Synchrotron X-Ray Radiation-Induced Bystander Effect: An Impact of the Scattered Radiation, Distance From the Irradiated Site and p53 Cell Status.

Synchrotron radiation, especially microbeam radiotherapy (MRT), has a great potential to improve cancer radiotherapy, but non-targeted effects of synchrotron radiation have not yet been sufficiently explored. We have previously demonstrated that scattered synchrotron radiation induces measurable γ-H2AX foci, a biomarker of DNA double-strand breaks, at biologically relevant distances from the irradiated field that could contribute to the apparent accumulation of bystander DNA damage detected in cells and tissues outside of the irradiated area. Here, we quantified an impact of scattered radiation to DNA damage response in "naïve" cells sharing the medium with the cells that were exposed to synchrotron radiation. To understand the effect of genetic alterations in naïve cells, we utilised p53-null and p53-wild-type human colon cancer cells HCT116. The cells were grown in two-well chamber slides, with only one of nine zones (of equal area) of one well irradiated with broad beam or MRT. γ-H2AX foci per cell values induced by scattered radiation in selected zones of the unirradiated well were compared to the commensurate values from selected zones in the irradiated well, with matching distances from the irradiated zone. Scattered radiation highly impacted the DNA damage response in both wells and a pronounced distance-independent bystander DNA damage was generated by broad-beam irradiations, while MRT-generated bystander response was negligible. For p53-null cells, a trend for a reduced response to scattered irradiation was observed, but not to bystander signalling. These results will be taken into account for the assessment of genotoxic effects in surrounding non-targeted tissues in preclinical experiments designed to optimise conditions for clinical MRT and for cancer treatment in patients.

Abscopal Gene Expression in Response to Synchrotron Radiation Indicates a Role for Immunological and DNA Damage Response Genes.

Abscopal effects are an important aspect of targeted radiation therapy due to their implication in normal tissue toxicity from chronic inflammatory responses and mutagenesis. Gene expression can be used to determine abscopal effects at the molecular level. Synchrotron microbeam radiation therapy utilizing high-intensity X rays collimated into planar microbeams is a promising cancer treatment due to its reported ability to ablate tumors with less damage to normal tissues compared to conventional broadbeam radiation therapy techniques. The low scatter of synchrotron radiation enables microbeams to be delivered to tissue effectively, and is also advantageous for out-of-field studies because there is minimal interference from scatter. Mouse legs were irradiated at a dose rate of 49 Gy/s and skin samples in the out-of-field areas were collected. The out-of-field skin showed an increase in Tnf expression and a decrease in Mdm2 expression, genes associated with inflammation and DNA damage. These expression effects from microbeam exposure were similar to those found with broadbeam exposure. In immune-deficient Ccl2 knockout mice, we identified a different gene expression profile which showed an early increase in Mdm2, Tgfb1, Tnf and Ccl22 expression in out-of-field skin that was not observed in the immune-proficient mice. Our results suggest that the innate immune system is involved in out-of-field tissue responses and alterations in the immune response may not eliminate abscopal effects, but could change them.

A Functional Immune System Is Required for the Systemic Genotoxic Effects of Localized Irradiation.

PURPOSE: Nontargeted effects of ionizing radiation, by which unirradiated cells and tissues are also damaged, are a relatively new paradigm in radiobiology. We recently reported radiation-induced abscopal effects (RIAEs) in normal tissues; namely, DNA damage, apoptosis, and activation of the local and systemic immune responses in C57BL6/J mice after irradiation of a small region of the body. High-dose-rate, synchrotron-generated broad beam or multiplanar x-ray microbeam radiation therapy was used with various field sizes and doses. This study explores components of the immune system involved in the generation of these abscopal effects. METHODS AND MATERIALS: The following mice with various immune deficiencies were irradiated with the microbeam radiation therapy beam: (1) SCID/IL2γR-/- (NOD SCID gamma, NSG) mice, (2) wild-type C57BL6/J mice treated with an antibody-blocking macrophage colony-stimulating factor 1 receptor, which depletes and alters the function of macrophages, and (3) chemokine ligand 2/monocyte chemotactic protein 1 null mice. Complex DNA damage (ie, DNA double-strand breaks), oxidatively induced clustered DNA lesions, and apoptotic cells in tissues distant from the irradiation site were measured as RIAE endpoints and compared with those in wild-type C57BL6/J mice. RESULTS: Wild-type mice accumulated double-strand breaks, oxidatively induced clustered DNA lesions, and apoptosis, enforcing our RIAE model. However, these effects were completely or partially abrogated in mice with immune disruption, highlighting the pivotal role of the immune system in propagation of systemic genotoxic effects after localized irradiation. CONCLUSIONS: These results underline the importance of not only delineating the best strategies for tumor control but also mitigating systemic radiation toxicity.

