HDR brachytherapy decreases proliferation rate and cellular progression of a radioresistant human squamous cell carcinoma in vitro

Purpose: To investigate the effects of high dose rate (HDR) brachytherapy on cellular progression of a radioresistant human squamous cell carcinoma in vitro, based on clinical parameters.

Materials and methods: An acrylic platform was designed to attach tissue culture flasks and assure source positioning during irradiation. At exponential phase, A431cells, a human squamous cell carcinoma, were irradiated twice up to 1100 cGy. Cellular proliferation was assessed by Trypan blue exclusion assay and survival fraction was calculated by clonogenic assay. DNA content analysis and cell cycle phases were assessed by flow cytometry and gel electrophoresis, respectively. Cellular death patterns were measured by HOPI double-staining method.

Results: Significant decreasing cellular proliferation rate (p?<?0.05) as well as reduced survival fraction (p?<?0.001) in irradiated cells were observed. Moreover, increased percentage of cells arrested in the G₂/M phase (32.3?±?1.5%) in the irradiated group as compared with untreated cells (8.22?±?1.2%) was detected. Also, a significant (p?<?0.0001) nuclei shrinking in irradiated cells without evidence of necrosis or apoptosis was found.

Conclusion: HDR brachytherapy led to a decreased proliferation rate and cell survival and also hampered cellular progression to mitosis suggesting that tumor cell death mainly occurred due to mitotic death and G₂/M cell cycle arrest.     

Ionizing radiation induces EphA2 S897 phosphorylation in a MEK/ERK/RSK-dependent manner

Purpose: The EphA2 tyrosine kinase is frequently overexpressed in human tumors that are also treated with radiation. However, few studies have examined the effect of radiation on the EphA2 receptor itself. The purpose of this project was to investigate the impact of radiation on EphA2 to better understand mechanisms of radioresistance.

Materials and methods: Cell lines were exposed to X-rays and assayed for changes in EphA2 protein levels and phosphorylation over time by Western blotting. HEK293 cells stably expressing wild-type EphA2 or the S897A mutant were analyzed for cell survival from X-rays.

Results: Treatment of different cancer cell lines with 2?Gy of X-rays induced the phosphorylation of EphA2 on S897 but no changes were found in EphA2 total levels or its tyrosine phosphorylation. Radiation-induced S897 phosphorylation was unaffected by an AKT inhibitor but blocked by a MEK or RSK inhibitor. HEK293 cells expressing the EphA2 S897A mutant had a nearly 2-fold lower level of cell survival from X-rays than cells expressing wild-type EphA2.

Conclusions: These findings show that radiation induces S897 EphA2 phosphorylation, an event associated with increased cell survival. Therefore, targeting pathways that mediate EphA2 S897 phosphorylation may be a beneficial strategy to reduce radioresistance.     

Differing effects of breast cancer 1, early onset (BRCA1) and ataxia‐telangiectasia mutated (ATM) mutations on cellular responses to ionizing radiation

Purpose: The ataxia‐telangiectasia mutated (ATM) gene encodes a protein kinase, the activation of which is an early event in the cellular response to ionizing radiation. One of the many substrates of ATM is BRCA1 (breast cancer 1, early onset gene), which has been associated with susceptibility to breast and ovarian cancer, and has been implicated in DNA repair processes. Various cellular responses to radiation were analysed in cells with mutations in ATM or BRCA1 in an attempt to clarify which effects of ATM can be mediated through BRCA1.

Materials and methods: The response to radiation of cells with mutations in ATM or BRCA1 was examined, as were BRCA1‐mutant tumour cells transfected with an exogenous wild‐type BRCA1 allele. Assays included cell‐survival curves, studies of potentially lethal damage repair, measurement of chromosomal aberrations and of G1 arrest, and Western blot analysis of lysates of irradiated cells to determine the phosphorylation of the product of the human Mdm2 gene (HDM2).

