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May 2009

Upregulated Protein Jumpstarts DNA Repair Process

Team discovers calmodulin levels reflect a specific cellular response to low-level radiation

Visualization of DNA repair complexes (green) within DAPI-stained nucleus (blue) of macrophage cell, demonstrating augmentation of repair pathways associated with the upregulation of the regulatory protein calmodulin. Visualization of DNA repair complexes (green) within DAPI-stained nucleus (blue) of macrophage cell, demonstrating augmentation of repair pathways associated with the upregulation of the regulatory protein calmodulin. Enlarged View

Results: Students taught by Dr. Heather Smallwood at Washington State University working side-by-side with scientists at Pacific Northwest National Laboratory identified a new pathway that cells take when exposed to low levels of radiation, such as those used to shrink tumors. The newly identified cellular pathway upregulates, or increases the cellular components of, DNA repair pathways in response to low-dose radiological exposures. Cell survival is enhanced through pathways involving phosphorylated histone H2AX—the addition of phosphorous to the chief protein components of the complex combination of DNA, RNA, and protein that makes up chromosomes. These DNA repair pathways are distinct from other protein complex pathways tested that enhance cell death in an attempt to remove damaged cells in response to radiation. Such removal systems prevent the damaged cells from sapping further nutrients from an organism and act to halt further spread of infection.

As part of the mammalian immune system, macrophages, or white blood cells, normally recognize and remove dead cells and pathogens while further stimulating an immune response. This capability makes them a first line of defense against disease. Identifying a dose-dependent increase in the expression level of the calcium signaling protein, calmodulin (CaM), in irradiated mouse macrophage cells indicates that such increases are part of a specific radiation-dependent cellular response that can help expedite DNA damage repair.

Why it matters: Understanding the molecular mechanisms that modulate macrophage resistance to radiation is necessary for developing effective radiation therapies, because tumor-associated macrophages promote processes that enhance the spread of cancer. The increases in CaM abundance in response to the lower radiation doses used in the study suggest a possible role of CaM in mediating cellular response pathways to clinically relevant doses in radiation therapy. Because phosphorylated histone H2AX acts as a universal organizing center that both anchors chromosomal ends and mediates DNA repair, these results are broadly significant for understanding the radioresistance mechanisms of macrophages and other blood-generating cell types in response to radiation exposure.

The results suggest that therapeutic treatments that target CaM in conjunction with traditional radiotherapies may help kill tumor-associated macrophages, thereby restricting cancer cell proliferation and tumor metastisis.  Such treatments are likely to have other beneficial effects because CaM antagonists are known to prevent cancer invasiveness.

Methods: To better understand the molecular basis for the sensitivity of macrophages to low therapeutic doses of ionizing radiation, the research team investigated the possible role of CaM in modulating double-stranded DNA damage. CaM is suggested to be a principal mechanism of radiation-induced cellular death and transformation. They grew and irradiated mouse macrophage leukemia cells (i.e., RAW 264.7), then assayed cell survival to identify cells undergoing programmed death, or apoptosis, and to assess DNA damage and repair pathways, changes in the expression level of CaM, and total protein content.

After macrophage irradiation, increases in CaM abundance resulted in an increase in the number of phosphorylated histone H2AX foci, associated with DNA repair, with no change in the extent of double-stranded DNA damage. Measurement of the radiation sensitivity of RAW 264.7 macrophages, using a well-established survival assay for studying the effectiveness of specific agents on the survival and proliferation of cells, showed that macrophages are more resistant to  high radiation doses than low. Altering CaM levels and protein complex NFκB-dependent pathways was found to have multiple effects, some of which may disrupt DNA damage response pathways and cellular apoptosis. CaM overexpression reduced radiation-dependent cell killing and disrupted the adaptive cellular response to low-dose radiation, while radiation-induced DNA damage was shown to be insensitive to CaM. Upregulation of CaM abundance enhanced DNA repair pathways after irradiation. Hence, Laboratory Fellow Thomas Squier emphasized that "the medical significance of the finding is that specifically inhibiting repair mechanisms in tumor cells and tumor associated macrophages before radiation damage occurs will enable more rapid and targeted killing of these cells upon treatment, which is the main challenge in successful radiation therapies."

What's next: Future measurements will need to identify the components of the CaM-dependent pathway that leads to histone H2AX phosphorylation, thereby activating DNA repair and enhancing macrophage survival following radiation exposure.

Acknowledgments: This work was completed as part of the student radiobiology laboratory associated with Washington State University Tri-Cites, Richland. A portion of the research was performed using an LTQ-Orbitrap mass spectrometer and a custom-built liquid chromatography system at the Department of Energy's Environmental Molecular Sciences Laboratory, a national scientific user facility located at Pacific Northwest National Laboratory. Funded as part of DOE's Office of Biological and Environmental Research, EMSL provides integrated experimental and computational resources for discovery and technological innovation in the environmental molecular sciences to support the needs of DOE and the nation.

The authors of the paper are Heather Smallwood, formerly of PNNL; Daniel Lopez-Ferrer and Thomas Squier, PNNL; and Elis Eberlein and David Watson, WSU-Tri Cities. Other PNNL researchers involved in the work include Diana Bigelow, James Morris, William Chrisler, Sue Karagiosis, David Stenoien, Colette Sacksteder and Katrina Walters.

Reference: Smallwood HS, D Lopez-Ferrer, PE Eberlein, DJ Watson, and TC Squier. 2009. "Calmodulin Mediates DNA Repair Pathways Involving H2AX in Response to Low-Dose Radiation Exposure of RAW 264.7 Macrophages."  Chemical Research in Toxicology 22(3):460-470.  doi:DOI: 10.1021/tx800236r

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