Kinetic complexity of the global response to glucocorticoid receptor action.

We have characterized the kinetic response of gene targets throughout the murine genome to transcriptional modulation by the glucocorticoid receptor (GR). In contrast to a model in which multiple genes are either repressed or activated during the GR response, the vast majority of responsive genes are subject to complex regulation profiles, frequently with alternate activation and repression phases. We also observe that GR binding at response elements does not always correlate with the target gene response profile. Thus, the cellular response to GR stimulation involves a highly orchestrated series of regulatory actions and not simply a binary response to hormone.

Integrating global gene expression and radiation survival parameters across the 60 cell lines of the National Cancer Institute Anticancer Drug Screen.

The 60 cell lines of the National Cancer Institute Anticancer Drug Screen (NCI-60) constitute the most extensively characterized in vitro cancer cell model. They have been tested for sensitivity to more than 100,000 potential chemotherapy agents and have been profiled extensively at the DNA, RNA, protein, functional, and pharmacologic levels. We have used the NCI-60 cell lines and three additional lines to develop a database of responses of cancer cells to ionizing radiation. We compared clonogenic survival, apoptosis, and gene expression response by microarray. Although several studies have profiled relative basal gene expression in the NCI-60, this is the first comparison of large-scale gene expression changes in response to genotoxic stress. Twenty-two genes were differentially regulated in cells with low survival after 2-Gy gamma-rays; 14 genes identified lines more sensitive to 8 Gy. Unlike reported basal gene expression patterns, changes in expression in response to radiation showed little tissue-of-origin effect, except for differentiating the lymphoblastoid cell lines from other cell types. Basal expression patterns, however, discriminated well between radiosensitive and more resistant lines, possibly being more informative than radiation response signatures. The most striking patterns in the radiation data were a set of genes up-regulated preferentially in the p53 wild-type lines and a set of cell cycle regulatory genes down-regulated across the entire NCI-60 panel. The response of those genes to gamma-rays seems to be unaffected by the myriad of genetic differences across this diverse cell set; it represents the most penetrant gene expression response to ionizing radiation yet observed.

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.

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.

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.