Results: Both ATM and BRCA1 mutations were associated with sensitivity to ionizing radiation, deficient repair of potentially lethal damage and markedly increased chromosomal aberrations. A BRCA1‐mutated tumour cell line HCC1937, like ATM mutant cells, did not exhibit a normal G1 arrest but, unlike ATM mutant cells, did exhibit phosphorylation of HDM2. Expression of wild‐type BRCA1 in HCC1937 cells partially restored radioresistance, restored repair of potentially lethal damage and markedly reduced radiation‐induced chromosomal aberrations. G1 arrest, however, was not restored by expression of BRCA1.

Conclusions: The results are consistent with a model in which ATM phosphorylation of BRCA1 regulates DNA repair functions, particularly those involved in potentially lethal damage repair and chromosomal integrity, but not other aspects of the cellular response to radiation such as G1 cell cycle arrest. To the authors' knowledge, this is the first demonstration of the ability of exogenously expressed BRCA1 to restore the ability to perform potentially lethal damage repair and maintain chromosomal integrity in irradiated cells.     

Static electric fields interfere in the viability of cells exposed to ionising radiation

Purpose: The interference of electric fields (EF) with biological processes is an issue of considerable interest. No studies have as yet been reported on the combined effect of EF plus ionising radiation. Here we report studies on this combined effect using the prokaryote Microcystis panniformis, the eukaryote Candida albicans and human cells.

Materials and methods: Cultures of Microcystis panniformis (Cyanobacteria) in glass tubes were irradiated with doses in the interval 0.5–5 kGy, using a ⁶⁰Co gamma source facility. Samples irradiated with 3 kGy were exposed for 2 h to a 20 V · cm^?1 static electric field and viable cells were enumerated. Cultures of Candida albicans were incubated at 36°C for 20 h, gamma-irradiated with doses from 1–4 kGy, and submitted to an electric field of 180 V · cm^?1. Samples were examined under a fluorescence microscope and the number of unviable (red) and viable (apple green fluorescence) cells was determined. For crossing-check purposes, MRC5 strain of lung cells were irradiated with 2 Gy, exposed to an electric field of 1250 V/cm, incubated overnight with the anti-body anti-phospho-histone H2AX and examined under a fluorescence microscope to quantify nuclei with γ-H2AX foci.

Results: In cells exposed to EF, death increased substantially compared to irradiation alone. In C. albicans we observed suppression of the DNA repair shoulder. The effect of EF in growth of M. panniformis was substantial; the number of surviving cells on day-2 after irradiation was 12 times greater than when an EF was applied. By the action of a static electric field on the irradiated MRC5 cells the number of nuclei with γ-H2AX foci increased 40%, approximately.

Conclusions: Application of an EF following irradiation greatly increases cell death. The observation that the DNA repair shoulder in the survival curve of C. albicans is suppressed when cells are exposed to irradiation + EF suggests that EF likely inactivate cellular recovering processes. The result for the number of nuclei with γ-H2AX foci in MRC5 cells indicates that an EF interferes mostly in the DNA repair mechanisms. A molecular ad-hoc model is proposed.     

Is Biochemical Relapse-free Survival After Profoundly Hypofractionated Radiotherapy Consistent with Current Radiobiological Models?

AimsThe α/β ratio for prostate cancer is thought to be low and less than for the rectum, which is usually the dose-limiting organ. Hypofractionated radiotherapy should therefore improve the therapeutic ratio, increasing cure rates with less toxicity. A number of models for predicting biochemical relapse-free survival have been developed from large series of patients treated with conventional and moderately hypofractionated radiotherapy. The purpose of this study was to test these models when significant numbers of patients treated with profoundly hypofractionated radiotherapy were included.Materials and methodsA systematic review of the literature with regard to hypofractionated radiotherapy for prostate cancer was conducted, focussing on data recently presented on prostate stereotactic body radiotherapy. For the work described here, we have taken published biochemical control rates for a range of moderately and profoundly fractionated schedules and plotted these together with a range of radiobiological models, which are described.ResultsThe data reviewed show consistency between the various radiobiological model predictions and the currently observed data.ConclusionCurrent radiobiological models provide accurate predictions of biochemical relapse-free survival, even when profoundly hypofractionated patients are included in the analysis.     