Localized Synchrotron Irradiation of Mouse Skin Induces Persistent Systemic Genotoxic and Immune Responses.

The importance of nontargeted (systemic) effects of ionizing radiation is attracting increasing attention. Exploiting synchrotron radiation generated by the Imaging and Medical Beamline at the Australian Synchrotron, we studied radiation-induced nontargeted effects in C57BL/6 mice. Mice were locally irradiated with a synchrotron X-ray broad beam and a multiplanar microbeam radiotherapy beam. To assess the influence of the beam configurations and variations in peak dose and irradiated area in the response of normal tissues outside the irradiated field at 1 and 4 days after irradiation, we monitored oxidatively induced clustered DNA lesions (OCDL), DNA double-strand breaks (DSB), apoptosis, and the local and systemic immune responses. All radiation settings induced pronounced persistent systemic effects in mice, which resulted from even short exposures of a small irradiated area. OCDLs were elevated in a wide variety of unirradiated normal tissues. In out-of-field duodenum, there was a trend for elevated apoptotic cell death under most irradiation conditions; however, DSBs were elevated only after exposure to lower doses. These genotoxic events were accompanied by changes in plasma concentrations of macrophage-derived cytokine, eotaxin, IL10, TIMP1, VEGF, TGFß1, and TGFß2, along with changes in tissues in frequencies of macrophages, neutrophils, and T lymphocytes. Overall, our findings have implications for the planning of therapeutic and diagnostic radiation treatments to reduce the risk of radiation-related adverse systemic effects. Cancer Res; 77(22); 6389-99. ©2017 AACR.

N-cadherin identifies human endometrial epithelial progenitor cells by in vitro stem cell assays.

