Gene expression profiles in human cells submitted to genotoxic stress

Cell response to genotoxic agents is complex and involves the participation of different classes of genes (DNA repair, cell cycle control, signal transduction, apoptosis and oncogenesis). In this report, we present three approaches to document gene expression profiles, dealing with the evaluation of cellular responses to genotoxic agents (gamma-rays from 60Cobalt and cyclophosphamide). We used the method of cDNA arrays to analyze the differential gene expression profiles that were displayed by lymphocytes from radiation-exposed individuals, a human fibroblast cell line, and T lymphocytes from systemic lupus erythematosus (SLE) patients who were treated with cyclophosphamide. A preliminary analysis performed in lymphocytes from three radiation-workers showed that several induced genes can be associated with cell response to ionizing radiation: TRRAP (cell cycle regulation), Ligase IV (DNA repair), MAPK8IP1 and MAPK10 (signal transduction), RASSF2 (apoptosis induction/tumorigenesis), p53 (damage response/maintenance of genetic stability). The in vitro irradiated normal VH16 cell line (primary) showed a complex response to the genotoxic stress at the molecular level. Many apoptotic pathways were concomitantly induced. In addition, several genes involved in signaling and cell cycle arrest/control were significantly modulated after irradiation. Many genes involved in oxidative damage were also induced, indicating that this mechanism seems to be an important component of cell response. After treatment of the SLE patients with cyclophosphamide, 154 genes were differentially and significantly induced. Among them, we identified those associated with drug detoxification, cell cycle control, apoptosis, and tumor-suppressor. These findings indicate that at least two apoptotic pathways were induced after cyclophosphamide treatment. The induction of APAF1 and two genes coding for two subunits of cytochrome c supports a previous report showing increased apoptosis in lymphocytes from SLE patients. The present study provides new information on the molecular mechanism underlying the cell response to genotoxic stress, with relevance to basic and clinical research.     

Comparison of the expression profiles induced by genotoxic and nongenotoxic carcinogens in rat liver

Application of recently developed gene expression techniques using microarrays in toxicological studies (toxicogenomics) facilitate the interpretation of a toxic compound's mode of action and may also allow the prediction of selected toxic effects based on gene expression changes. In order to test this hypothesis, we investigated whether carcinogens at doses known to induce liver tumors in the 2-year rat bioassay deregulate characteristic sets of genes in a short term in vivo study and whether these deregulated genes represent defined biological pathways. Male Wistar rats were dosed with the four nongenotoxic hepatocarcinogens methapyrilene (MPy, 60 mg/kg/day), diethylstilbestrol (DES, 10 mg/kg/day), Wy-14643 (Wy, 60 mg/kg/day), and piperonylbutoxide (PBO, 1200 mg/kg/day). After 1, 3, 7, and 14 days, the livers were taken for histopathological evaluation and for analysis of the gene expression profiles on Affymetrix RG_U34A arrays. The expression profile of the four nongenotoxic carcinogens were compared to the profiles of the four genotoxic carcinogens 2-nitrofluorene (2-NF), dimethylnitrosamine (DMN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and aflatoxin B1 (AB1) from a similar study reported previously. By using statistical and clustering tools characteristically deregulated genes were extracted and functionally classified. Distinct cellular pathways were affected by the nongenotoxic carcinogens compared to the genotoxic carcinogens which at least partly correlated with the two-stage model of carcinogenesis. Characteristic to genotoxic carcinogens were a DNA damage response and the activation of proliferative and survival signaling. Nongenotoxic carcinogens showed responses to oxidative DNA or protein damage, as well as cell cycle progression and signs of regeneration. Many of the gene alterations found with the nongenotoxic carcinogens imply compound-specific mechanisms. Although neither a single gene nor a single pathway will be sufficient to discriminate the two classes of carcinogens, it became evident that combinations of pathway-associated gene expression profiles may be used to predict a genotoxic or nongenotoxic carcinogenic potential of a compound in short-term studies.     

