Lara Barazzuol

Evaluation of poly (ADP-ribose) polymerase inhibitor ABT-888 combined with radiotherapy and temozolomide in glioblastoma

BackgroundThe cytotoxicity of radiotherapy and chemotherapy can be enhanced by modulating DNA repair. PARP is a family of enzymes required for an efficient base-excision repair of DNA single-strand breaks and inhibition of PARP can prevent the repair of these lesions. The current study investigates the trimodal combination of ABT-888, a potent inhibitor of PARP1-2, ionizing radiation and temozolomide(TMZ)-based chemotherapy in glioblastoma (GBM) cells.MethodsFour human GBM cell lines were treated for 5 h with 5 μM ABT-888 before being exposed to X-rays concurrently with TMZ at doses of 5 or 10 μM for 2 h. ABT-888′s PARP inhibition was measured using immunodetection of poly(ADP-ribose) (pADPr). Cell survival and the different cell death pathways were examined via clonogenic assay and morphological characterization of the cell and cell nucleus.ResultsCombining ABT-888 with radiation yielded enhanced cell killing in all four cell lines, as demonstrated by a sensitizer enhancement ratio at 50% survival (SER50) ranging between 1.12 and 1.37. Radio- and chemo-sensitization was further enhanced when ABT-888 was combined with both X-rays and TMZ in the O6-methylguanine-DNA-methyltransferase (MGMT)-methylated cell lines with a SER50 up to 1.44. This effect was also measured in one of the MGMT-unmethylated cell lines with a SER50 value of 1.30. Apoptosis induction by ABT-888, TMZ and X-rays was also considered and the effect of ABT-888 on the number of apoptotic cells was noticeable at later time points. In addition, this work showed that ABT-888 mediated sensitization is replication dependent, thus demonstrating that this effect might be more pronounced in tumour cells in which endogenous replication lesions are present in a larger proportion than in normal cells.ConclusionsThis study suggests that ABT-888 has the clinical potential to enhance the current standard treatment for GBM, in combination with conventional chemo-radiotherapy. Interestingly, our results suggest that the use of PARP inhibitors might be clinically significant in those patients whose tumour is MGMT-unmethylated and currently derive less benefit from TMZ.

A mathematical model of brain tumour response to radiotherapy and chemotherapy considering radiobiological aspects

Glioblastoma is the most frequent and malignant brain tumour. For many years, the conventional treatment has been maximal surgical resection followed by radiotherapy (RT), with a median survival time of less than 10 months. Previously, the use of adjuvant chemotherapy (given after RT) has failed to demonstrate a statistically significant survival advantage. Recently, a randomized phase III trial has confirmed the benefit of temozolomide (TMZ) and has defined a new standard of care for the treatment of patients with high-grade brain tumours. The results showed an increase of 2.5 months in median survival, and 16.1% in 2 year survival, for patients receiving RT with TMZ compared with RT alone. It is not clear whether the major benefit of TMZ comes from either concomitant administration of TMZ with RT, or from six cycles of adjuvant TMZ, or both. The objectives were to develop our original model, which addressed survival after RT, to construct a new module to assess the potential role of TMZ from clinical data, and to explore its synergistic contribution in addition to radiation. The model has been extended to include radiobiological parameters. The addition of the linear quadratic equation to describe cellular response to treatment has enabled us to quantify the effects of radiation and TMZ in radiobiological terms. The results indicate that the model achieves an excellent fit to the clinical data, with the assumption that TMZ given concomitantly with RT synergistically increases radiosensitivity. The alternative, that the effect of TMZ is due only to direct cell killing, does not fit the clinical data so well. The addition of concomitant TMZ appears to change the radiobiological parameters. This aspect of our results suggests possible treatment developments. Our observations need further evaluations in real clinical trials, may suggest treatment strategies for new trials, and inform their design.

