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Sally A Amundson

Publications and source records attributed to Sally A Amundson.

14 recordsLinked to original sources
PubMed2005

Mechanism of radiation-induced bystander effect: role of the cyclooxygenase-2 signaling pathway.

The radiation-induced bystander effect is defined as "the induction of biological effects in cells that are not directly traversed by a charged particle but are in close proximity to cells that are." Although these bystander effects have been demonstrated with a variety of biological endpoints in both human and rodent cell lines (as well as in 3D tissue samples), the mechanism of the phenomenon is not known. Although gap junction communication and the presence of soluble mediator(s) are both known to play important roles in the bystander response, the precise signaling molecules have yet to be identified. By using the Columbia University charged particle beam in conjunction with a strip dish design, we show here that the cyclooxygenase-2 (COX-2, also known as prostaglandin endoperoxide synthase-2) signaling cascade plays an essential role in the bystander process. Treatment of bystander cells with NS-398, which suppresses COX-2 activity, significantly reduced the bystander effect. Because the critical event of the COX-2 signaling is the activation of the mitogen-activated protein kinase pathways, our finding that inhibition of the extracellular signal-related kinase phosphorylation suppressed bystander response further confirmed the important role of mitogen-activated protein kinase signaling cascade in the bystander process. These results provide evidence that the COX-2-related pathway, which is essential in mediating cellular inflammatory response, is the critical signaling link for the bystander phenomenon.

Alpha Particles↗
PubMed2005

Biological effects in unirradiated human tissue induced by radiation damage up to 1 mm away.

A central tenet in understanding the biological effects of ionizing radiation has been that the initially affected cells were directly damaged by the radiation. By contrast, evidence has emerged concerning "bystander" responses involving damage to nearby cells that were not themselves directly traversed by the radiation. These long-range effects are of interest both mechanistically and for assessing risks from low-dose exposures, where only a small proportion of cells are directly hit. Bystander effects have been observed largely by using single-cell in vitro systems that do not have realistic multicellular morphology; no studies have as yet been reported in three-dimensional, normal human tissue. Given that the bystander phenomenon must involve cell-to-cell interactions, the relevance of such single-cell in vitro studies is questionable, and thus the significance of bystander responses for human health has remained unclear. Here, we describe bystander responses in a three-dimensional, normal human-tissue system. Endpoints were induction of micronucleated and apoptotic cells. A charged-particle microbeam was used, allowing irradiation of cells in defined locations in the tissue yet guaranteeing that no cells located more than a few micrometers away receive any radiation exposure. Unirradiated cells up to 1 mm distant from irradiated cells showed a significant enhancement in effect over background, with an average increase in effect of 1.7-fold for micronuclei and 2.8-fold for apoptosis. The surprisingly long range of bystander signals in human tissue suggests that bystander responses may be important in extrapolating radiation risk estimates from epidemiologically accessible doses down to very low doses where nonhit bystander cells will predominate.

Alpha Particles↗
PubMed2005

Stress-specific signatures: expression profiling of p53 wild-type and -null human cells.

Gene expression responses of human cell lines exposed to a diverse set of stress agents were compared by cDNA microarray hybridization. The B-lymphoblastoid cell line TK6 (p53 wild-type) and its p53-null derivative, NH32, were treated in parallel to facilitate investigation of p53-dependent responses. RNA was extracted 4 h after the beginning of treatment when no notable decrease in cell viability was evident in the cultures. Gene expression signatures were defined that discriminated between four broad general mechanisms of stress agents: Non-DNA-damaging stresses (heat shock, osmotic shock, and 12-O-tetradecanoylphorbol 13-acetate), agents causing mainly oxidative stress (arsenite and hydrogen peroxide), ionizing radiations (neutron and gamma-ray exposures), and other DNA-damaging agents (ultraviolet radiation, methyl methanesulfonate, adriamycin, camptothecin, and cis-Platinum(II)diammine dichloride (cisplatin)). Within this data set, non-DNA-damaging stresses could be discriminated from all DNA-damaging stresses, and profiles for individual agents were also defined. While DNA-damaging stresses showed a strong p53-dependent element in their responses, no discernible p53-dependent responses were triggered by the non-DNA-damaging stresses. A set of 16 genes did exhibit a robust p53-dependent pattern of induction in response to all nine DNA-damaging agents, however.

B-Lymphocytes↗
PubMed2004

Human in vivo radiation-induced biomarkers: gene expression changes in radiotherapy patients.

After initially identifying potential biomarkers of radiation exposure through microarray studies of ex vivo irradiated human peripheral white blood cells, we have now measured the in vivo responses of several of these biomarker genes in patients undergoing total body irradiation. Microarray analysis has identified additional in vivo radiation-responsive genes, although the general in vivo patterns of stress-gene induction appear similar to those obtained from ex vivo white blood cell experiments. Additional studies may reveal correlations between responses and either diagnosis or prognosis, and such in vivo validation marks an important step in the development of potentially informative radiation exposure biomarkers.

Adult↗
PubMed2004

Functional genomics of UV radiation responses in human cells.