Physiological function as regulation of large transcriptional programs: the cellular response to genotoxic stress

The responses to ionizing radiation and other genotoxic environmental stresses are complex and are regulated by a number of overlapping molecular pathways. One such stress signaling pathway involves p53, which regulates the expression of over 100 genes already identified. It is also becoming increasingly apparent that the pattern of stress gene expression has some cell type specificity. It may be possible to exploit these differences in stress gene responsiveness as molecular markers through the use of a combined informatics and functional genomics approach. The techniques of microarray analysis potentially offer the opportunity to monitor changes in gene expression across the entire set of expressed genes in a cell or organism. As an initial step in the development of a functional genomics approach to stress gene analysis, we have recently demonstrated the utility of cDNA microarray hybridization to measure radiation-stress gene responses and identified a number of previously unknown radiation-regulated genes. The responses of some of these genes to DNA-damaging agents vary widely in cell lines from different tissues of origin and different genetic backgrounds. While this again highlights the importance of a cellular context to genotoxic stress responses, it also raises the prospect of expression-profiling of cell lines, tissues, and tumors. Such profiles may have a predictive value if they can define regions of ‘expression space’ that correlate with important endpoints, such as response to cancer therapy regimens, or identification of exposures to environmental toxins.     

A genotoxic screen: rapid analysis of cellular dose-response to a wide range of agents that either damage DNA or alter genome maintenance pathways

SNP analysis has come to the forefront of genomics since the mouse and human genomes have been sequenced. High throughput functional screens are necessary to evaluate these sequence databases. Described here is a genotoxic screen: a rapid method that determines the cellular dose-response to a wide range of agents that either damage DNA or alter basic cellular pathways important for maintaining genomic integrity. Importantly, a single person utilizing standard tissue culture equipment may perform these assays composed of 20 agents that attack genomic integrity or maintenance at many different levels. Thus, a small lab may perform this screen to determine the integrity of a wide range of DNA repair, chromatin metabolism, and response pathways without the limitations of investigator bias. A genotoxic screen will be useful when analyzing cells with either known genetic alterations (generated directly by the investigator or derived from individuals with known mutations) or unknown genetic alterations (cells with spontaneous mutations such as cancer-derived cells). Screening many genotoxins at one time will aid in determining the biological importance of these altered genes. Here we show the dose-response curves of mouse embryonic stem (ES) cells and HeLa cells exposed to 20 genotoxic agents. ES cells were chosen since they are amenable to genetic alteration by the investigator. HeLa cells were chosen since they were derived from cancer and are commonly used. Comparing the dose-response curves of these two cell lines show their relative sensitivity to these agents and helps define their genotoxic profile. As a part of phenomics, a large genotoxic profile database for cancer-derived cells, when integrated with other databases such as expression profiles and comparative genomic hybridization, may aid in maximizing the effectiveness of developing anti-cancer protocols.     

Discrimination of genotoxic and non-genotoxic hepatocarcinogens by statistical analysis based on gene expression profiling in the mouse liver as determined by quantitative real-time PCR

