Sally A. Amundson

Fluorescent cDNA microarray hybridization reveals complexity and heterogeneity of cellular genotoxic stress responses

The fate of cells exposed to ionizing radiation (IR) may depend greatly on changes in gene expression, so that an improved view of gene induction profiles is important for understanding mechanisms of checkpoint control, repair and cell death following such exposures. We have used a quantitative fluorescent cDNA microarray hybridization approach to identify genes regulated in response to γ-irradiation in the p53 wild-type ML-1 human myeloid cell line. Hybridization of the array to fluorescently-labeled RNA from treated and untreated cells was followed by computer analysis to derive relative changes in expression levels of the genes present in the array, which agreed well with actual quantitative changes in expression. Forty-eight sequences, 30 not previously identified as IR-responsive, were significantly regulated by IR. Induction by IR and other stresses of a subset of these genes, including the previously characterized CIP1/WAF1, MDM2 and BAX genes, as well as nine genes not previously reported to be IR-responsive, was examined in a panel of 12 human cell lines. Responses varied widely in cell lines with different tissues of origin and different genetic backgrounds, highlighting the importance of cellular context to genotoxic stress responses. Two of the newly identified IR-responsive genes, FRA-1 and ATF3, showed a p53-associated component to their IR-induction, and this was confirmed both in isogenic human cell lines and in mouse thymus. The majority of the IR-responsive genes, however, showed no indication of p53-dependent regulation, representing a potentially important class of stress-responsive genes in leukemic cells.

Identification of Potential mRNA Biomarkers in Peripheral Blood Lymphocytes for Human Exposure to Ionizing Radiation

Abstract Amundson, S. A., Do, K. T., Shahab, S., Bittner, M., Meltzer, P., Trent, J. and Fornace, A. J., Jr. Identification of Potential mRNA Biomarkers in Peripheral Blood Lymphocytes for Human Exposure to Ionizing Radiation. Since early in the Atomic Age, biological indicators of radiation exposure have been sought, but currently available methods are not entirely satisfactory. Using cDNA microarray hybridization to discover new potential biomarkers, we have identified genes expressed at increased levels in human peripheral blood lymphocytes after ex vivo irradiation. We recently used this technique to identify a large set of ionizing radiation-responsive genes in a human cell line (Oncogene 18, 3666–3672, 1999). The present set of radiation markers in peripheral blood lymphocytes was identified 24 h after treatment, and while the magnitude of mRNA induction generally decreased over time, many markers were still significantly elevated up to 72 h after irradiation. In all donors, the most highly responsive gene identified was DDB2, which codes for the p48 subunit of XPE, a protein known to play a crucial role in repair of ultraviolet (UV) radiation damage in DNA. Induction of DDB2, CDKN1A (also known as CIP1/WAF1) and XPC showed a linear dose–response relationship between 0.2 and 2 Gy at 24 and 48 h after irradiation, with less linearity at earlier or later times. These results suggest that relative levels of gene expression in peripheral blood cells may provide estimates of environmental radiation exposures.

Induction of gene expression as a monitor of exposure to ionizing radiation.

Abstract Amundson, S. A., Bittner, M., Meltzer, P., Trent, J. and Fornace, A. J., Jr. Induction of Gene Expression as a Monitor of Exposure to Ionizing Radiation. Radiat. Res. 156, 657–661 (2001). The complex molecular responses to genotoxic stress are mediated by a variety of regulatory pathways. The transcription factor TP53 plays a central role in the cellular response to DNA-damaging agents such as ionizing radiation, but other pathways also play important roles. In addition, differences in radiation quality, such as the exposure to high-LET radiation that occurs during space travel, may influence the pattern of responses. The premise is developed that stress gene responses can be employed as molecular markers for radiation exposure using a combination of informatics and functional genomics approaches. Published studies from our laboratory have already demonstrated such transcriptional responses with doses of γ rays as low as 2 cGy, and in peripheral blood lymphocytes (PBLs) irradiated ex vivo with doses as low as 20 cGy. We have also found several genes elevated in vivo 24 h after whole-body irradiation of mice with 20 cGy. Such studies should provide insight into the molecular responses to physiologically relevant doses, which cannot necessarily be extrapolated from high-dose studies. In addition, ongoing experiments are identifying large numbers of potential biomarkers using microarray hybridization and various irradiation protocols including expression at different times after exposure to low- and high-LET radiation. Computation-intensive informatics analysis methods are also being developed for management of the complex gene expression profiles resulting from these experiments. With further development of these approaches, it may be feasible to monitor changes in gene expression after low-dose radiation exposure and other physiological stresses that may be encountered during manned space flight, such as the planned mission to Mars.

