Fractionated Radiation Exposure Research Articles

Combined photodynamic and radiodynamic therapy with a chlorine photosensitizer under fractionated radiation exposure to transplanted tumors in an <i>in vivo</i> experiment

Introduction. The current direction of scientific research in experimental and clinical oncology is radiodynamic therapy (RDT), a treatment method based on the activation of photosensitizers (PS) by ionizing radiation. As the results of in vitro / in vivo studies show, a number of photosensitizing agents have radiomodifying properties, and the method itself is characterized by high antitumor efficacy.Aim. To study the possibility of increasing the effectiveness of photodynamic therapy (PDT) through its combined use with RDT in an in vivo experiment.Materials and methods. The work was performed on 26 white non-linear rats weighing 180 ± 50 g. Pliss lymphosarcoma was used as a tumor model, which was transplanted subcutaneously. The chlorine-based PS (Belmedpreparaty, Republic of Belarus) was administered intravenously at a dose of 2.5 mg / kg of body weight. The RDT session was performed by the contact method (CRT) after 2.5–3, 24 and 48 hours after the end of PS administration on the microSelectron-HDR V3 Digital device (Elekta, Sweden) using γ-radiation (192Ir) in a single focal dose (SFD) of 2 Gy until the total focal dose of 6 Gy is reached. A PDT session was performed once immediately after the first exposure to ionizing radiation using a PDT diode laser (Imaf Axicon, Republic of Belarus, λ = 660 ± 5 nm) at an exposure dose of 100 J / cm² with a power density of 0.2 w / cm² and a power of 0.353 w. All rats were divided into 4 groups of 6–7 animals each: intact control, PS + PDT, PS + CRT, PS + CRT + PDT. The criteria for evaluating antitumor efficacy were: the average volume of tumors (Vav), the coefficient of absolute growth of tumors (K), the coefficient of tumor growth inhibition (TGI), the frequency of complete tumor regressions (CR), the proportion of cured rats, an increase in the average duration of dead rats. Differences were considered statistically significant at p <0.05.Results. On the 18th day of the experiment, Vav in groups was 65.98 ± 0.81 cm³; 24.02 ± 4.01 cm³ (р <0.00001); 17.11 ± 4.13 cm³ (р <0.00001); 4.42 ± 1.65 cm³ (р <0.00001), respectively. Coefficients K – 5497.33 OE; 1714.71 OE; 1424.83 OE; 367.33 OE. TGI coefficients – 63.60 % (PS + PDT); 74.07 % (PS + CRT); 93.30 % (PS + CRT + PDT). An in crease in the average duration of dead rats indicator – 25.31 % (PS + PDT); 55.06 % (PS + CRT); 158.22 % (PS + CRT + PDT). On the 60th and 90th days of the experiment, the frequency of CR and the proportion of cured rats were the same and amounted to 0; 16.7; 14.3 and 42.9 %, respectively.Conclusion. The results obtained indicate that the chlorine PS has, in addition to photosensitizing properties, also radiomodifying properties, and the developed method of combined therapy is characterized by high antitumor efficacy.

Fractionated radiation exposure enhances the DNA repair capacity to acquire radioresistance in HCT8 human colorectal cancer cells

Fractionated radiation exposure enhances the DNA repair capacity to acquire radioresistance in HCT8 human colorectal cancer cells

Effects of Fractionated Radiation Exposure on Vimentin Expression in Cervical Cancers: Analysis of Association with Cancer Stem Cell Response and Short-Term Prognosis

Elucidation of the mechanisms for the response of cancer stem cells (CSCs) to radiation exposure is of considerable interest for further improvement of radio- and chemoradiotherapy of cervical cancer (CC). The aim of this work is to evaluate the effects of fractionated radiation exposure on the expression of vimentin, which is one of the end-stage markers of epithelial-mesenchymal transition (EMT), and analyze its association with CSC radiation response and short-term prognosis of CC patients. The level of vimentin expression was determined in HeLa, SiHa cell lines, and scrapings from the cervix of 46 CC patients before treatment and after irradiation at a total dose of 10 Gy using real-time polymerase chain reaction (PCR) assay, flow cytometry, and fluorescence microscopy. The number of CSCs was assessed using flow cytometry. Significant correlations were shown between vimentin expression and postradiation changes in CSC numbers in both cell lines (R = 0.88, p = 0.04 for HeLa and R = 0.91, p = 0.01 for SiHa) and cervical scrapings (R = 0.45, p = 0.008). Associations were found at the level of tendency between postradiation increase in vimentin expression and unfavorable clinical outcome 3-6 months after treatment. The results clarify some of the relationships between EMT, CSCs, and therapeutic resistance that are needed to develop new strategies for cancer treatment.

