Vincent Favaudon

DNA double-strand break repair defects in syndromes associated with acute radiation response: at least two different assays to predict intrinsic radiosensitivity?

Purpose: Human diseases associated with acute radiation responses are rare genetic disorders with common clinical and biological features including radiosensitivity, genomic instability, chromosomal aberrations, and frequently immunodeficiency. To determine what molecular assays are predictive of cellular radiosensitivity whatever the genes mutations, the existence of a quantitative correlation between cellular radiosensitivity and unrepaired DNA double-strand breaks (DSB) repair defects was examined in a collection of 40 human fibroblasts representing 8 different syndromes. Materials and methods: A number of techniques such as pulsed-field gel electrophoresis, plasmid assay and immunofluorescence with antibodies against MRE11, MDC1, 53BP1 and phosphorylated forms of H2AX, DNA-PK were applied systematically. Results and conclusions: Survival fraction at 2 Gy was found to be inversely proportional to the amount of unrepaired DSB, whatever the genes mutations and the assay applied. However, no single assay discriminates the full range of human radiosensitivity. Particularly, nuclear foci formed by the phosphorylation of H2AX do not predict well moderate radiosensitivities. Our findings suggest the existence of an ATM-dependent interplay between the activation of DNA-PK and MRE11. A classification of diseases according their cellular radiosensitivity, their molecular response to radiation and the functional assays permitting their evaluation is proposed.

Constitutive Association of BRCA1 and c-Abl and Its ATM-Dependent Disruption after Irradiation

ABSTRACT BRCA1 plays an important role in mechanisms of response to double-strand breaks, participating in genome surveillance, DNA repair, and cell cycle checkpoint arrests. Here, we identify a constitutive BRCA1-c-Abl complex and provide evidence for a direct interaction between the PXXP motif in the C terminus of BRCA1 and the SH3 domain of c-Abl. Following exposure to ionizing radiation (IR), the BRCA1-c-Abl complex is disrupted in an ATM-dependent manner, which correlates temporally with ATM-dependent phosphorylation of BRCA1 and ATM-dependent enhancement of the tyrosine kinase activity of c-Abl. The BRCA1-c-Abl interaction is affected by radiation-induced modification to both BRCA1 and c-Abl. We show that the C terminus of BRCA1 is phosphorylated by c-Abl in vitro. In vivo, BRCA1 is phosphorylated at tyrosine residues in an ATM-dependent, radiation-dependent manner. Tyrosine phosphorylation of BRCA1, however, is not required for the disruption of the BRCA1-c-Abl complex. BRCA1-mutated cells exhibit constitutively high c-Abl kinase activity that is not further increased on exposure to IR. We suggest a model in which BRCA1 acts in concert with ATM to regulate c-Abl tyrosine kinase activity.

Radiosensitization by the poly(ADP-ribose) polymerase inhibitor 4-amino-1,8-naphthalimide is specific of the S phase of the cell cycle and involves arrest of DNA synthesis

Radiosensitization caused by the poly(ADP-ribose) polymerase (PARP) inhibitor 4-amino-1,8-naphthalimide (ANI) was investigated in 10 asynchronously growing rodent (V79, CHO-Xrs6, CHO-K1, PARP-1+/+ 3T3, and PARP-1−/− 3T3) or human (HeLa, MRC5VI, IMR90, M059J, and M059K) cell lines, either repair proficient or defective in DNA-PK (CHO-Xrs6 and M059J) or PARP-1 (PARP-1−/− 3T3). Pulse exposure to ANI (1-hour contact) potentiated radiation response in rodent cells except in PARP-1−/− 3T3 fibroblasts. In contrast, ANI did not significantly enhance radiation susceptibility in asynchronously dividing human cells; yet, single-strand break rejoining was lengthened by ca. 7-fold in all but mouse PARP-1−/− 3T3s. Circumstantial evidence suggested that radiosensitization by ANI occurs in rapidly dividing cells only. Experiments using synchronized HeLa cells consistently showed that ANI-induced radiosensitization is specific of the S phase of the cell cycle and involves stalled replication forks. Under these conditions, prolonged contact with ANI ended in the formation of de novo DNA double-strand breaks hours after irradiation, evoking collision with uncontrolled replication forks of DNA lesions whose repair was impaired by inhibition of the PARP catalytic activity. The data suggest that increased response to radiotherapy by PARP inhibitors may be achieved only in rapidly growing tumors with a high S-phase content. [Mol Cancer Ther 2006;5(3):564–74]

Synthesis and in vitro evaluation of novel 2,6,9-trisubstituted purines acting as cyclin-dependent kinase inhibitors