STUDY QUESTION: Is there a specific surface marker that identifies human endometrial epithelial progenitor cells with adult stem cell activity using in vitro assays? SUMMARY ANSWER: N-cadherin isolates clonogenic, self-renewing human endometrial epithelial progenitor cells with high proliferative potential that differentiate into cytokeratin+ gland-like structures in vitro and identifies their location in some cells of gland profiles predominantly in basalis endometrium adjacent to the myometrium. WHAT IS KNOWN ALREADY: Human endometrium contains a small population of clonogenic, self-renewing epithelial cells with high proliferative potential that differentiate into large gland-like structures, but their identity and location is unknown. Stage-specific embryonic antigen-1 (SSEA-1) distinguishes the epithelium of basalis from functionalis and is a marker of human post-menopausal (Post-M) endometrial epithelium. STUDY DESIGN, SIZE, DURATION: Prospective observational study of endometrial epithelial cells obtained from hysterectomy samples taken from 50 pre-menopausal (Pre-M) and 24 Post-M women, of which 4 were from women who had taken daily estradiol valerate 2 mg/day for 8 weeks prior. PARTICIPANTS/MATERIALS, SETTING, METHODS: Gene profiling was used to identify differentially expressed surface markers between fresh EpCAM (Epithelial Cell Adhesion Molecule)-magnetic bead-selected basalis-like epithelial cells from Post-M endometrium compared with predominantly functionalis epithelial cells from Pre-M endometrium and validated by qRT-PCR. In vitro clonogenicity and self-renewal assays were used to assess the stem/progenitor cell properties of magnetic bead-sorted N-cadherin+ and N-cadherin- epithelial cells. The cellular identity, location and phenotype of N-cadherin+ cells was assessed by dual colour immunofluorescence and confocal microscopy for cytokeratin, proliferative status (Ki-67), ERα, SSEA-1, SOX9 and epithelial mesenchymal transition (EMT) markers on full thickness human endometrium. MAIN RESULTS AND THE ROLE OF CHANCE: CDH2 (N-cadherin gene) was one of 11 surface molecules highly expressed in Post-M compared to Pre-M endometrial epithelial cells. N-cadherin+ cells comprise a median 16.7% (n = 8) and 20.2% (n = 5) of Pre-M endometrial epithelial cells by flow cytometry and magnetic bead sorting, respectively. N-cadherin+ epithelial cells from Pre-M endometrium were more clonogenic than N-cadherin- cells (n = 12, P = 0.003), underwent more population doublings (n = 7), showed greater capacity for serial cloning (n = 7) and differentiated into cytokeratin+ gland-like organoids. N-cadherin immunolocalised to the lateral and apical membrane of epithelial cells in the bases of glands in the basalis of Pre-M endometrium and Post-M gland profiles, co-expressing cytokeratin, ERα but not SSEA-1 or SOX9, which localized on gland profiles proximal to N-cadherin+ cells. N-cadherin+ cells were quiescent (Ki-67-) in the basalis and in Post-M endometrial glands and co-localized with EMT markers vimentin and E-cadherin. LARGE SCALE DATA: The raw and processed data files from the gene microarray have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus data set with accession number GSE35221. LIMITATIONS, REASONS FOR CAUTION: This is a descriptive study in human endometrium only using in vitro stem cell assays. The differential ability of N-cadherin+ and N-cadherin-cells to generate endometrial glands in vivo was not determined. A small number of uterine tissues analysed contained adenomyosis for which N-cadherin has been implicated in epithelial-EMT. WIDER IMPLICATIONS OF THE FINDINGS: A new marker enriching for human endometrial epithelial progenitor cells identifies a different and potentially more primitive cell population than SSEA-1, suggesting a potential hierarchy of epithelial differentiation in the basalis. Using N-cadherin as a marker, the molecular and cellular characteristics of epithelial progenitor cells and their role in endometrial proliferative disorders including endometriosis, adenomyosis and thin dysfunctional endometrium can be investigated. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by Cancer Council Victoria grant 491079 (C.E.G.) and Australian National Health and Medical Research Council grants 1021127 (C.E.G.), 1085435 (C.E.G., J.A.D.), 145780 and 288713 (C.N.S.), RD Wright Career Development Award 465121 (C.E.G.), Senior Research Fellowship 1042298 (C.E.G.), the Victorian Government's Operational Infrastructure Support and an Australian Postgraduate Award (HPTN), and China Council Scholarship (L.X.). The authors have nothing to declare.

Follistatin attenuates radiation-induced fibrosis in a murine model.

PURPOSE: Fibrosis can be a disabling, severe side effect of radiotherapy that can occur in patients, and for which there is currently no effective treatment. The activins, proteins which are members of the TGFß superfamily, have a major role in stimulating the inflammatory response and subsequent fibrosis. Follistatin is an endogenous protein that binds the activins virtually irreversibly and inhibits their actions. These studies test if follistatin can attenuate the fibrotic response using a murine model of radiation-induced fibrosis. EXPERIMENTAL DESIGN: C57BL/6 mice were subcutaneously injected with follistatin 24 hours prior to irradiation. Mice were irradiated in a 10 x 10 mm square area of the right hind leg with 35 Gy and were given follistatin 24 hours before radiation and three times a week for six months following. Leg extension was measured, and tissue was collected for histological and molecular analysis to evaluate the progression of the radiation-induced fibrosis. RESULTS: Leg extension was improved in follistatin treated mice compared to vehicle treated mice at six months after irradiation. Also, epidermal thickness and cell nucleus area of keratinocytes were decreased by the follistatin treatment compared to the cells in irradiated skin of control mice. Finally, the gene expression of transforming growth factor ß1 (Tgfb1), and smooth muscle actin (Acta2) were decreased in the irradiated skin and Acta2 and inhibin ßA subunit (Inhba) were decreased in the irradiated muscle of the follistatin treated mice. CONCLUSIONS: Follistatin attenuated the radiation-induced fibrotic response in irradiated mice. These studies provide the data to support further investigation of the use of follistatin to reduce radiation-induced fibrosis in patients undergoing radiotherapy for cancer.