The general aim of the present study is to discriminate between mouse genotoxic and non-genotoxic hepatocarcinogens via selected gene expression patterns in the liver as analyzed by quantitative real-time PCR (qPCR) and statistical analysis. qPCR was conducted on liver samples from groups of 5 male, 9-week-old B6C3F(1) mice, at 4 and 48h following a single intraperitoneal administration of chemicals. We quantified 35 genes selected from our previous DNA microarray studies using 12 different chemicals: 8 genotoxic hepatocarcinogens (2-acetylaminofluorene, 2,4-diaminotoluene, diisopropanolnitrosamine, 4-dimethylaminoazobenzene, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, N-nitrosomorpholine, quinoline and urethane) and 4 non-genotoxic hepatocarcinogens (1,4-dichlorobenzene, dichlorodiphenyltrichloroethane, di(2-ethylhexyl)phthalate and furan). A considerable number of genes exhibited significant changes in their gene expression ratios (experimental group/control group) analyzed statistically by the Dunnett's test and Welch's t-test. Finally, we distinguished between the genotoxic and non-genotoxic hepatocarcinogens by statistical analysis using principal component analysis (PCA) of the gene expression profiles for 7 genes (Btg2, Ccnf, Ccng1, Lpr1, Mbd1, Phlda3 and Tubb2c) at 4h and for 12 genes (Aen, Bax, Btg2, Ccnf, Ccng1, Cdkn1a, Gdf15, Lrp1, Mbd1, Phlda3, Plk2 and Tubb2c) at 48h. Seven major biological processes were extracted from the gene ontology analysis: apoptosis, the cell cycle, cell proliferation, DNA damage, DNA repair, oncogenes and tumor suppression. The major, biologically relevant gene pathway suggested was the DNA damage response pathway, resulting from signal transduction by a p53-class mediator leading to the induction of apoptosis. Eight genes (Aen, Bax, Btg2, Ccng1, Cdkn1a, Gdf15, Phlda3 and Plk2) that are directly associated with Trp53 contributed to the PCA. The current findings demonstrate a successful discrimination between genotoxic and non-genotoxic hepatocarcinogens, using qPCR and PCA, on 12 genes associated with a Trp53-mediated signaling pathway for DNA damage response at 4 and 48 h after a single administration of chemicals.     

Gene expression patterns associated with infertility in humans and rodent models

Modern genomic technologies such as DNA arrays provide the means to investigate molecular interactions at an unprecedented level, and arrays have been used to carry out gene expression profiling as a means of identifying candidate genes involved in molecular mechanisms underlying a variety of phenotypes. By comparing gene expression profiles from normal and abnormal human testes with those from comparable infertile mouse models, we endeavored to identify genes and gene networks critical for male fertility. We used commercially available filter-based DNA arrays to analyze testicular gene expression from eight human testis biopsies and three different infertile mouse models (atrichosis mutation, ataxia telangiectasia knockout and CREMtau knockout). Forty-seven mouse genes exhibited differential testicular gene expression (P <0.01) associated with male infertility. These included genes involved in DNA repair (Vim, Rad23A, Rad23B), glutathione metabolism (Gsr, Gstp 1, Mgst1), proteolysis (Ace, Casp1, Ctsd), spermatogenesis (Prlr, Tmsb4 and Zfp-37) and stress response (Hsp 1, Osp94). The expression of 19 human genes was different (P<0.05) between normal and abnormal samples, including those associated with apoptosis (GADD45), gonad development (SOX9), proteolysis (PSMC3, SPINK2, TIMP3, UBE213) and signal transduction (DLK1, NAP4, S100A10). Direct comparison of differentially expressed human and mouse genes identified glucose phosphate isomerase, and the highly similar human tissue inhibitor of metalloproteinase 3 (TIMP3) and mouse Timp2. Using DNA microarrays to profile gene expression in testes from infertile animal models and humans will be useful for understanding congenital infertility, and also infertility caused by environmental exposures where the same genes and molecular mechanisms are involved.     

The nuclear guanine nucleotide exchange factors Ect2 and Net1 regulate RhoB-mediated cell death after DNA damage

