Olga A. Martin

Use of the γ-H2AX assay to monitor DNA damage and repair in translational cancer research

Formation of γ-H2AX in response to DNA double stranded breaks (DSBs) provides the basis for a sensitive assay of DNA damage in human biopsies. The review focuses on the application of γ-H2AX-based methods to translational studies to monitor the clinical response to DNA targeted therapies such as some forms of chemotherapy, external beam radiotherapy, radionuclide therapy or combinations thereof. The escalating attention on radiation biodosimetry has also highlighted the potential of the assay including renewed efforts to assess the radiosensitivity of prospective radiotherapy patients. Finally the γ-H2AX response has been suggested as a basis for an in vivo imaging modality.

γH2AX foci as a measure of DNA damage: a computational approach to automatic analysis.

The γH2AX focus assay represents a fast and sensitive approach for the detection of one of the critical types of DNA damage - double-strand breaks (DSB) induced by various cytotoxic agents including ionising radiation. Apart from research applications, the assay has a potential in clinical medicine/pathology, such as assessment of individual radiosensitivity, response to cancer therapies, as well as in biodosimetry. Given that generally there is a direct relationship between numbers of microscopically visualised γH2AX foci and DNA DSB in a cell, the number of foci per nucleus represents the most efficient and informative parameter of the assay. Although computational approaches have been developed for automatic focus counting, the tedious and time consuming manual focus counting still remains the most reliable way due to limitations of computational approaches. We suggest a computational approach and associated software for automatic focus counting that minimises these limitations. Our approach, while using standard image processing algorithms, maximises the automation of identification of nuclei/cells in complex images, offers an efficient way to optimise parameters used in the image analysis and counting procedures, optionally invokes additional procedures to deal with variations in intensity of the signal and background in individual images, and provides automatic batch processing of a series of images. We report results of validation studies that demonstrated correlation of manual focus counting with results obtained using our computational algorithm for mouse jejunum touch prints, mouse tongue sections and human blood lymphocytes as well as radiation dose response of γH2AX focus induction for these biological specimens.

The role of miRNA in the direct and indirect effects of ionizing radiation.

This review focuses on a number of recent studies that have examined changes in microRNA (miRNA) expression profiles in response to ionizing radiation and other forms of oxidative stress. In both murine and human cells and tissues, a number of miRNAs display significant alterations in expression levels in response to both direct and indirect radiation exposure. In terms of direct irradiation, or exposure to agents that induce oxidative stress, miRNA array analyses indicate that a number of miRNAs are up- and down-regulated and, in particular, the let-7 family of miRNAs may well be critical in the cellular response to oxidative stress. In bystander cells that are not directly irradiated, but close to, or share media with directly irradiated cells or tissues, the miRNA expression profiles are also altered, but are somewhat distinct from the directly irradiated cells. Based on the results of these numerous studies, as well as our own data presented here, we conclude that miRNA regulation is a critical step in the cellular response to radiation and oxidative stress and that future studies should elucidate the mechanisms through which this altered regulation affects cell metabolism.

p21: A Two-Faced Genome Guardian

Upon DNA damage or other stressors, the tumor suppressor p53 is activated, leading to transient expression of the cyclin-dependent kinase inhibitor (CKI) p21. This either triggers momentary G1 cell cycle arrest or leads to a chronic state of senescence or apoptosis, a form of genome guardianship. In the clinic, the presence of p21 has been considered an indicator of wildtype p53 activity. However, recent evidence suggests that p21 also acts as an oncogenic factor in a p53-deficient environment. Here, we discuss the controversial aspects of the two-faced involvement of p21 in cancer and speculate on how this new information may increase our understanding of its role in cancer pathogenesis. Prevailing notions indicate that p21 might also act as antiapoptotic agent, which may have relevant implications for future therapeutic strategies.

Susceptibility to bystander DNA damage is influenced by replication and transcriptional activity.

Direct cellular DNA damage may lead to genome destabilization in unexposed, bystander, cells sharing the same milieu with directly damaged cells by means of the bystander effect. One proposed mechanism involves double strand break (DSB) formation in S phase cells at sites of single strand lesions in the DNA of replication complexes, which has a more open structure compared with neighboring DNA. The DNA in transcription complexes also has a more open structure, and hence may be susceptible to bystander DSB formation from single strand lesions. To examine whether transcription predisposes non-replicating cells to bystander effect-induced DNA DSBs, we examined two types of primary cells that exhibit high levels of transcription in the absence of replication, rat neurons and human lymphocytes. We found that non-replicating bystander cells with high transcription rates exhibited substantial levels of DNA DSBs, as monitored by γ-H2AX foci formation. Additionally, as reported in proliferating cells, TGF-β and NO were found to mimic bystander effects in cell populations lacking DNA synthesis. These results indicate that cell vulnerability to bystander DSB damage may result from transcription as well as replication. The findings offer insights into which tissues may be vulnerable to bystander genomic destabilization in vivo.