Findings from international archived data: Fractionation reduces mortality risk of ionizing radiation for total doses below 4 Gray in rodents.

Ionizing radiation is omnipresent and unavoidable on Earth; nevertheless, the range of doses and modes of radiation delivery that represent health risks remain controversial. Radiation protection policy for civilians in US is set at 1 mSv per year. Average persons from contemporary populations are exposed to several hundred milliSieverts (mSv) over their lifetimes from both natural and human made sources such as radon, cosmic rays, CT-scans (20-50 mSv partial body exposure per scan), etc. Health risks associated with these and larger exposures are focus of many epidemiological studies, but uncertainties of these estimates coupled with individual and environmental variation make it is prudent to attempt to use animal models and tightly controlled experimental conditions to supplement our evaluation of radiation risk question. Data on 11,528 of rodents of both genders exposed to x-ray or gamma-ray radiation in facilities in US and Europe were used for this analysis; animal mortality data argue that fractionated radiation exposures have about 2 fold less risk per Gray than acute radiation exposures in the range of doses between 0.25 and 4Gy.

TGF-β/Akt/Smad signaling regulates ionizing radiation-induced epithelial-mesenchymal transition in acquired radioresistant lung cancer cells

ObjectiveTo define the properties of lung cancer cells that resisted conventionally fractionated radiation exposure. MethodsAcquired radioresistant lung cancer cell line A549 was constructed by X-ray irradiation with a clinical conventional fraction dose of 2 ​Gy daily during 30 fractions. Cell morphology, molecular markers, migration capacity and invasion potential were evaluated by the microscope, Western blot, immunofluorescence, wound healing test and transwell chamber assay, respectively. ResultsRadioresistant A549 ​cells shifted from an epithelial to a mesenchymal morphology, termed as epithelial-mesenchymal transition (EMT), and was accompanied by decreased expressions of epithelial markers (F ​= ​4.568, P ​< ​0.05) and increased expression of mesenchymal markers (F ​= ​4.270, P ​< ​0.05), greater migratory and invasive capabilities (t ​= ​6.386, 5.644, P ​< ​0.05). The expression of TGF-β, and phosphorylated levels of Akt and Smad3 were also enhanced (F ​= ​6.496, 4.685, 3.370, P ​< ​0.05). Furthermore, the EMT phenotype induced by radiation could be reversed through inhibition of TGF-β, Akt or Smad3, indicating a functional relationship between them. ConclusionsEMT mediates acquired radioresistance of lung cancer cells induced by IR with clinical parameters, and the crosstalk mode of TGF-β/Akt/Smad signaling plays a critical regulatory role in this process.

Updated Standardized Mortality Ratio Evaluation of Disease Risks of Shipyard Workers Exposed to Low Dose Ionizing Radiation.

To examine the risk of diseases among industrial workers with low and fractionated radiation exposures. The 372,047 US male shipyard radiation and non-radiation workers were followed for 54 years and compared with US men using standardized mortality ratio (SMR) method. SMRs for both radiation and non-radiation workers had lower risks of death from all causes (0.74; 95% confidence interval [CI] 0.74 to 0.75 and 0.77; 95% Cl 0.77 to 0.78, respectively) and from all cancers (0.92; 95% CI 0.91 to 0.93 and 0.90; 95% CI 0.89 to 0.91, respectively) compared with US men. Asbestos-related diseases including pleural cancers, mesothelioma, and asbestosis, but not lung cancers, were statistically higher in both radiation and non-radiation workers compared with the US men. US shipyard male radiation and non-radiation workers did not show any elevated mortality risks that might be associated with radiation exposure.