Novel C-2, C-6, N-9 trisubstituted purines derived from the olomoucine/roscovitine lead structure were synthesized and evaluated for their ability to inhibit starfish oocyte CDK1/cyclin B, neuronal CDK5/p35 and erk1 kinases in purified extracts. Structure activity relationship studies showed that increased steric bulk at N-9 reduces the inhibitory potential whereas substitution of the aminoethanol C-2 side chain by various groups of different size (methyl, propyl, butyl, phenyl, benzyl) only slightly decreases the activity when compared to (R)-roscovitine. Optimal inhibitory activity against CDK5, CDK1 and CDK2, with IC50 values of 0.16, 0.45 and 0.65 microM, respectively, was obtained with compound 21 containing a (2R)-pyrrolidin-2-yl-methanol substituent at the C-2 and a 3-iodobenzylamino group at the C-6 of the purine. Compound 21 proved cytotoxic against human tumor HeLa cells (LD50-6.7 microM versus 42.7 microM for olomoucine, 24-h contact). Furthermore, unlike olomoucine, compound 21 was effective upon short exposure (LD50= 25.3 microM, 2-h contact). The available data suggest that the affinity for CDKs and the cytotoxic potential of the drugs are inter-related. However, no straightforward cell cycle phase specificity of the cytotoxic response to 21 was observed in synchronized HeLa cells. With the noticeable exception of pronounced lengthening of the S-phase transit by 21 applied during early-S in synchronized HeLa cells, and in striking contrast with earlier reports on studies using plant or echinoderm cells. olomoucilnc and compound 21 were unable to reversibly arrest cell cycle progression in asynchronous growing HeLa cells. Some irreversible hlock in GI and G2 phase occurred at high olomoucine concentration, correlated with induced cell death. Moreover, chmronic exposure to lethal doses of compound 21 resulted in massive nuclear fragmentation, evocative of mitotic catastrophe with minour amounts of apoptosis only. It was also found that olomoucine and compound 21 reversibly block the intracellular uptake of nuicleosides with high efficiency.

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.

Ultrahigh dose-rate FLASH irradiation increases the differential response between normal and tumor tissue in mice

Pulsed, ultrahigh dose-rate irradiation is safer than standard radiotherapy for the treatment of lung cancer and selectively spares normal tissue. Safer Radiation for the Lung Radiation is used to treat a variety of tumor types, including lung cancer. Unfortunately, radiation-induced damage to the surrounding healthy lung is a major problem, which can cause long-term complications and limits the amount of radiation that can be safely delivered to the tumor. Favaudon et al. now present a technology called FLASH, which allows the delivery of pulsed, ultrahigh dose-rate irradiation, which causes less damage to the healthy lung than conventional radiotherapy in mouse models. The authors confirmed that FLASH is effective against tumor cells but causes little damage to normal tissue. These results suggest that FLASH radiation may be a viable option for treating lung tumors, although this will need to be confirmed in human patients. In vitro studies suggested that sub-millisecond pulses of radiation elicit less genomic instability than continuous, protracted irradiation at the same total dose. To determine the potential of ultrahigh dose-rate irradiation in radiotherapy, we investigated lung fibrogenesis in C57BL/6J mice exposed either to short pulses (≤500 ms) of radiation delivered at ultrahigh dose rate (≥40 Gy/s, FLASH) or to conventional dose-rate irradiation (≤0.03 Gy/s, CONV) in single doses. The growth of human HBCx-12A and HEp-2 tumor xenografts in nude mice and syngeneic TC-1 Luc+ orthotopic lung tumors in C57BL/6J mice was monitored under similar radiation conditions. CONV (15 Gy) triggered lung fibrosis associated with activation of the TGF-β (transforming growth factor–β) cascade, whereas no complications developed after doses of FLASH below 20 Gy for more than 36 weeks after irradiation. FLASH irradiation also spared normal smooth muscle and epithelial cells from acute radiation-induced apoptosis, which could be reinduced by administration of systemic TNF-α (tumor necrosis factor–α) before irradiation. In contrast, FLASH was as efficient as CONV in the repression of tumor growth. Together, these results suggest that FLASH radiotherapy might allow complete eradication of lung tumors and reduce the occurrence and severity of early and late complications affecting normal tissue.

On the mechanism of reductive activation in the mode of action of some anticancer drugs

Bioactivation of a number of DNA-specific antitumor drugs depends on oxidoreduction. Bleomycin, neocarzinostatin and anthracycline glycosides are the best known among such drugs in terms of reductive activation processes. Their reduction results in short-lived radical or electrophilic intermediates attacking DNA stereospecifically. The physico-chemical properties of these drugs and the nature of DNA damage are reviewed. Models for DNA-intercalation, electron-donor systems involved in drug metabolisation, and the role of oxygen in radical reactions, are discussed in the light of recent reports.