Potential strategies to ameliorate risk of radiotherapy-induced second malignant neoplasms.

This review is aimed at the issue of radiation-induced second malignant neoplasms (SMN), which has become an important problem with the increasing success of modern cancer radiotherapy (RT). It is imperative to avoid compromising the therapeutic ratio while addressing the challenge of SMN. The dilemma is illustrated by the role of reactive oxygen species in both the mechanisms of tumor cell kill and of radiation-induced carcinogenesis. We explore the literature focusing on three potential routes of amelioration to address this challenge. An obvious approach to avoiding compromise of the tumor response is the use of radioprotectors or mitigators that are selective for normal tissues. We also explore the opportunities to avoid protection of the tumor by topical/regional radioprotection of normal tissues, although this strategy limits the scope of protection. Finally, we explore the role of the bystander/abscopal phenomenon in radiation carcinogenesis, in association with the inflammatory response. Targeted and non-targeted effects of radiation are both linked to SMN through induction of DNA damage, genome instability and mutagenesis, but differences in the mechanisms and kinetics between targeted and non-targeted effects may provide opportunities to lessen SMN. The agents that could be employed to pursue each of these strategies are briefly reviewed. In many cases, the same agent has potential utility for more than one strategy. Although the parallel problem of chemotherapy-induced SMN shares common features, this review focuses on RT associated SMN. Also, we avoid the burgeoning literature on the endeavor to suppress cancer incidence by use of antioxidants and vitamins either as dietary strategies or supplementation.

Assessment and Implications of Scattered Microbeam and Broadbeam Synchrotron Radiation for Bystander Effect Studies.

Synchrotron radiation is an excellent tool for investigating bystander effects in cell and animal models because of the well-defined and controllable configuration of the beam. Although synchrotron radiation has many advantages for such studies compared to conventional radiation, the contribution of dose exposure from scattered radiation nevertheless remains a source of concern. Therefore, the influence of scattered radiation on the detection of bystander effects induced by synchrotron radiation in biological in vitro models was evaluated. Radiochromic XRQA2 film-based dosimetry was employed to measure the absorbed dose of scattered radiation in cultured cells at various distances from a field exposed to microbeam radiotherapy and broadbeam X-ray radiation. The level of scattered radiation was dependent on the distance, dose in the target zone and beam mode. The number of γ-H2AX foci in cells positioned at the same target distances was measured and used as a biodosimeter to evaluate the absorbed dose. A correlation of absorbed dose values measured by the physical and biological methods was identified. The γ-H2AX assay successfully quantitated the scattered radiation in the range starting from 10 mGy and its contribution to the observed radiation-induced bystander effect.

Immunological markers that predict radiation toxicity.

Radiotherapy is a major modality of cancer treatment responsible for a large proportion of cancer that is cured. Radiation exposure induces an inflammatory response which can be influenced by genetic, epigenetic, tumour, health and other factors which can lead to very different treatment outcomes between individuals. Molecules involved in the immunological response provide excellent potential biomarkers for the prediction of radiation-induced toxicity. The known molecular and cellular immunological responses in relation to radiation and the potential to improve cancer treatment are presented in this review. In particular, immunological biomarkers of radiation-induced fibrosis and pneumonitis in cancer radiotherapy patients are discussed.

Oxidative DNA damage caused by inflammation may link to stress-induced non-targeted effects.