In vitro evaluation of combined temozolomide and radiotherapy using X-rays and high-linear energy transfer radiation for glioblastoma

High-linear energy transfer radiation offers superior biophysical properties over conventional radiotherapy and may have a great potential for treating radioresistant tumors, such as glioblastoma. However, very little pre-clinical data exists on the effects of high-LET radiation on glioblastoma cell lines and on the concomitant application of chemotherapy. This study investigates the in vitro effects of temozolomide in combination with low-energy protons and α particles. Cell survival, DNA damage and repair, and cell growth were examined in four human glioblastoma cell lines (LN18, T98G, U87 and U373) after treatment with either X rays, protons (LET 12.91 keV/μm), or α particles (LET 99.26 keV/μm) with or without concurrent temozolomide at clinically-relevant doses of 25 and 50 μM. The relative biological effectiveness at 10% survival (RBE10) increased as LET increased: 1.17 and 1.06 for protons, and 1.84 and 1.68 for α particles in the LN18 and U87 cell lines, respectively. Temozolomide administration increased cell killing in the O6-methylguanine DNA methyltransferase-methylated U87 and U373 cell lines. In contrast, temozolomide provided no therapeutic enhancement in the methylguanine DNA methyltransferase-unmethylated LN18 and T98G cell lines. In addition, the residual number of γ-H2AX foci at 24 h after treatment with radiation and concomitant temozolomide was found to be lower than or equal to that expected by DNA damage with either of the individual treatments. Kinetics of foci disappearance after X-ray and proton irradiation followed similar time courses; whereas, loss of γ-H2AX foci after α particle irradiation occurred at a slower rate than that by low-LET radiation (half-life 12.51–16.87 h). The combination of temozolomide with different radiation types causes additive rather than synergistic cytotoxicity. Nevertheless, particle therapy combined with chemotherapy may offer a promising alternative with the additional benefit of superior biophysical properties. It is also possible that new fractionation schedules could be designed to exploit the change in DNA repair kinetics when MGMT-methylated cells respond to high-LET radiation.

Increased apoptosis and DNA double-strand breaks in the embryonic mouse brain in response to very low-dose X-rays but not 50 Hz magnetic fields

The use of X-rays for medical diagnosis is enhancing exposure to low radiation doses. Exposure to extremely low-frequency electromagnetic or magnetic fields is also increasing. Epidemiological studies show consistent associations of childhood leukaemia with exposure to magnetic fields but any causal relationship is unclear. A limitation in assessing the consequence of such exposure is the availability of sensitive assays. The embryonic neuronal stem and progenitor cell compartments are radiosensitive tissues. Using sensitive assays, we report a statistically significant increase in DNA double-strand break (DSB) formation and apoptosis in the embryonic neuronal stem cell compartment following in utero exposure to 10–200 mGy X-rays. Both endpoints show a linear response. We also show that DSB repair is delayed following exposure to doses below 50 mGy compared with 100 mGy. Thus, we demonstrate in vivo consequences of low-dose radiation. In contrast to these impacts, we did not observe any significant induction of DSBs or apoptosis following exposure to 50 Hz magnetic fields (100 or 300 µT). We conclude that any DSB induction by treatment with magnetic fields is lower than following exposure to 10 mGy X-rays. For comparison, certain procedures involving computed tomography scanning are equivalent to 1–5 mGy X-rays.

Radiosensitization of glioblastoma cells using a histone deacetylase inhibitor (SAHA) comparing carbon ions with X-rays