Does the mobilization of circulating tumour cells during cancer therapy cause metastasis

Despite progressive improvements in the management of patients with locoregionally confined, advanced-stage solid tumours, distant metastasis remains a very common — and usually fatal — mode of failure after attempted curative treatment. Surgery and radiotherapy are the primary curative modalities for these patients, often combined with each other and/or with chemotherapy. Distant metastasis occurring after treatment can arise from previously undetected micrometastases or, alternatively, from persistent locoregional disease. Another possibility is that treatment itself might sometimes cause or promote metastasis. Surgical interventions in patients with cancer, including biopsies, are commonly associated with increased concentrations of circulating tumour cells (CTCs). High CTC numbers are associated with an unfavourable prognosis in many cancers. Radiotherapy and systemic antitumour therapies might also mobilize CTCs. We review the preclinical and clinical data concerning cancer treatments, CTC mobilization and other factors that might promote metastasis. Contemporary treatment regimens represent the best available curative options for patients who might otherwise die from locally confined, advanced-stage cancers; however, if such treatments can promote metastasis, this process must be understood and addressed therapeutically to improve patient survival.

A Pattern of Early Radiation-Induced Inflammatory Cytokine Expression Is Associated with Lung Toxicity in Patients with Non-Small Cell Lung Cancer

Purpose Lung inflammation leading to pulmonary toxicity after radiotherapy (RT) can occur in patients with non-small cell lung cancer (NSCLC). We investigated the kinetics of RT induced plasma inflammatory cytokines in these patients in order to identify clinical predictors of toxicity. Experimental Design In 12 NSCLC patients, RT to 60 Gy (30 fractions over 6 weeks) was delivered; 6 received concurrent chemoradiation (chemoRT) and 6 received RT alone. Blood samples were taken before therapy, at 1 and 24 hours after delivery of the 1st fraction, 4 weeks into RT, and 12 weeks after completion of treatment, for analysis of a panel of 22 plasma cytokines. The severity of respiratory toxicities were recorded using common terminology criteria for adverse events (CTCAE) v4.0. Results Twelve cytokines were detected in response to RT, of which ten demonstrated significant temporal changes in plasma concentration. For Eotaxin, IL-33, IL-6, MDC, MIP-1α and VEGF, plasma concentrations were dependent upon treatment group (chemoRT vs RT alone, all p-values <0.05), whilst concentrations of MCP-1, IP-10, MCP-3, MIP-1β, TIMP-1 and TNF-α were not. Mean lung radiation dose correlated with a reduction at 1 hour in plasma levels of IP-10 (r2 = 0.858, p<0.01), MCP-1 (r2 = 0.653, p<0.01), MCP-3 (r2 = 0.721, p<0.01), and IL-6 (r2 = 0.531, p = 0.02). Patients who sustained pulmonary toxicity demonstrated significantly different levels of IP-10 and MCP-1 at 1 hour, and Eotaxin, IL-6 and TIMP-1 concentration at 24 hours (all p-values <0.05) when compared to patients without respiratory toxicity. Conclusions Inflammatory cytokines were induced in NSCLC patients during and after RT. Early changes in levels of IP-10, MCP-1, Eotaxin, IL-6 and TIMP-1 were associated with higher grade toxicity. Measurement of cytokine concentrations during RT could help predict lung toxicity and lead to new therapeutic strategies.

Building immunity to cancer with radiation therapy.

Over the last decade there has been a dramatic shift in the focus of cancer research toward understanding how the bodys immune defenses can be harnessed to promote the effectiveness of cytotoxic anti-cancer therapies. The ability of ionizing radiation to elicit anti-cancer immune responses capable of controlling tumor growth has led to the emergence of promising combination-based radio-immunotherapeutic strategies for the treatment of cancer. Herein we review the immunoadjuvant properties of localized radiation therapy and discuss how technological advances in radio-oncology and developments in the field of tumor-immunotherapy have started to revolutionize the therapeutic application of radiotherapy.

Para-inflammation mediates systemic DNA damage in response to tumor growth.

The radiation induced bystander effect is a well accepted consequence of ionizing radiation exposure. However, it has become clear that bystander responses in vitro can result from a number of stress stimuli. We had reported that media conditioned on tumor cell cultures induced a bystander effect in recipient normal cell cultures and asked whether an analogous process could occur in vivo - could the presence of a tumor induce DNA damage in distant tissues. We recently demonstrated the presence of a distant bystander DNA damage response in vivo in the gastrointestinal organs and skin of mice implanted with subcutaneous tumors. The activation of inflammatory macrophages through the cytokine CCL2 was found to be required for this distant genotoxic response. These results shed new light on the consequences of tumor growth to distant parts of the body and highlight the potential for possible medical interventions to mitigate the effect of cancers.

Where did they come from? The origin of endogenous γ-H2AX foci in tumor cells

Comment on: Telomere-dependent and telomere-independent origins of endogenous DNA damage in tumor cells. Asako J. Nakamura, Christophe E. Redon, William M. Bonner and Olga A. Sedelnikova. Aging 2009; 212-8.