Chromosome aberration dynamics in breast cancer patients treated with radiotherapy: Implications for radiation biodosimetry

Although radiological accidents often result in partial-body radiation exposure, most biodosimetry studies focus on estimating whole-body exposure doses. We have evaluated time-dependent changes in chromosomal aberrations before, during, and after localized fractionated radiotherapy. Twelve patients with carcinoma in situ of the breast who underwent identical adjuvant radiation therapy (50 Gy in 25 fractions) were included in the study. Lymphocytes were collected from patients before, during, and after radiotherapy, to measure chromosome aberrations, such as dicentric chromosomes and translocations. Chromosome aberrations were then used to calculate whole- and partial-body biological absorbed doses of radiation. Dicentric chromosome frequencies in all study participants increased during radiotherapy (p < 0.05 in Kruskal-Wallis test). Increases of translocation frequencies during radiotherapy were observed in seven of the twelve patients. The increased levels of dicentric chromosomes and translocations persisted throughout our 1-year follow-up, and evidence of partial-body exposure (such as Papworth’s U-value > 1.96) was observed more than 1 year after radiotherapy. We found that cytogenetic biomarkers reflected partial-body fractionated radiation exposure more than 1 year post-exposure. Our findings suggest that chromosome aberrations can be used to estimate biological absorbed radiation doses and can inform medical intervention for individuals suspected of fractionated or partial-body radiation exposure.

Alpha Radiation as a Way to Target Heterochromatic and Gamma Radiation-Exposed Breast Cancer Cells.

Compact chromatin is linked to a poor tumour prognosis and resistance to radiotherapy from photons. We investigated DNA damage induction and repair in the context of chromatin structure for densely ionising alpha radiation as well as its therapeutic potential. Chromatin opening by histone deacetylase inhibitor trichostatin A (TSA) pretreatment reduced clonogenic survival and increased γH2AX foci in MDA-MB-231 cells, indicative of increased damage induction by free radicals using gamma radiation. In contrast, TSA pretreatment tended to improve survival after alpha radiation while γH2AX foci were similar or lower; therefore, an increased DNA repair is suggested due to increased access of repair proteins. MDA-MB-231 cells exposed to fractionated gamma radiation (2 Gy × 6) expressed high levels of stem cell markers, elevated heterochromatin H3K9me3 marker, and a trend towards reduced clonogenic survival in response to alpha radiation. There was a higher level of H3K9me3 at baseline, and the ratio of DNA damage induced by alpha vs. gamma radiation was higher in the aggressive MDA-MB-231 cells compared to hormone receptor-positive MCF7 cells. We demonstrate that heterochromatin structure and stemness properties are induced by fractionated radiation exposure. Gamma radiation-exposed cells may be targeted using alpha radiation, and we provide a mechanistic basis for the involvement of chromatin in these effects.

The Disparity of Impairment of Neurogenesis and Cognition After Acute or Fractionated Radiation Exposure in Adolescent BALB/c Mice.

The effect of acute X-ray irradiation with 2 Gy or fractionated exposure with 0.2 Gy continuously for 10 days (0.2 Gy × 10 = 2 Gy) was evaluated in the postnatal day 21 (P21) BALB/c mouse model. Both acute and fractionated irradiation induced impairment of cell proliferation and neurogenesis in the subgranular zone of the dentate gyrus labeled by Ki67 and doublecortin, respectively. Parvalbumin immunopositive interneurons in the subgranular zone were also reduced significantly. However, the 2 patterns of irradiation did not affect animal weight gain when measured at ages of P90 and P180 or 69 and 159 days after irradiation. Behavioral tests indicated that neither acute nor fractionated irradiation with a total dose of 2 Gy induced deficits in the contextual fear or spatial memory and memory for novel object recognition. Animal motor activity was also not affected in the open-field test. The disparity of the impairment of neurogenesis and unaffected cognition suggests that the severity of impairment of neurogenesis induced by acute or fractionated irradiation with a total dose of 2 Gy at P21 may not be worse enough to induce the deficit of cognition.

Fractionated radiation exposure amplifies the radioresistant nature of prostate cancer cells.