Abstract Purpose: Prognosis for patients with glioblastoma (GBM) remains poor, and new treatments are needed. Here we used a combination of two novel treatment modalities: Carbon ions and a histone deacetylase inhibitor (HDACi). We compared these to conventional X-rays, measuring the increased effectiveness of carbon ions as well as radiosensitization using HDACi. Materials and methods: Suberoylanilide hydroxamic acid (SAHA) was used at a non-toxic concentration of 0.5 μM in combination with 85 keV μm−1 carbon ions, and 250 kVp X-rays for comparison. Effects were assayed using clonogenic survival, γH2AX foci repair kinetics and measuring chromatin decondensation. Results: Dose toxicity curves showed that human GBM LN18 cells were more sensitive to SAHA compared to U251 cells at higher doses, but there was little effect at low doses. When combined with radiation, clonogenic assays showed that the Sensitizer Enhancement Ratio with carbon ions at 50% survival (SER50) was about 1.2 and 1.5 for LN18 and U251, respectively, but was similar for X-rays at about 1.3. The repair half-life of γH2AX foci was slower for cells treated with SAHA and was most noticeable in U251 cells treated with carbon ions where after 24 h, more than double the number of foci remained in comparison to the untreated cells. Hoechst fluorescent dye incorporation into the nucleus showed significant chromatin decondensation and density homogenization with SAHA treatment for both cell lines. Conclusion: Our results suggest a vital role of histone deacetylases (HDAC) in the modulation of DNA damage response and support the use of SAHA for the treatment of GBM through the combination with heavy ion therapy.

Low levels of endogenous or X-ray-induced DNA double-strand breaks activate apoptosis in adult neural stem cells.

ABSTRACT The embryonic neural stem cell compartment is characterised by rapid proliferation from embryonic day (E)11 to E16.5, high endogenous DNA double-strand break (DSB) formation and sensitive activation of apoptosis. Here, we ask whether DSBs arise in the adult neural stem cell compartments, the sub-ventricular zone (SVZ) of the lateral ventricles and the sub-granular zone (SGZ) of the hippocampal dentate gyrus, and whether they activate apoptosis. We used mice with a hypomorphic mutation in DNA ligase IV (Lig4Y288C), ataxia telangiectasia mutated (Atm−/−) and double mutant Atm−/−/Lig4Y288C mice. We demonstrate that, although DSBs do not arise at a high frequency in adult neural stem cells, the low numbers of DSBs that persist endogenously in Lig4Y288C mice or that are induced by low radiation doses can activate apoptosis. A temporal analysis shows that DSB levels in Lig4Y288C mice diminish gradually from the embryo to a steady state level in adult mice. The neonatal SVZ compartment of Lig4Y288C mice harbours diminished DSBs compared to its differentiated counterpart, suggesting a process selecting against unfit stem cells. Finally, we reveal high endogenous apoptosis in the developing SVZ of wild-type newborn mice. Summary: This study shows that endogenous and radiation-induced DNA damage sensitively activates apoptosis in the adult sub-ventricular zone (SVZ). Developmentally regulated apoptosis is shown in the neonatal SVZ.

The rate of X-ray-induced DNA double-strand break repair in the embryonic mouse brain is unaffected by exposure to 50 Hz magnetic fields

Abstract Purpose: Following in utero exposure to low dose radiation (10–200 mGy), we recently observed a linear induction of DNA double-strand breaks (DSB) and activation of apoptosis in the embryonic neuronal stem/progenitor cell compartment. No significant induction of DSB or apoptosis was observed following exposure to magnetic fields (MF). In the present study, we exploited this in vivo system to examine whether exposure to MF before and after exposure to 100 mGy X-rays impacts upon DSB repair rates. Materials and methods: 53BP1 foci were quantified following combined exposure to radiation and MF in the embryonic neuronal stem/progenitor cell compartment. Embryos were exposed in utero to 50 Hz MF at 300 μT for 3 h before and up to 9 h after exposure to 100 mGy X-rays. Controls included embryos exposed to MF or X-rays alone plus sham exposures. Results: Exposure to MF before and after 100 mGy X-rays did not impact upon the rate of DSB repair in the embryonic neuronal stem cell compartment compared to repair rates following radiation exposure alone. Conclusions: We conclude that in this sensitive system MF do not exert any significant level of DNA damage and do not impede the repair of X-ray induced damage.