A spectrum of radiation-induced non-targeted effects has been reported during the last two decades since Nagasawa and Little first described a phenomenon in cultured cells that was later called the "bystander effect". These non-targeted effects include radiotherapy-related abscopal effects, where changes in organs or tissues occur distant from the irradiated region. The spectrum of non-targeted effects continue to broaden over time and now embrace many types of exogenous and endogenous stressors that induce a systemic genotoxic response including a widely studied tumor microenvironment. Here we discuss processes and factors leading to DNA damage induction in non-targeted cells and tissues and highlight similarities in the regulation of systemic effects caused by different stressors.

Identification of a radiation sensitivity gene expression profile in primary fibroblasts derived from patients who developed radiotherapy-induced fibrosis.

BACKGROUND AND PURPOSE: During radiotherapy, normal tissue is unavoidably exposed to radiation which results in severe normal tissue reactions in a small fraction of patients. Because those who are sensitive cannot be determined prior to radiotherapy, the doses are limited to all patients to avoid an unacceptable number of severe adverse normal tissue responses. This limitation restricts the optimal treatment for individuals who are more tolerant to radiation. Genetic variation is a likely source for the normal tissue radiosensitivity variation observed between individuals. Therefore, understanding the radiation response at the genomic level may provide knowledge to develop individualized treatment and improve radiotherapy outcomes. MATERIAL AND METHODS: Exon arrays were utilized to compare the basal expression profile between cell lines derived from six cancer patients with and without severe fibrosis. These data were supported by qRT-PCR and RNA-Seq techniques. RESULTS: A set of genes (FBN2, FST, GPRC5B, NOTCH3, PLCB1, DPT, DDIT4L and SGCG) were identified as potential predictors for radiation-induced fibrosis. Many of these genes are associated with TGFß or retinoic acid both having known links to fibrosis. CONCLUSION: A combinatorial gene expression approach provides a promising strategy to predict fibrosis in cancer patients prior to radiotherapy.

Intragenic controls utilizing radiation-induced alternative transcript regions improves gene expression biodosimetry.

Ionizing-radiation exposure can be life threatening if given to the whole body. In addition, whole body radiation exposure can affect large numbers of people such as after a nuclear reactor accident, a nuclear explosion or a radiological terrorist attack. In these cases, an accurate biodosimeter is essential for triage management. One of the problems for biodosimetry in general is the interindividual variation before and after exposure, which can make it challenging to assign an accurate dose. To begin to address this challenge, lymphocyte cell lines were exposed to 0, 1, 2 and 5 Gy ionizing radiation from a ¹³7Cs source at a dose rate of 0.6 Gy/min. Alternative transcripts with regions showing large differential responses to ionizing radiation were determined from exon array data. Gene expression analysis was then performed on isolated mRNA using qRT-PCR with normalization to intergenic (PGK1, GAPDH) and novel intragenic regions for candidate radiation-responsive genes, PPM1D and MDM2. Our studies show that the use of a cis-associated expression reference improved the potential dose prediction approximately 2.3-8.3 fold and provided an advantage for dose prediction compared to distantly or trans-located control ionizing radiation nonresponsive genes. This approach also provides an alternative gene expression normalization method to potentially reduce interindividual variations when untreated basal gene expression levels are unavailable. Using associated noninduced regions of ionizing radiation-induced genes provides a way to estimate basal gene expression in the irradiated sample. This strategy can be utilized as a biodosimeter on its own or to enhance other gene expression candidates for biodosimetry. This normalization strategy may also be generally applicable for other quantitative PCR strategies where normalization is required for a particular response.

Follistatin is induced by ionizing radiation and potentially predictive of radiosensitivity in radiation-induced fibrosis patient derived fibroblasts.