The risk of recurrence following radiation therapy remains high for a significant number of prostate cancer patients. The development of in vitro isogenic models of radioresistance through exposure to fractionated radiation is an increasingly used approach to investigate the mechanisms of radioresistance in cancer cells and help guide improvements in radiotherapy standards. We treated 22Rv1 prostate cancer cells with fractionated 2 Gy radiation to a cumulative total dose of 60 Gy. This process selected for 22Rv1-cells with increased clonogenic survival following subsequent radiation exposure but increased sensitivity to Docetaxel. This RR-22Rv1 cell line was enriched in S-phase cells, less susceptible to DNA damage, radiation-induced apoptosis and acquired enhanced migration potential, when compared to wild type and aged matched control 22Rv1 cells. The selection of radioresistant cancer cells during fractionated radiation therapy may have implications in the development and administration of future targeted therapy in conjunction with radiation therapy.

γH2AX formation kinetics in PBMCs of rabbits exposed to acute and fractionated radiation and attenuation of focus frequency through preadministration of a combination of podophyllotoxin and rutin hydrate

DNA damage can be assessed by the quantitation of γH2AX foci that form at DSB sites. This study examines the generation and persistence of γH2AX foci, variability in foci size after acute and fractionated radiation exposure, and the effect of pretreatment with a safe radioprotective formulation termed G-003M on foci generation and persistence. G-003M contains a combination of podophyllotoxin and rutin hydrate, and was administered intramuscularly to rabbits 1 hr prior to Co(60) gamma irradiation. Rabbits were assigned to one of the following treatment groups: untreated, G-003M alone, irradiated (single dose 8 Gy, fractionated 2 Gy/day for 4 days or single dose 2 Gy) or G-003M preadministration followed by radiation exposure. Foci continuously persisted for a week in peripheral blood mononuclear cells of rabbits exposed to a single 8 Gy dose. However, the number of foci gradually decreased after reaching a maximum at 1 h. In rabbits exposed to fractionated radiation, foci detected 1 hr after the final exposure were significantly larger (P < 0.001) than in rabbits exposed to a single 8 Gy dose, but disappeared completely after 24 h. In both groups, foci reappeared on days 11-15 in terminally ill animals. G-003M pretreatment significantly (P < 0.05) attenuated the formation of γH2AX foci in all irradiated rabbits. This study reveals that γH2AX focus assessment could be used to confirm radiation exposure, that focus size reflects the type of radiation exposure (acute or fractionated), that the re-appearance of foci is a strong indicator of imminent death in animals, and that G-003M provides protection against radiation. Environ. Mol. Mutagen. 57:455-468, 2016. © 2016 Wiley Periodicals, Inc.

Fractionated Radiation Exposure of Rat Spinal Cords Leads to Latent Neuro-Inflammation in Brain, Cognitive Deficits, and Alterations in Apurinic Endonuclease 1.

Ionizing radiation causes degeneration of myelin, the insulating sheaths of neuronal axons, leading to neurological impairment. As radiation research on the central nervous system has predominantly focused on neurons, with few studies addressing the role of glial cells, we have focused our present research on identifying the latent effects of single/ fractionated -low dose of low/ high energy radiation on the role of base excision repair protein Apurinic Endonuclease-1, in the rat spinal cords oligodendrocyte progenitor cells’ differentiation. Apurinic endonuclease-1 is predominantly upregulated in response to oxidative stress by low- energy radiation, and previous studies show significant induction of Apurinic Endonuclease-1 in neurons and astrocytes. Our studies show for the first time, that fractionation of protons cause latent damage to spinal cord architecture while fractionation of HZE (28Si) induce increase in APE1 with single dose, which then decreased with fractionation. The oligodendrocyte progenitor cells differentiation was skewed with increase in immature oligodendrocytes and astrocytes, which likely cause the observed decrease in white matter, increased neuro-inflammation, together leading to the observed significant cognitive defects.

