs γ-rays, but did not depend on the proton energy or the position in the SOBP. CONCLUSIONS Until now, little attention has been paid to the variation of RBE with depth in the SOBP as a function of the nominal energy of the primary proton beam and the molecular nature of the DNA damage. The RBE increase in the 76-MeV SOBP implies that the tumor tissues at the distal end receives a higher biologically equivalent dose than at the proximal end, despite a homogeneous physical dose. This is not the case for the 201-MeV energy beam. The precise determination of the effects of incident beam energy, modulation, and depth in tissues on the linear energy transfer-RBE relationship is essential for treatment planning.

The impact of cyclin-dependent kinase 5 depletion on poly(ADP-ribose) polymerase activity and responses to radiation

Cyclin-dependent kinase 5 (Cdk5) has been identified as a determinant of sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors. Here, the consequences of its depletion on cell survival, PARP activity, the recruitment of base excision repair (BER) proteins to DNA damage sites, and overall DNA single-strand break (SSB) repair were investigated using isogenic HeLa stably depleted (KD) and Control cell lines. Synthetic lethality achieved by disrupting PARP activity in Cdk5-deficient cells was confirmed, and the Cdk5KD cells were also found to be sensitive to the killing effects of ionizing radiation (IR) but not methyl methanesulfonate or neocarzinostatin. The recruitment profiles of GFP-PARP-1 and XRCC1-YFP to sites of micro-irradiated Cdk5KD cells were slower and reached lower maximum values, while the profile of GFP-PCNA recruitment was faster and attained higher maximum values compared to Control cells. Higher basal, IR, and hydrogen peroxide-induced polymer levels were observed in Cdk5KD compared to Control cells. Recruitment of GFP-PARP-1 in which serines 782, 785, and 786, potential Cdk5 phosphorylation targets, were mutated to alanines in micro-irradiated Control cells was also reduced. We hypothesize that Cdk5-dependent PARP-1 phosphorylation on one or more of these serines results in an attenuation of its ribosylating activity facilitating persistence at DNA damage sites. Despite these deficiencies, Cdk5KD cells are able to effectively repair SSBs probably via the long patch BER pathway, suggesting that the enhanced radiation sensitivity of Cdk5KD cells is due to a role of Cdk5 in other pathways or the altered polymer levels.

Acetate stimulates flux through the tricarboxylic acid cycle in rabbit renal proximal tubules synthesizing glutamine from alanine: a 13C NMR study.

Although glutamine synthesis has a major role in the control of acid-base balance and ammonia detoxification in the kidney of herbivorous species, very little is known about the regulation of this process. We therefore studied the influence of acetate, which is readily metabolized by the kidney and whose metabolism is accompanied by the production of bicarbonate, on glutamine synthesis from variously labelled [(13)C]alanine and [(14)C]alanine molecules in isolated rabbit renal proximal tubules. With alanine as sole exogenous substrate, glutamine and, to a smaller extent, glutamate and CO(2), were the only significant products of the metabolism of this amino acid, which was removed at high rates. Absolute fluxes through the enzymes involved in alanine conversion into glutamine were assessed by using a novel model describing the corresponding reactions in conjunction with the (13)C NMR, and to a smaller extent, the radioactive and enzymic data. The presence of acetate (5 mM) led to a large stimulation of fluxes through citrate synthase and alpha-oxoglutarate dehydrogenase. These effects were accompanied by increases in the removal of alanine, in the accumulation of glutamate and in flux through the anaplerotic enzyme pyruvate carboxylase. Acetate did not alter fluxes through glutamate dehydrogenase and glutamine synthetase; as a result, acetate did not change the accumulation of ammonia, which was negligible under both experimental conditions. We conclude that acetate, which seems to be an important energy-provider to the rabbit renal proximal tubule, simultaneously traps as glutamate the extra nitrogen removed as alanine, thus preventing the release of additional ammonia by the glutamate dehydrogenase reaction.