Follistatin is a potent regulator of the inflammatory response and binds to and inhibits activin A action. Activin A is a member of the TGFß protein superfamily which has regulatory roles in the inflammatory response and in the fibrotic process. Fibrosis can occur following cell injury and cell death induced by agents such as ionizing radiation (IR). IR is used to treat cancer and marked fibrotic response is a normal tissue (non-tumour) consequence in a fraction of patients under the current dose regimes. The discovery and development of a therapeutic to abate fibrosis in these radiosensitive patients would be a major advance for cancer radiotherapy. Likewise, prediction of which patients are susceptible to fibrosis would enable individualization of treatment and provide an opportunity for pre-emptive fibrosis control and better tumour treatment outcomes. The levels of activin A and follistatin were measured in fibroblasts derived from patients who developed severe radiation-induced fibrosis following radiotherapy and compared to fibroblasts from patients who did not. Both follistatin and activin A gene expression levels were increased following IR and the follistatin gene expression level was lower in the fibroblasts from fibrosis patients compared to controls at both basal levels and after IR. The major follistatin transcript variants were found to have a similar response to IR and both were reduced in fibrosis patients. Levels of follistatin and activin A secreted in the fibroblast culture medium also increased in response to IR and the relative follistatin protein levels were significantly lower in the samples derived from fibrosis patients. The decrease in the follistatin levels can lead to an increased bioactivity of activin A and hence may provide a useful measurement to identify patients at risk of a severe fibrotic response to IR. Additionally, follistatin, by its ability to neutralise the actions of activin A may be of value as an anti-fibrotic for radiation induced fibrosis.

Genome-wide analysis using exon arrays demonstrates an important role for expression of extra-cellular matrix, fibrotic control and tissue remodelling genes in Dupuytren's disease.

Dupuytren's disease (DD) is a classic example of pathological fibrosis which results in a debilitating disorder affecting a large sector of the human population. It is characterized by excessive local proliferation of fibroblasts and over-production of collagen and other components of extracellular matrix (ECM) in the palmar fascia. The fibrosis progressively results in contracture of elements between the palmar fascia and skin causing flexion deformity or clawing of the fingers and a severe reduction in hand function. While much is known about the pathogenesis and surgical treatment of DD, little is known about the factors that cause its onset and progression, despite many years of research. Gene expression patterns in DD patients now offers the potential to identify genes that direct the pathogenesis of DD. In this study we used primary cultures of fibroblasts derived from excisional biopsies of fibrotic tissue from DD patients to compare the gene expression profiles on a genome-wide basis with normal control fibroblasts. Our investigations have identified genes that may be involved with DD pathogenesis including some which are directly relevant to fibrosis. In particular, these include significantly reduced expression levels of three matrix metallopeptidases (MMP1, MMP3, MMP16), follistatin, and STAT1, and significantly increased expression levels of fibroblast growth factors (FGF9, FGF11), a number of collagen genes and other ECM genes in DD patient samples. Many of these gene products are known to be involved in fibrosis, tumour formation and in the normal processes of tissue remodelling. In addition, alternative splicing was identified in some DD associated genes. These highly sensitive genomic investigations provide new insight into the molecular mechanisms that may underpin the development and progression of DD.

Genome-wide transcription responses to synchrotron microbeam radiotherapy.

The majority of cancer patients achieve benefit from radiotherapy. A significant limitation of radiotherapy is its relatively low therapeutic index, defined as the maximum radiation dose that causes acceptable normal tissue damage to the minimum dose required to achieve tumor control. Recently, a new radiotherapy modality using synchrotron-generated X-ray microbeam radiotherapy has been demonstrated in animal models to ablate tumors with concurrent sparing of normal tissue. Very little work has been undertaken into the cellular and molecular mechanisms that differentiate microbeam radiotherapy from broad beam. The purpose of this study was to investigate and compare the whole genome transcriptional response of in vivo microbeam radiotherapy versus broad beam irradiated tumors. We hypothesized that gene expression changes after microbeam radiotherapy are different from those seen after broad beam. We found that in EMT6.5 tumors at 4-48 h postirradiation, microbeam radiotherapy differentially regulates a number of genes, including major histocompatibility complex (MHC) class II antigen gene family members, and other immunity-related genes including Ciita, Ifng, Cxcl1, Cxcl9, Indo and Ubd when compared to broad beam. Our findings demonstrate molecular differences in the tumor response to microbeam versus broad beam irradiation and these differences provide insight into the underlying mechanisms of microbeam radiotherapy and broad beam.