Tumor Induction in Mice After Localized Single- or Fractionated-Dose Irradiation: Differences in Tumor Histotype and Genetic Susceptibility Based on Dose Scheduling

To investigate differences in tumor histotype, incidence, latency, and strain susceptibility in mice exposed to single-dose or clinically relevant, fractioned-dose γ-ray radiation. C3Hf/Kam and C57BL/6J mice were locally irradiated to the right hindlimb with either single large doses between 10 and 70 Gy or fractionated doses totaling 40 to 80 Gy delivered at 2-Gy/d fractions, 5 d/wk, for 4 to 8 weeks. The mice were closely evaluated for tumor development in the irradiated field for 800 days after irradiation, and all tumors were characterized histologically. A total of 210 tumors were induced within the radiation field in 788 mice. An overall decrease in tumor incidence was observed after fractionated irradiation (16.4%) in comparison with single-dose irradiation (36.1%). Sarcomas were the predominant postirradiation tumor observed (n=201), with carcinomas occurring less frequently (n=9). The proportion of mice developing tumors increased significantly with total dose for both single-dose and fractionated schedules, and latencies were significantly decreased in mice exposed to larger total doses. C3Hf/Kam mice were more susceptible to tumor induction than C57BL/6J mice after single-dose irradiation; however, significant differences in tumor susceptibilities after fractionated radiation were not observed. For both strains of mice, osteosarcomas and hemangiosarcomas were significantly more common after fractionated irradiation, whereas fibrosarcomas and malignant fibrous histiocytomas were significantly more common after single-dose irradiation. This study investigated the tumorigenic effect of acute large doses in comparison with fractionated radiation in which both the dose and delivery schedule were similar to those used in clinical radiation therapy. Differences in tumor histotype after single-dose or fractionated radiation exposures provide novel in vivo evidence for differences in tumor susceptibility among stromal cell populations.

MicroRNA expression profiling altered by variant dosage of radiation exposure.

Various biological effects are associated with radiation exposure. Irradiated cells may elevate the risk for genetic instability, mutation, and cancer under low levels of radiation exposure, in addition to being able to extend the postradiation side effects in normal tissues. Radiation-induced bystander effect (RIBE) is the focus of rigorous research as it may promote the development of cancer even at low radiation doses. Alterations in the DNA sequence could not explain these biological effects of radiation and it is thought that epigenetics factors may be involved. Indeed, some microRNAs (or miRNAs) have been found to correlate radiation-induced damages and may be potential biomarkers for the various biological effects caused by different levels of radiation exposure. However, the regulatory role that miRNA plays in this aspect remains elusive. In this study, we profiled the expression changes in miRNA under fractionated radiation exposure in human peripheral blood mononuclear cells. By utilizing publicly available microRNA knowledge bases and performing cross validations with our previous gene expression profiling under the same radiation condition, we identified various miRNA-gene interactions specific to different doses of radiation treatment, providing new insights for the molecular underpinnings of radiation injury.

Human epidermal keratinocytes death and expression of protein markers of apoptosis after ionizing radiation exposure

Purpose: Knowledge of the pathophysiology of the irradiated skin is important to understand the tolerance and cosmetic response of the human skin to radiation. There are limited studies on the effect of radiotherapy dosage and fraction size in inducing apoptotic cell death in human skin. The expression of apoptotic biomarkers within a controlled population in different fractionation schemes has also never been studied. This study aims to investigate radiation induced apoptotic cell death in human skin cells after fractionated radiation exposure and the expression of unique biomarkers that reflect cell death or biology using multiplexed immunoassays. Methods : Breast skin biopsies were obtained from a single individual and divided into small pieces. Each piece was irradiated under different radiotherapy treatment fractionation schedules to a total dose of 50Gy. The irradiated skin tissues were analysed using Tunnel, immunohistochemistry and Western blot assays for expression of apoptotic keratinocytes and biomarkers (p53, p21, and PCNA). Haematoxylin and eosin (H&amp;E) immunostaining was performed to study the morphological changes in the skin cells. Results: Radiation is mostly absorbed by the epidermal layers and observed to damage the epidermal keratinocytes leading to the activation of apoptotic proteins. Apoptotic proteins (p53, p21 and PCNA) were confirmed to be up-regulated in radiation exposed skin cells as compared to normal skin cells with no radiation. There is strong correlation of apoptotic protein expressions with increased radiation dosage and dose fractionation. Statistical analysis with ANOVA revealed a significant increase of PCNA and p21 expression with increased radiation dosage and dose fractionation (p &lt; 0.05). Immunohistochemically, 14 % (range 10.71% to 17.29%) of the keratinocytes were positive for PCNA and 22.5% (range 18.28% to 27.2%) for p21 after 2Gy of irradiation. The most widespread, intense and uniform staining for PCNA and p21 was observed in skin that had received 50Gy of irradiation. The maximum expression of p53 (range 37.09% to 50.91%) was reached at 10Gy. Conclusion : Findings from this study will assist clinicians in predicting radiation induced skin toxicity with the current changes in radiation fractionation protocols. ----------------------------------- Cite this article as: Wong S, Chor HH, Moothy S, Ong CT, Phan TT, Lu JJ. Human epidermal keratinocytes death and expression of protein markers of apoptosis after ionizing radiation exposure. Int J Cancer Ther Oncol 2013; 1 (2):01027. DOI : http://dx.doi.org/10.14319/ijcto.0102.7