The antiapoptotic decoy receptor TRID/TRAIL-R3 is a p53-regulated DNA damage-inducible gene that is overexpressed in primary tumors of the gastrointestinal tract.

Both DR4 and DR5 have recently been identified as membrane death receptors that are activated by their ligand TRAIL to engage the intracellular apoptotic machinery. TRID (also named as TRAIL-R3) is an antagonist decoy receptor and lacks the cytoplasmic death domain. TRID protects from TRAIL-induced apoptosis by competing with DR4 and DR5 for binding to TRAIL. TRID has been shown to be overexpressed in normal human tissues but not in malignantly transformed cell lines. DR5 is a p53-regulated gene and we have recently reported that DR5 expression is induced in response to genotoxic stress in both a p53-dependent and independent manner (Sheikh et al., 1998). In the current study, we demonstrate that TRID gene expression is also induced by the genotoxic agents ionizing radiation and methyl methanesulfonate (MMS) in predominantly p53 wild-type cells, whereas UV-irradiation does not induce TRID gene expression. Consistent with these results, exogenous wild-type p53 also upregulates the expression of endogenous TRID in p53-null cells. Thus, TRID appears to be a p53 target gene that is regulated by genotoxic stress in a p53-dependent manner. Using primary gastrointestinal tract (GIT) tumors and their matching normal tissue, we also demonstrate for the first time that TRID expression is enhanced in primary tumors of the GIT. It is, therefore, possible that TRID overexpressing GIT tumors may gain a selective growth advantage by escaping from TRAIL-induced apoptosis.

CHAPTER 298 – Complexity of Stress Signaling and Responses

Many different kinds of stress can be encountered by a cell or organism. One important category of stressors is genotoxic agents that cause damage to DNA. These include ultraviolet and ionizing radiations as well as many chemical mutagens and carcinogens. Organisms must also defend against a myriad of physical stresses acting mainly through mechanisms other than DNA damage. Such stresses may include shear stress, wounding, infection, nutrient deprivation, osmotic stress, hypoxia, and heat shock. In all organisms, sensor systems are needed to detect stress and its resultant damage. Signals are then exchanged between different cellular compartments, and even between cells, resulting in changes in expression and function of specific transcripts and proteins. This in turn impacts various cellular processes such as cell cycle progression, DNA repair, and activation of the apoptotic program, ultimately resulting in either recovery and repair or in death. This chapter attempts to provide an overview of these interwoven signaling pathways and processes.

Stress gene expression: analysis by informatics and functional genomics approaches

Molecular responses to genotoxic stress are complex and are mediated by a variety of regulatory pathways. One key element in cellular response is the stress gene transcription factor p53, which can regulate nearly 200 genes that have already been identified. Although p53 has a central role in the cellular response to DNAdamaging agents such as ionizing radiation (IR), other pathways can also have important roles. One example is the transcriptional responses associated with IRinduced apoptosis, where induction of some genes is limited to p53 wild-type cells that also have the ability to undergo rapid apoptosis after irradiation. In contrast, other genes are triggered after IR in lines undergoing rapid apoptosis regardless of p53 status. From this and other examples, it is apparent that the pattern of stress gene expression is cell-type specific in both primary and transformed lines. The premise will be developed that such differences in stress gene responsiveness can be employed as molecular markers using a combination of informatics and functional genomics approaches. An example will be given using the panel of lines of the NCI anticancer drug screen where both the p53 status and sensitivity to a large collection of cytotoxic agents have been determined. The use of cDNA microarray hybridization to measure IR-stress gene responses has recently been demonstrated and a large number of additional IR-stress genes have been identified. The basal expression and responses of some of these genes to DNA-damaging agents varied widely in cell lines from different tissues of origin and different genetic backgrounds, highlighting the importance of cellular context to genotoxic stress responses; this also highlights the need for informatics approaches to discover and prioritize hypotheses regarding the importance of particular cellular factors. The presentation will focus on the use of combining an informatics approach with functional genomics in the study of stress responses.