Differential expression of Wnt signaling molecules between pre- and postmenopausal endometrial epithelial cells suggests a population of putative epithelial stem/progenitor cells reside in the basalis layer.

The human endometrium undergoes extensive monthly regeneration in response to fluctuating levels of circulating estrogen and progesterone in premenopausal (Pre-M) women. In contrast, postmenopausal (Post-M) endometrium is thin and quiescent with low mitotic activity, similar to the Pre-M endometrial basalis layer. Clonogenic epithelial stem/progenitor (ESP) cells, likely responsible for regenerating endometrial epithelium, have been identified in Pre-M and Post-M endometrium, but their location is unknown. We undertook transcriptional profiling of highly purified epithelial cells from full-thickness Pre-M and Post-M endometrium to identify differentially regulated genes that may indicate a putative ESP cell population resides in the basalis of Pre-M and basalis-like Post-M endometrium. Of 1077 differentially expressed genes identified, the Wnt signaling pathway, important in endometrial development and stem cell regulation, was one of the main gene families detected, including 22 Wnt-associated genes. Twelve genes were validated using quantitative RT-PCR, and all were concordant with microarray data. Immunostaining showed glandular epithelial location of Wnt-regulated genes, Axin-related protein 2 and ß-catenin. Axin2 localized to the nucleus of basalis Pre-M and Post-M and cytoplasm of functionalis Pre-M endometrium, suggesting that it regulates ß-catenin. Comparison of our Post-M gene profile with published gene microarray datasets revealed similarities to Pre-M basalis epithelial profiles. This differential expression of multiple Wnt-associated genes in human Pre-M and Post-M endometrial epithelial cells and the similar gene profile of Post-M and Pre-M basalis epithelium suggests that a population of putative endometrial ESP may reside in the basalis of Pre-M endometrium, which may be responsible for regenerating glandular epithelium each month.

DNA repair genes: alternative transcription and gene expression at the exon level in response to the DNA damaging agent, ionizing radiation.

DNA repair is an essential cellular process required to maintain genomic stability. Every cell is subjected to thousands of DNA lesions daily under normal physiological conditions. Ionizing radiation (IR) is a major DNA damaging agent that can be produced by both natural and man-made sources. A common source of radiation exposure is through its use in medical diagnostics or treatments such as for cancer radiotherapy where relatively high doses are received by patients. To understand the detailed DNA repair gene transcription response to high dose IR, gene expression exon array studies have been performed and the response to radiation in two divergent cell types, lymphoblastoid cell lines and primary fibroblasts, has been examined. These exon arrays detect expression levels across the entire gene, and have the advantage of high sensitivity and the ability to identify alternative transcripts. We found a selection of DNA repair genes, including some not previously reported, that are modulated in response to radiation. Detailed dose and time course kinetics of DNA repair transcription was conducted and results have been validated utilizing PCR methods. Alternative transcription products in response to IR were identified in several DNA repair genes including RRM2B and XPC where alternative initiation sites were found. These investigations have advanced the knowledge about the transcriptional response of DNA repair.

DNA damage and repair kinetics after microbeam radiation therapy emulation in living cells using monoenergetic synchrotron X-ray microbeams.