Human epidermal keratinocytes death and expression of protein markers of apoptosis after ionizing radiation exposure

Purpose: Knowledge of the pathophysiology of the irradiated skin is important to understand the tolerance and cosmetic response of the human skin to radiation. There are limited studies on the effect of radiotherapy dosage and fraction size in inducing apoptotic cell death in human skin. The expression of apoptotic biomarkers within a controlled population in different fractionation schemes has also never been studied. This study aims to investigate radiation induced apoptotic cell death in human skin cells after fractionated radiation exposure and the expression of unique biomarkers that reflect cell death or biology using multiplexed immunoassays. Methods : Breast skin biopsies were obtained from a single individual and divided into small pieces. Each piece was irradiated under different radiotherapy treatment fractionation schedules to a total dose of 50Gy. The irradiated skin tissues were analysed using Tunnel, immunohistochemistry and Western blot assays for expression of apoptotic keratinocytes and biomarkers (p53, p21, and PCNA). Haematoxylin and eosin (HE 1 (2):01027. DOI : http://dx.doi.org/10.14319/ijcto.0102.7

Sarcoma risk after radiation exposure

Sarcomas were one of the first solid cancers to be linked to ionizing radiation exposure. We reviewed the current evidence on this relationship, focusing particularly on the studies that had individual estimates of radiation doses. There is clear evidence of an increased risk of both bone and soft tissue sarcomas after high-dose fractionated radiation exposure (10 + Gy) in childhood, and the risk increases approximately linearly in dose, at least up to 40 Gy. There are few studies available of sarcoma after radiotherapy in adulthood for cancer, but data from cancer registries and studies of treatment for benign conditions confirm that the risk of sarcoma is also increased in this age-group after fractionated high-dose exposure. New findings from the long-term follow-up of the Japanese atomic bomb survivors suggest, for the first time, that sarcomas can be induced by acute lower-doses of radiation (<5 Gy) at any age, and the magnitude of the risk is similar to that observed for other solid cancers. While there is evidence that individuals with certain rare familial genetic syndromes predisposing to sarcoma, particularly Nijmegen Breakage Syndrome, are particularly sensitive to the effects of high dose radiation, it is unclear whether this is also true in very low-dose settings (<0.1 Gy). The effects of common low-penetrance alleles on radiosensitivity in the general population have not been well-characterized. Some evidence suggests that it may be possible to identify radiation-induced sarcomas by a distinct molecular signature, but this work needs to be replicated in several dose settings, and the potential role of chemotherapy and tumor heterogeneity needs to be examined in more detail. In summary, radiation exposure remains one of the few established risk factors for both bone and soft tissue sarcomas. Similar to many other cancers children have the highest risks of developing a radiation-related sarcoma. Efforts to limit unnecessary high-dose radiation exposure, particularly in children, therefore remain important given the high fatality rates associated with this disease.

TGFβ1 Inhibition Increases the Radiosensitivity of Breast Cancer CellsIn Vitroand Promotes Tumor Control by RadiationIn Vivo

