Concerning the first project, we are in the process of determining the limits of DSB detection after whole body exposure of animals to ionizing radiation using H2AX phosphorylation as a model for triage during a radiation-related emergency. The results show that the amount of exposure to ionizing radiation in the sub-lethal to lethal range can be measured up to four days post exposure. We are also investigating the usefulness of gamma-H2AX detection in cases of partial body exposure. Concerning chemotherapeutic agents, two recent developments have given considerable impetus to this project. The first development is the finding by us and others that gamma-H2AX is formed in tissue culture cells as a response not only to agents that directly cause DSBs but also to those that indirectly induce DSBs. As many of the agents used for cancer treatment fall into this latter category, this finding greatly increases possible roles for gamma-H2AX as a biomarker for drug responses. The second important development is the formation of a multi-disiplinary NCI team to expedite the clinical evaluation of new therapeutic and imaging agents, so-called phase zero trials. gamma-H2AX formation is being examined as a possible biodosimeter in these studies utilizing several possible surrogate tissues. We are also examining gamma-H2AX formation in mouse models and are involved in several clinical protocols either approved or being approved in the phase zero and phase one trials. This work is important because it will permit clinicians to obtain immediate feedback from the cells of an individual patient, feedback which can then be used to tailor the treatment to that patient, thereby improving their survival. The ultimate goal of this initiative is to develop gamma-H2AX detection into a useful tool for human health. Concerning our second project, we are utilizing gamma-H2AX to probe for interactions, even distant communication, among cells in animals. In vitro studies by us and others have shown that cells that have been exposed to ionizing radiation lead to the presence of DNA damage products in unexposed cells that contact the exposed cells or media from the exposed cells and affect their viability, a phenomenon known as the bystander effect. We have shown that a robust bystander effect is present in artificial human skin tissue. Working from the hypothesis that the bystander effect is an example of a larger phenomenon of communication among damaged and healthy cells, we have demonstrated that media from tumor cells induces responses in bystander cells indistinguishable from those induced by media from irradiated cells, and are currently demonstrating the ability of other stresses to induce bystander responses. In addition, we are finding that this communication can be demonstrated in the intact animal. The presence of a human tumors implanted into syngeneic mice leads to higher levels of DNA damage in certain other cells in the mouse. We have determined that macrophages are involved in this communication and proliferating tissues are most sensitive. We are continuing to investigate when and how this communication takes place in the animal and how this might be useful for human health. This work is important because it may increase the ways information can be obtained from individual patients. It is possible that by monitoring certain easily obtained tissues of a patient, information can be obtained about events in other less accessible tissues, perhaps leading to early detection of disease and/or optimization of treatment. Concerning the third project, our goal is to gain insight into the function of these foci both in terms of their growth and disappearance, and their roles in DSB repair. H2AX is essential for genome stability and the rapid phase of DNA DSB repair. We are working from a hypothesis that robust genome stability depends on the speed of the fast repair phase, which in turn depends on the rate of focal growth and accumulation of repair proteins at those foci. Thus measuring the kinetics of focal growth and protein accumulation when various proteins are absent or altered may open a novel window by which to gain insights into the mechanism of early DSB repair processes, and which proteins are rate limiting in this process. We are developing tools that will permit us to study focal substructure and how it changes with time. We have shown for example, that in yeast, Mre11 does not bind to the whole gamma-H2AX domain, but is concentrated next to the break site. Studies such as these will complement other findings concerning the interactions of various proteins with gamma-H2AX, leading to a greater understanding of DNA DSB repair and the maintenance of genome stability. This work is important as it will provide the basis for understanding the important parameters involved in utilizing gamma-H2AX foci as a biodosimeter.
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