In vivo sensitivity of the embryonic and adult neural stem cell compartments to low-dose radiation

The embryonic brain is radiation-sensitive, with cognitive deficits being observed after exposure to low radiation doses. Exposure of neonates to radiation can cause intracranial carcinogenesis. To gain insight into the basis underlying these outcomes, we examined the response of the embryonic, neonatal and adult brain to low-dose radiation, focusing on the neural stem cell compartments. This review summarizes our recent findings. At E13.5–14.5 the embryonic neocortex encompasses rapidly proliferating stem and progenitor cells. Exploiting mice with a hypomorphic mutation in DNA ligase IV (Lig4Y288C), we found a high level of DNA double-strand breaks (DSBs) at E14.5, which we attribute to the rapid proliferation. We observed endogenous apoptosis in Lig4Y288C embryos and in WT embryos following exposure to low radiation doses. An examination of DSB levels and apoptosis in adult neural stem cell compartments, the subventricular zone (SVZ) and the subgranular zone (SGZ) revealed low DSB levels in Lig4Y288C mice, comparable with the levels in differentiated neuronal tissues. We conclude that the adult SVZ does not incur high levels of DNA breakage, but sensitively activates apoptosis; apoptosis was less sensitively activated in the SGZ, and differentiated neuronal tissues did not activate apoptosis. P5/P15 mice showed intermediate DSB levels, suggesting that DSBs generated in the embryo can be transmitted to neonates and undergo slow repair. Interestingly, this analysis revealed a stage of high endogenous apoptosis in the neonatal SVZ. Collectively, these studies reveal that the adult neural stem cell compartment, like the embryonic counterpart, can sensitively activate apoptosis.

A coordinated DNA damage response promotes adult quiescent neural stem cell activation

Stem and differentiated cells frequently differ in their response to DNA damage, which can determine tissue sensitivity. By exploiting insight into the spatial arrangement of subdomains within the adult neural subventricular zone (SVZ) in vivo, we show distinct responses to ionising radiation (IR) between neural stem and progenitor cells. Further, we reveal different DNA damage responses between neonatal and adult neural stem cells (NSCs). Neural progenitors (transit amplifying cells and neuroblasts) but not NSCs (quiescent and activated) undergo apoptosis after 2 Gy IR. This response is cell type- rather than proliferation-dependent and does not appear to be driven by distinctions in DNA damage induction or repair capacity. Moreover, exposure to 2 Gy IR promotes proliferation arrest and differentiation in the adult SVZ. These 3 responses are ataxia telangiectasia mutated (ATM)-dependent and promote quiescent NSC (qNSC) activation, which does not occur in the subdomains that lack progenitors. Neuroblasts arising post-IR derive from activated qNSCs rather than irradiated progenitors, minimising damage compounded by replication or mitosis. We propose that rather than conferring sensitive cell death, apoptosis is a form of rapid cell death that serves to remove damaged progenitors and promote qNSC activation. Significantly, analysis of the neonatal (P5) SVZ reveals that although progenitors remain sensitive to apoptosis, they fail to efficiently arrest proliferation. Consequently, their repopulation occurs rapidly from irradiated progenitors rather than via qNSC activation.

Influence of the nucleus area distribution on the survival fraction after charged particles broad beam irradiation

It is well known that broad beam irradiation with heavy ions leads to variation in the number of hit(s) received by each cell as the distribution of particles follows the Poisson statistics. Although the nucleus area will determine the number of hit(s) received for a given dose, variation amongst its irradiated cell population is generally not considered. In this work, we investigate the effect of the nucleus areas distribution on the survival fraction. More specifically, this work aims to explain the deviation, or tail, which might be observed in the survival fraction at high irradiation doses. For this purpose, the nucleus area distribution was added to the beam Poisson statistics and the Linear-Quadratic model in order to fit the experimental data. As shown in this study, nucleus size variation, and the associated Poisson statistics, can lead to an upward survival trend after broad beam irradiation. The influence of the distribution parameters (mean area and standard deviation) was studied using a normal distribution, along with the Linear-Quadratic model parameters (α and β). Finally, the model proposed here was successfully tested to the survival fraction of LN18 cells irradiated with a 85 keV µm(- 1) carbon ion broad beam for which the distribution in the area of the nucleus had been determined.