To determine whether inhibition of TGFβ signaling prior to irradiation sensitizes human and murine cancer cells in vitro and in vivo. TGFβ-mediated growth and Smad phosphorylation of MCF7, Hs578T, MDA-MB-231, and T47D human breast cancer cell lines were examined and correlated with clonogenic survival following graded radiation doses with and without pretreatment with LY364947, a small molecule inhibitor of the TGFβ type I receptor kinase. The DNA damage response was assessed in irradiated MDA-MB-231 cells pretreated with LY364947 in vitro and LY2109761, a pharmacokinetically stable inhibitor of TGFβ signaling, in vivo. The in vitro response of a syngeneic murine tumor, 4T1, was tested using a TGFβ neutralizing antibody, 1D11, with single or fractionated radiation doses in vivo. Human breast cancer cell lines pretreated with TGFβ small molecule inhibitor were radiosensitized, irrespective of sensitivity to TGFβ growth inhibition. Consistent with increased clonogenic cell death, radiation-induced phosphorylation of H2AX and p53 was significantly reduced in MDA-MB-231 triple-negative breast cancer cells when pretreated in vitro or in vivo with a TGFβ type I receptor kinase inhibitor. Moreover, TGFβ neutralizing antibodies increased radiation sensitivity, blocked γH2AX foci formation, and significantly increased tumor growth delay in 4T1 murine mammary tumors in response to single and fractionated radiation exposures. These results show that TGFβ inhibition prior to radiation attenuated DNA damage responses, increased clonogenic cell death, and promoted tumor growth delay, and thus may be an effective adjunct in cancer radiotherapy.

Abstract 2721: Comparative responses of HPV-positive vs HPV-negative head and neck cancer cells to radiation and/or chemotherapeutic drugs

Abstract Purpose: HPV is associated with a growing proportion of head and neck squamous cell carcinomas (HNSCCs), which show improved clinical outcome compared with non-HPV associated HNSCC. Whether this improved outcome reflects enhanced responsiveness to radiation and/or chemotherapy remains unclear. There is little data available regarding the radio- and chemo-sensitivity of HPV-associated HNSCC. We provide detailed analysis of radiation response profiles in verified HPV-positive HNSCC lines. Methods: Degenerate PCR and Southern blot were used to confirm HPV status in HNSCC human tumor cell lines. Clonogenic survival assays were used to assess radiation sensitivity. Proliferation assays were used to assess sensitivity to chemotherapy. Western blot was used to assess EGFR expression, activity, and the effect of EGFR inhibitor treatment. Results: Clonal expansion of existing HPV-positive HNSCC cell lines was performed to confirm the presence of a homogeneous population of cells and HPV-integration status confirmed by Southern Blot. HPV-positive cells were more sensitive to radiation-induced cell death compared to HPV-negative cells using single dose and fractionated radiation exposure regimens. Of the HPV+ cells tested to date, we have seen further radiosensitization with exposure to EGFR inhibitors (e.g., enhancement ratio 1.27 for UM-SCC47 with erlotinib) despite similar response to the EGFR inhibitor alone. HPV+ tumor xenografts have been raised in immunocompromised mice for in vivo radiation and chemotherapy response assessment. Conclusions: Consistent with clinical reports, HPV-associated HNSCC cells exhibit increased sensitivity to radiation. The in vitro sensitivity of HPV-positive HNSCC to chemotherapy is in progress, as is the response evaluation of HPV+ xenografts to radiation and chemotherapy. Underlying molecular mechanisms for altered response profiles in HPV+ HNSCC are considered as they pertain to future clinical trial designs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2721. doi:10.1158/1538-7445.AM2011-2721

The Modified High-Density Survival Assay is the Useful Tool to Predict the Effectiveness of Fractionated Radiation Exposure

The high-density survival (HDS) assay was originally elaborated to assess cancer cell responses to therapeutic agents under the influence of intercellular communication. Here, we simplified the original HDS assay and studied its applicability for the detection of cellular radioresistance. We have recently defined clinically relevant radioresistant (CRR) cells, which continue to proliferate with daily exposure to 2 gray (Gy) of X-rays for more than 30 days in vitro. We established human CRR cell lines, HepG2-8960-R from HepG2, and SAS-R1 and -R2 from SAS, respectively. In an attempt to apply the HDS assay to detect radioresistance with clinical relevance, we simplified the original HDS assay by scoring the total number of surviving cells after exposure to X-rays. The modified HDS assay successfully detected radioresistance with clinical relevance. The modified HDS assay detected CRR phenotype, which is not always detectable by clonogenic assay. Therefore, we believe that the modified HDS assay presented in this study is a powerful tool to predict the effectiveness of fractionated radiotherapy against malignant tumors.