The gene expression responses of MCF-7, a p53 wild-type (wt) human cell line, were monitored by cDNA microarray hybridization after exposure to different wavelengths of UV irradiation. Equitoxic doses of UVA, UVB, and UVC radiation were used to reduce survival to 37%. The effects of suramin, a signal pathway inhibitor, on the gene expression responses to the three UV wavelengths were also compared in this model system. UVB radiation triggered the broadest gene expression responses, and 172 genes were found to be consistently responsive in at least two-thirds of independent UVB experiments. These UVB radiation-responsive genes encode proteins with diverse cellular roles including cell cycle control, DNA repair, signaling, transcription, protein synthesis, protein degradation, and RNA metabolism. The set of UVB-responsive genes included most of the genes responding to an equitoxic dose of UVC radiation, plus additional genes that were not strongly triggered by UVC radiation. There was also some overlap with genes responding to an equitoxic dose of UVA radiation, although responses to this lower energy UV radiation were overall weaker. Signaling through growth factor receptors and other cytokine receptors was shown to have a major role in mediating UV radiation stress responses, as suramin, which inhibits such receptors, attenuated responses to UV radiation in nearly all the cases. Inhibition by suramin was greater for UVC than for UVB irradiation. This probably reflects the more prominent role in UVB damage response of signaling by reactive oxygen species, which would not be affected by suramin. Our results with suramin demonstrate the power of cDNA microarray hybridization to illuminate the global effects of a pharmacologic inhibitor on cell signaling.

Cell Line, Tumor↗
PubMed2003

Functional genomics as a window on radiation stress signaling.

Exposure to ionizing radiation, as well as other stresses, results in the activation of complex signal transduction pathways, which eventually shape the response of cells and organisms. Some of the important pathways responding to radiation include the ATM/P53 pathway, MAPK cascades and NF-kappaB activation, as well as signaling events initiated at the cell membrane and within the cytoplasm. Alterations in gene expression play roles both as intermediaries in signaling and as downstream effector genes. Differences in cell type, interindividual genetic differences and crosstalk occurring between signaling pathways may help to channel radiation stress signals between cell cycle delay, enhanced DNA repair, and apoptosis. These differences may in turn help determine the likelihood of late effects of radiation exposure, including carcinogenesis and fibrosis. The tools of the postgenomic era enable high-throughput studies of the multiple changes resulting from the interplay of radiation signaling pathways. Gene expression profiling, in particular shows great promise, both in terms of insight into basic molecular mechanisms and for the future hope of biomarker development and individual tailoring of cancer therapy.

Animals↗
PubMed2003

Monitoring human radiation exposure by gene expression profiling: possibilities and pitfalls.

Advances in high throughput analysis of mRNA expression have made it possible to establish gene expression profiles for different cells, tissues, diseases and exposure states. For instance, recent studies have demonstrated the utility of such an approach to classify sub-types of cancers with more detail than was previously possible. In addition, gene expression studies of ionizing radiation exposure both in vitro and in vivo are affording insight into the molecular mechanisms of mammalian radiation response. We have demonstrated that radiation expression profiles are a good predictor of p53 function in cell lines, and such profiles also indicate a major role for p53-regulated genes in the in vivo radiation response. Gene expression can be a sensitive indicator of radiation response as we have shown linear dose-responses for induction of several genes down to doses as low as 2 cGy. As profiles are established from radiation studies, it is hoped that they may be useful for identifying individuals with specific exposures or predisposition to negative outcome of exposure. Although this technology holds great promise, some obstacles remain to be overcome before it can be successfully applied to population studies.

Cyclin-Dependent Kinase Inhibitor p21↗
PubMed2003

Differential responses of stress genes to low dose-rate gamma irradiation.

In the past, most mechanistic studies of ionizing radiation response have employed very large doses, then extrapolated the results down to doses relevant to human exposure. It is becoming increasingly apparent, however, that this does not give an accurate or complete picture of the effects of most environmental exposures, which tend to be of low dose and protracted over time. We have initiated direct studies of low dose exposures, and using the relatively responsive ML-1 cell line, have shown that changes in gene expression can be triggered by doses of gamma-rays of 10 cGy and less in human cells. We have now extended these studies to investigate the effects on gene induction of reducing the rate of irradiation. In the ML-1 human myeloid leukemia cell line, we have found that reducing the dose rate over three orders of magnitude results in some protection against the induction of apoptosis, but still causes linear induction of the p53-regulated genes CDKN1A, GADD45A, and MDM2 between 2 and 50 cGy. Reducing the rate of exposure reduces the magnitude of induction of CDKN1A and GADD45A, but not the magnitude or duration of cell cycle delay. In contrast, MDM2 is induced to the same extent regardless of the rate of dose delivery. Microarray analysis has identified additional low dose-rate-inducible genes, and indicates the existence of two general classes of low dose-rate responders in ML-1. One group of genes is induced in a dose rate-dependent fashion, similar to GADD45A and CDKN1A. Functional annotation of this gene cluster indicates a preponderance of genes with known roles in apoptosis regulation. Similarly, a group of genes with dose rate-independent induction, such as seen for MDM2, was also identified. The majority of genes in this group are involved in cell cycle regulation. This apparent differential regulation of stress signaling pathways and outcomes in response to protracted radiation exposure has implications for carcinogenesis and risk assessment, and could not have been predicted from classical high dose studies.