The farnesyl transferase inhibitor RPR-130401 does not alter radiation susceptibility in human tumor cells with a K-Ras mutation in spite of large changes in ploidy and lamin B distribution

BackgroundGrowth inhibition by RPR-130401, a non-peptidomimetic farnesyltransferase inhibitor, was investigated without or with combined exposure to ionizing radiation in three human tumor cell lines (HCT-116, MiAPaCa-2 and A-549) bearing a point mutation in the K-Ras gene.ResultsRPR-130401 inhibited cell growth with an IC50 of 50 nM (HCT-116), 120 nM (MiAPaCa-2) and 710 nM (A-549), with a poor incidence of apoptosis. The drug brought about G1 and S phase depletion together with arrest of cells in G2 phase and induced a significant accumulation of hyperploid cells showing active S phase DNA synthesis, with HCT-116 and A-549 cells being the most and least responsive, respectively. The drug also produced dramatic changes of the nuclear lamin B pattern, without lamin B cleavage and perturbation of the actin cytoskeleton. On the other hand, RPR-130401 elicited strictly additive interaction in combined treatment with ionizing radiation with regard to cell kill, altered cell cycle progression and induced hyperploidy.ConclusionsThe data suggest that disruption of orderly progression through mitosis and cytokinesis, is a major outcome of drug action and that this effect proceeds from inhibition of lamin B farnesylation. It is anticipated from the strict additivity of RPR-130401 and radiation that neither induced radiation resistance nor acute or late complications of radiotherapy, should occur in combined treatment with RPR-130401.

Concurrent administration of anti-HER2 therapy and radiotherapy: Systematic review

BACKGROUND Over the past few years, anti-HER2 targeted therapies have proven to be a key treatment in the management of human epidermal growth receptor 2 (HER2)-positive breast cancers, as well as gastrointestinal tract tumors and head and neck tumors. Anti-HER2 therapies administered alone or in combination with chemotherapy have been extensively studied, but only limited robust data are available concerning the safety and efficacy of anti-HER2 molecules in combination with radiotherapy. METHODS We searched on Medline, Embase and Cochrane databases the articles providing data on the concomitant association between the antiHER2 therapies used in clinical practice (trastuzumab, pertuzumab, lapatinib and T-DM1) with radiotherapy. The articles were selected according to their pre-clinical and clinical relevance. RESULTS The trastuzumab-irradiation combination is the most studied, with a focus on the cardiac toxicity. The combination of lapatinib-irradiation was particularly studied in the context of cerebral metastases of HER2-positive breast cancer. The data on pertuzumab and T-DM1 were poor and are mainly case reports. CONCLUSION To date, reliable conclusions about the toxicity and/or efficacy of concomitant irradiation with anti-HER2 therapies are difficult to make due to the heterogeneity of the data in the literature and need to be confirmed on a larger scale and long term follow-up. Nevertheless, no serious adverse events are reported and the toxicity profile seems to be manageable.

Involvement of the Artemis Protein in the Relative Biological Efficiency Observed With the 76-MeV Proton Beam Used at the Institut Curie Proton Therapy Center in Orsay

PURPOSE Previously we showed that the relative biological efficiency for induced cell killing by the 76-MeV beam used at the Institut Curie Proton Therapy Center in Orsay increased with depth throughout the spread-out Bragg peak (SOBP). To investigate the repair pathways underlying this increase, we used an isogenic human cell model in which individual DNA repair proteins have been depleted, and techniques dedicated to precise measurements of radiation-induced DNA single-strand breaks (SSBs) and double-strand breaks (DSBs). METHODS AND MATERIALS The 3-Gy surviving fractions of HeLa cells individually depleted of Ogg1, XRCC1, and PARP1 (the base excision repair/SSB repair pathway) or of ATM, DNA-PKcs, XRCC4, and Artemis (nonhomologous end-joining pathway) were determined at the 3 positions previously defined in the SOBP. Quantification of incident SSBs and DSBs by the alkaline elution technique and 3-dimensional (3D) immunofluorescence of γ-H2AX foci, respectively, was performed in SQ20 B cells. RESULTS We showed that the amount of SSBs and DSBs depends directly on the particle fluence and that the increase in relative biological efficiency observed in the distal part of the SOBP is due to a subset of lesions generated under these conditions, leading to cell death via a pathway in which the Artemis protein plays a central role. CONCLUSIONS Because therapies like proton or carbon beams are now being used to treat cancer, it is even more important to dissect the mechanisms implicated in the repair of the lesions generated by these particles. Additionally, alteration of the expression or activity of the Artemis protein could be a novel therapeutic tool before high linear energy transfer irradiation treatment.