Could hyperthermia with proton therapy mimic carbon ion therapy? Exploring a thermo-radiobiological rationale

Hyperthermia has been conventionally used in conjunction with photon beam irradiation. With a gradual increase in particle therapy facilities worldwide, this paper explores the physical, thermal and radiobiological implications of using a combination of hyperthermia with proton beam therapy. Hyperthermia is known to exhibit radiobiological features similar to those of high linear energy transfer radiation. Protons have many of the physical dose distribution properties of ¹²C ion therapy. Thus, the thermo-radiobiological advantages of hyperthermia coupled with the physical dose distribution advantages of proton beams could possibly mimic ¹²C ion therapy.     

Radiation Track Structure: How the Spatial Distribution of Energy Deposition Drives Biological Response

Ionising radiation is incredibly effective at causing biological effects. This is due to the unique way energy is deposited along highly structured tracks of ionisation and excitation events, which results in correlation with sites of DNA damage from the nanometre to the micrometre scale. Correlation of these events along the track on the nanometre scale results in clustered damage, which not only results in the formation of DNA double-strand breaks (DSB), but also more difficult to repair complex DSB, which include additional damage within a few base pairs. The track structure varies significantly with radiation quality and the increase in relative biological effectiveness observed with increasing linear energy transfer in part corresponds to an increase in the probability and complexity of clustered DNA damage produced. Likewise, correlation over larger scales, associated with packing of DNA and associated chromosomes within the cell nucleus, can also have a major impact on the biological response. The proximity of the correlated damage along the track increases the probability of miss-repair through pairwise interactions resulting in an increase in probability and complexity of DNA fragments/deletions, mutations and chromosomal rearrangements. Understanding the mechanisms underlying the biological effectiveness of ionising radiation can provide an important insight into ways of increasing the efficacy of radiotherapy, as well as the risks associated with exposure. This requires a multi-scale approach for modelling, not only considering the physics of the track structure from the millimetre scale down to the nanometre scale, but also the structural packing of the DNA within the nucleus, the resulting chemistry in the context of the highly reactive environment of the nucleus, together with the subsequent biological response.     

PET identifies transitional metabolic change in the spinal cord following a subthreshold dose of irradiation

Positron emission tomographic (PET) investigations were performed to obtainin vivo information on symptomless radiation-induced pathological changes in the human spinal cord. PET investigations were carried out prior to radiotherapy and during the regular follow-up in an early hypopharyngeal cancer patient (the spinal cord was irradiated with a biologically effective dose of 80 Gy₂), with [¹⁸F]fluorodeoxyglucose (FDG), [¹¹C]methionine and [¹⁵O]butanol as tracers; radiosensitivity and electroneuronographic (ENG) studies were also performed. A very low background FDG accumulation (mean standardized uptake values, i.e. SUV: 0.84) was observed in the spinal cord before the initiation of radiotherapy. An increased FDG uptake was measured 2 months after the completion of radiotherapy (mean SUV: 1.69), followed by a fall-off, as measured 7 months later (mean SUV: 1.21). By 44 months after completion of irradiation, the FDG accumulation in the irradiated segments of the spinal cord had decreased to a level very close to the initial value (mean SUV: 1.11). The simultaneous [¹⁵O]butanol uptake results demonstrated a set of perfusion changes similar to those observed in connection with the FDG accumulation. The patient exhibited an extremely low [¹¹C]methionine uptake within the irradiated and the nonirradiated spinal cord during the clinical course. She has not had any neurological symptoms, and the results of central ENG measurements before radiotherapy and 2 months following its completion proved normal. Radiobiological investigations did not reveal unequivocal signs of an increased radiosensitivity. A transitory increased spinal cord FDG uptake following radiotherapy may be related to the posttherapeutic mild inflammatory and regenerative processes. The normal [¹¹C]methionine accumulation observed is strong evidence against intensive cell proliferation. The high degree of normalization of the temporarily increased FDG uptake of the irradiated spinal cord segments by 44 months is in good agreement with the results of monkey studies, which demonstrated a nearly complete recovery from radiation-induced spinal cord injury. This work was supported in part by grants from the Hungarian Research Fund (OTKA T-032499 and T-046128), ETT 395/KO/2003, 37/2003 and the Ministry of Education „Széchenyi” (OM 1/008/2001).