Apoptosis↗
PubMed2002

ATF3 induction following DNA damage is regulated by distinct signaling pathways and over-expression of ATF3 protein suppresses cells growth.

Mammalian cells have a remarkable diverse repertoire of response to genotoxic stress that damage DNA. Cellular responses to DNA damaging agents will initially exhibit gene induction, which is regulated by complex mechanism(s) and probably involves multiple signaling pathways. In this paper, we demonstrate that induction of ATF3 protein, a member of the ATF/CREB family of transcription factors, by ionizing radiation (IR) requires normal cellular p53 function. In contrast, induction of ATF3 after UV radiation (UV) or Methyl methanesulphonate (MMS) is independent of p53 status. Induction of ATF3 by DNA damage is rapid, transient, and through a transcriptional mechanism. The ATF3 promoter is induced by UV and MMS, but not by IR. In addition, ATF3 promoter can be activated by MEKK1, an upstream activator of the ERK and JNK kinase pathway, but not induced following p53 expression. Those results indicate that regulation of ATF3 induction after DNA damage utilizes both the p53-dependent and -independent pathways, and may also involve MAP kinase signaling pathways. Using the tetracycline-inducible system (tet-off), we have found that over-expression of ATF3 protein moderately suppresses cell growth. Interestingly, over-expression of ATF3 protein is able to slow down progression of cells from G1 to S phase, indicating that ATF3 protein might play a negative role in the control of cell cycle progression.

Activating Transcription Factor 3↗
PubMed2002

Amplification of PPM1D in human tumors abrogates p53 tumor-suppressor activity.

Expression of oncogenic Ras in primary human cells activates p53, thereby protecting cells from transformation. We show that in Ras-expressing IMR-90 cells, p53 is phosphorylated at Ser33 and Ser46 by the p38 mitogen-activated protein kinase (MAPK). Activity of p38 MAPK is regulated by the p53-inducible phosphatase PPM1D, creating a potential feedback loop. Expression of oncogenic Ras suppresses PPM1D mRNA induction, leaving p53 phosphorylated at Ser33 and Ser46 and in an active state. Retrovirus-mediated overexpression of PPM1D reduced p53 phosphorylation at these sites, abrogated Ras-induced apoptosis and partially rescued cells from cell-cycle arrest. Inactivation of p38 MAPK (the product of Mapk14) in vivo by gene targeting or by PPM1D overexpression expedited tumor formation after injection of mouse embryo fibroblasts (MEFs) expressing E1A+Ras into nude mice. The gene encoding PPM1D (PPM1D, at 17q22/q23) is amplified in human breast-tumor cell lines and in approximately 11% of primary breast tumors, most of which harbor wildtype p53. These findings suggest that inactivation of the p38 MAPK through PPM1D overexpression resulting from PPM1D amplification contributes to the development of human cancers by suppressing p53 activation.

Animals↗
PubMed2002

p38 and Chk1 kinases: different conductors for the G(2)/M checkpoint symphony.

The mechanism controlling G(2)/M checkpoint activation after DNA damage was thought to be mediated primarily by nuclear Chk1/Chk2 kinases. Recent evidence indicates that this checkpoint is more complex, involving at least two different biochemical systems that target the Cdc25B and Cdc25C phosphatases. Following genotoxic stress, different kinases integrate signaling from the damaged DNA and other damaged cellular components to regulate Cdc25 inactivation. Our current model for G(2)/M checkpoint activation after genotoxic stress is discussed emphasizing the roles for Chk1 and p38 kinases in checkpoint regulation.

Animals↗
PubMed2002

Stress-gene induction by low-dose gamma irradiation.

Using a human myeloid tumor cell line (ML-1), we detected induction of mRNA expression of several stress-responsive genes by doses of gamma rays as low as 2 cGy. For instance, the dose response for induction of CIP1/WAF1 and GADD45 appears to be linear over the range of 2 to 50 cGy and shows no evidence of a threshold for induction. Although 2 and 5 cGy exposures did not result in any detectable reduction in cloning efficiency nor in increased apoptosis in ML-1 cells, these exposures did produce a brief cell-cycle delay. We also used fluorescent cDNA microarray hybridization to investigate transcriptional stress responses following low doses of gamma rays and to identify additional radiation-responsive genes for inclusion in a stress-specific microarray we are developing. These studies provide insight into the molecular responses to physiologically relevant doses, which cannot necessarily be extrapolated from high-dose studies. The use of high throughput arrays will allow the identification of multiple stress-responsive genes that are radiation inducible in a variety of cell types and tissues. The expectation is that transcriptional stress responses will provide a molecular approach to monitoring for radiation exposure and detecting interindividual differences.

Gamma Rays↗