Poly(ADP-ribose) polymerase, a major determinant of early cell response to ionizing radiation.

Purpose : To determine whether DNA-dependent protein kinase (DNA-PK) and poly(ADP-ribose) polymerase (PARP-1) are involved in eliciting the rapid fluctuations of radiosensitivity that have been observed when cells are exposed to short pulses of ionizing radiation. Materials and methods : The effect of DNA-PK and PARP-1 inhibitors on the survival of cells to split-dose irradiation was investigated using Chinese hamster V79 fibroblasts and human carcinoma SQ-20B cells. The responses of PARP-1 proficient and PARP-1 knockout mouse 3T3 fibroblasts were compared in a similar split-dose assay. Results : Inactivation of DNA-PK by wortmannin potentiated radiation-induced cell kill but it did not alter the oscillatory, W-shaped pattern of early radiation response. In contrast, oscillatory radiation response was abolished by 3-aminobenzamide, a reversible inhibitor of enzymes containing a PARP catalytic domain. The oscillatory response was also lacking in PARP-1 knockout mouse 3T3 fibroblasts. Conclusion : The results show that PARP-1 plays a key role in the earliest steps of cell response to ionizing radiation with clonogenic ability or growth as endpoint. It is hypothesized that rapid poly(ADP-ribosylation) of target proteins, or recruitment of repair proteins by activated PARP-1 at the sites of DNA damage, bring about rapid chromatin remodelling that may affect the incidence of chromosomal damage upon re-irradiation.PURPOSE To determine whether DNA-dependent protein kinase (DNA-PK) and poly(ADP-ribose) polymerase (PARP-1) are involved in eliciting the rapid fluctuations of radiosensitivity that have been observed when cells are exposed to short pulses of ionizing radiation. MATERIALS AND METHODS The effect of DNA-PK and PARP-1 inhibitors on the survival of cells to split-dose irradiation was investigated using Chinese hamster V79 fibroblasts and human carcinoma SQ-20B cells. The responses of PARP-1 proficient and PARP-1 knockout mouse 3T3 fibroblasts were compared in a similar split-dose assay. RESULTS Inactivation of DNA-PK by wortmannin potentiated radiation-induced cell kill but it did not alter the oscillatory, W-shaped pattern of early radiation response. In contrast, oscillatory radiation response was abolished by 3-aminobenzamide, a reversible inhibitor of enzymes containing a PARP catalytic domain. The oscillatory response was also lacking in PARP-1 knockout mouse 3T3 fibroblasts. CONCLUSION The results show that PARP-1 plays a key role in the earliest steps of cell response to ionizing radiation with clonogenic ability or growth as endpoint. It is hypothesized that rapid poly(ADP-ribosylation) of target proteins, or recruitment of repair proteins by activated PARP-1 at the sites of DNA damage, bring about rapid chromatin remodelling that may affect the incidence of chromosomal damage upon re-irradiation.

Physicochemical properties of flavodoxin from Desulfovibrio vulgaris

Reductive titration curves of flavodoxin from Desulfovibrio vulgaris displayed two one-electron steps. The redox potential E-2 for the couple oxidized flavodoxin/flavodoxin semiquinone was determined by direct titration with dithionite. E-2 was -149 plus or minus 3 mV (pH 7.78, 25 degrees C). The redox potential E-1 for the couple flavodoxin semiquinone/fully reduced flavodoxin was deduced from the equilibrium concentration of these species in the presence of hydrogenase and H-2. E-1 was -438 plus or minus 8 mV (pH 7.78, 25 degrees C). Light-absorption and fluorescence spectra of flavodoxin in its three redox states have been recorded. Both the rate and extent of reduction of flavodoxin semiguinone with dithionite were found to depend on pH. An equilibrium between the semiquinone and hydroquinone forms occurred at pH values close to the neutrality, even in the presence of a large excess of dithionite, suggesting an ionization in fully reduced flavodoxin with a pK-a = 6.6. The association constants K for the three FMN redox forms with the apoprotein were deduced from the value of K (K = 8 times 10-7 M-1) measured with oxidized EMN at pH 7.0. Oxidized flavodoxin was found to comproportionate with the fully reduced protein (k-comp = 4.3 times 10-3 M-1 times s-1, pH 9.0, 22 degrees C) and with reduced free FMN (K-comp = 44 M-1 times s-1, pH 8.1, 20 degrees C). Fast oxidation of reduced flavodoxin occurred in the presence of O-2. Slower oxidation of semiquinone was dependent on pH in a drastic way.