A novel synchrotron-based approach, known as microbeam radiation therapy (MRT), currently shows considerable promise in increased tumour control and reduced normal tissue damage compared with conventional radiotherapy. Different microbeam widths and separations were investigated using a controlled cell culture system and monoenergetic (5.35 keV) synchrotron X-rays in order to gain further insight into the underlying cellular response to MRT. DNA damage and repair was measured using fluorescent antibodies against phosphorylated histone H2AX, which also allowed us to verify the exact location of the microbeam path. Beam dimensions that reproduced promising MRT strategies were used to identify useful methods to study the underpinnings of MRT. These studies include the investigation of different spatial configurations on bystander effects. γH2AX foci number were robustly induced in directly hit cells and considerable DNA double-strand break repair occurred by 12 h post-10 Gy irradiation; however, many cells had some γH2AX foci at the 12 h time point. γH2AX foci at later time points did not directly correspond with the targeted regions suggesting cell movement or bystander effects as a potential mechanism for MRT effectiveness. Partial irradiation of single nuclei was also investigated and in most cases γH2AX foci were not observed outside the field of irradiation within 1 h after irradiation indicating very little chromatin movement in this time frame. These studies contribute to the understanding of the fundamental radiation biology relating to the MRT response, a potential new therapy for cancer patients.

Lymphoid and fibroblastic cell lineages from radiosensitive cancer patients: molecular analysis of DNA double strand break repair by major non-homologous end-joining sub-pathways.

AIMS: Radiation therapy (RT) is used in the treatment of approximately half of all cancer patients. Although there have been great improvements in tumor localization and the technical accuracy of RT delivery, some RT patients still have idiosyncratic hypersensitivity to ionizing radiation (IR) in their normal tissues. Although much effort has been expended in the search for assays that could detect radiosensitive individuals prior to treatment and facilitate tailored therapy; a suitable and clinically practical predictive assay has yet to be realized. Since DNA double-strand breaks (DSB) are a major lesion caused by IR, we hypothesized that radiation hypersensitive individuals might be deficient in the repair of such lesions. METHODS: To test this hypothesis we quantitatively and functionally characterized DSB repair of the two major non-homologous end-joining (NHEJ) sub-pathways in a pilot study using a plasmid repair reconstitution assay in lymphoblastoid and fibroblast cell lines from radiosensitive cancer patients and controls. Experiments using well-characterized mammalian DSB repair mutants demonstrated the ability of the assay to distinguish NHEJ sub-pathways. The proportion of direct end-joining repair compared with that of microhomology-directed repair was used as a functional end-point of DSB repair competence in the different cell lines. RESULTS: We found that the overall level of NHEJ sub-pathway repair competency was similar in cell lines from radiosensitive patients and controls. CONCLUSION: These data suggest that this assay in these cell lineages has limited usefulness as a predictive screen for the endogenous DNA DSB repair competency of radiosensitive cancer patients' cells but can usefully characterize major cellular DSB repair phenotypes.

Protection by methylproamine of irradiated human keratinocytes correlates with reduction of DNA damage.

PURPOSE: The therapeutic ratio for ionising radiation treatment of tumour is a trade-off between normal tissue side-effects and tumour control. Application of a radioprotector to normal tissue can reduce side-effects. Here we study the effects of a new radioprotector on the cellular response to radiation. Methylproamine is a DNA-binding radioprotector which, on the basis of published pulse radiolysis studies, acts by repair of transient radiation-induced oxidative species on DNA. To substantiate this hypothesis, we studied protection by methylproamine at both clonogenic survival and radiation-induced DNA damage, assessed by γH2AX (histone 2AX phosphorylation at serine 139) focus formation endpoints. MATERIALS AND METHODS: The human keratinocyte cell line FEP1811 was used to study clonogenic survival and yield of γH2AX foci following irradiation (¹³7Cs γ-rays) of cells exposed to various concentrations of methylproamine. Uptake of methylproamine into cell nuclei was measured in parallel. RESULTS: The extent of radioprotection at the clonogenic survival endpoint increased with methylproamine concentration up to a maximum dose modification factor (DMF) of 2.0 at 10 µM. At least 0.1 fmole/nucleus of methylproamine is required to achieve a substantial level of radioprotection (DMF of 1.3) with maximum protection (DMF of 2.0) achieved at 0.23 fmole/nucleus. The γH2AX focus yield per cell nucleus 45 min after irradiation decreased with drug concentration with a DMF of 2.5 at 10 µM. CONCLUSIONS: These results are consistent with the hypothesis that radioprotection by methylproamine is mediated by attenuation of the extent of initial DNA damage.