Commonly used antitumor treatments, including radiation and chemotherapy, function by damaging the DNA of rapidly proliferating cells. However, resistance to these agents is a predominant clinical problem. A member of the Rho family of small GTPases, RhoB has been shown to be integral in mediating cell death after ionizing radiation (IR) or other DNA damaging agents in Ras-transformed cell lines. In addition, RhoB protein expression increases after genotoxic stress, and loss of RhoB expression causes radio- and chemotherapeutic resistance. However, the signaling pathways that govern RhoB-induced cell death after DNA damage remain enigmatic. Here, we show that RhoB activity increases in human breast and cervical cancer cell lines after treatment with DNA damaging agents. Furthermore, RhoB activity is necessary for DNA damage-induced cell death, as the stable loss of RhoB protein expression using shRNA partially protects cells and prevents the phosphorylation of c-Jun N-terminal kinases (JNKs) and the induction of the pro-apoptotic protein Bim after IR. The increase in RhoB activity after genotoxic stress is associated with increased activity of the nuclear guanine nucleotide exchange factors (GEFs), Ect2 and Net1, but not the cytoplasmic GEFs p115 RhoGEF or Vav2. Importantly, loss of Ect2 and Net1 via siRNA-mediated protein knock-down inhibited IR-induced increases in RhoB activity, reduced apoptotic signaling events, and protected cells from IR-induced cell death. Collectively, these data suggest a mechanism involving the nuclear GEFs Ect2 and Net1 for activating RhoB after genotoxic stress, thereby facilitating cell death after treatment with DNA damaging agents.     

Protein kinase C delta regulates Ser46 phosphorylation of p53 tumor suppressor in the apoptotic response to DNA damage

The p53 tumor suppressor is activated in the cellular response to genotoxic stress. Transactivation of p53 target genes dictates cell cycle arrest and DNA repair or induction of apoptosis; however, a molecular mechanism responsible for these distinct functions remains unclear. Recent studies revealed that phosphorylation of p53 on Ser(46) was associated with induction of p53AIP1 expression, resulting in the commitment of the cell fate into apoptotic cell death. Moreover, upon exposure to genotoxic stress, p53DINP1 was expressed and recruited a kinase(s) to p53 that specifically phosphorylated Ser(46). Here, we show that the pro-apoptotic kinase, protein kinase C delta (PKCdelta), is involved in phosphorylation of p53 on Ser(46). PKCdelta-mediated phosphorylation is required for the interaction of PKCdelta with p53. The results also demonstrate that p53DINP1 associates with PKCdelta upon exposure to genotoxic agents. Consistent with these results, PKCdelta potentiates p53-dependent apoptosis by Ser(46) phosphorylation in response to genotoxic stress. These findings indicate that PKCdelta regulates p53 to induce apoptotic cell death in the cellular response to DNA damage.     

Potassium bromate treatment predominantly causes large deletions, but not GC>TA transversion in human cells

Potassium bromate (KBrO(3)) is strongly carcinogenic in rodents and mutagenic in bacteria and mammalian cells in vitro. The proposed genotoxic mechanism for KBrO(3) is oxidative DNA damage. KBrO(3) can generate high yields of 8-hydroxydeoxyguanosine (8OHdG) DNA adducts, which cause GC>TA transversions in cell-free systems. In this study, we investigated the in vitro genotoxicity of KBrO(3) in human lymphoblastoid TK6 cells using the comet (COM) assay, the micronucleus (MN) test, and the thymidine kinase (TK) gene mutation assay. After a 4h treatment, the alkaline and neutral COM assay demonstrated that KBrO(3) directly yielded DNA damages including DNA double strand breaks (DSBs). KBrO(3) also induced MN and TK mutations concentration-dependently. At the highest concentration (5mM), KBrO(3) induced MN and TK mutation frequencies that were over 30 times the background level. Molecular analysis revealed that 90% of the induced mutations were large deletions that involved loss of heterozygosity (LOH) at the TK locus. Ionizing-irradiation exhibited similar mutational spectrum in our system. These results indicate that the major genotoxicity of KBrO(3) may be due to DSBs that lead to large deletions rather than to 8OHdG adducts that lead to GC>TA transversions, as is commonly believed. To better understand the genotoxic mechanism of KBrO(3), we analyzed gene expression profiles of TK6 cells using Affymetrix Genechip. Some genes involved in stress, apoptosis, and DNA repair were up-regulated by the treatment of KBrO(3). However, we could not observe the similarity of gene expression profile in the treatment of KBrO(3) to ionizing-irradiation as well as oxidative damage inducers.