Abstract 4428: Pre-clinical studies of the therpaeutic effect of a PARP inhibitor combined with radiotherapy for breast cancer treatment.

Introduction: Triple negative breast cancer (TNBC) is an aggressive disease associated with a high risk of distant recurrence and poor overall survival and, as for other BC sub-types, loco-regional treatment relies on surgery and radiotherapy (RT). Small molecules inhibitors of poly(ADP-ribose) polymerases (PARP) can potentially be exploited to sensitise breast tumour cells either when used in combination with chemo- and radiotherapy or in certain genetic backgrounds. In order to test this hypothesis, pre-clinical studies of the therapeutic effects of the combination of a PARP inhibitor and RT in human breast cancer xenograft models have been initiated using two TNBC models. Experimental design: Two human TNBC models, HBCx-17 and HBCx-12A xenografts were subcutaneously transplanted into the flanks of nude mice. The PARP inhibitor, 4-[3-(4-Cyclopropanecarbonyl-piperazine-1-carbonyl)-4-fluoro-benzyl]-2H-phthalazin-1-one (PARPi), suspended in 0.5% methylcellulose was administered by tube feeding at 100 mg/kg for 3 or 28 days. After tumors reached a calculated average volume of 125 mm3 (HBCx-12A) or 300 mm3 (HBCx-17), the animals were randomized into 8 treatment groups (10 mice/group): (1, 2) methylcellulose alone for 3 or 28 days, (3,4) methylcellulose for 3 or 28 days with 3 days radiotherapy, (5, 6) PARPi alone for 3 or 28 days, (7) PARPi combined with RT for 3 days and (8) PARPi for 3 days, then PARPi combined with 3 days RT followed by 22 days of PARPi. To mimic the clinical application of RT, the tumors were locally irradiated with X-Rays (200 kV, mean energy 80 keV) with a fractionated (3.25 Gy per fraction) schedule. Preliminary Results: In both TNBC models, individual group comparisons showed that after 28 days of treatment with PARPi alone tumour growth was markedly slowed, an effect that has persisted to day 60. Treatment with RT alone or RT combined with PARPi also resulted in significant growth inhibition over the same period. Animals are being followed to assess tumour regrowth and determine the long-term outcome of these treatment protocols. Conclusion: Our preclinical results show the susceptibility of TNBCs to the PARP inhibitor alone or combined with RT. However whether the response seen when the PARP inhibitor was combined with RT is due exclusively to impaired DNA damage responses or whether tumor re-oxygenation via the vasoactive effects of the PARP inhibitors contributes remains to be fully determined in further preclinical and clinical studies. Acknowledgments: This project is in-part financially supported by Institut Curie9s (IC) CEST program. V.P. was supported by an IC translational studentship (MD-Master2 science) and T.Z. by IC9s International Postodoctoral fellowship program and the Fondation PGG. Citation Format: Frederic Pouzoulet, Victor Pernin, Christophe Roulin, Helene Alcade, Franck Assayag, Frederique Megnin-Chanet, Laurence Vaslin-Lepetit, Sophie Heinrich, Florence Mahuteau-Betzer, Aurelie Thuleau, Tomasz Zaremba, Vincent Favaudon, Elisabetta Marangoni, Youlia Kirova, Alain Fourquet, Janet Hall, Didier Decaudin. Pre-clinical studies of the therpaeutic effect of a PARP inhibitor combined with radiotherapy for breast cancer treatment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4428. doi:10.1158/1538-7445.AM2013-4428