J Am Soc Nephrol 14: S284–S289, 2003 Application of High-Density DNA to Study Smoke- and Hydrogen Peroxide–Induced Injury and Repair in Human Bronchial Epithelial Cells

KEN YONEDA, MARY MANN-JONG CHANG, KEN CHMIEL, YIN CHEN, and REEN WU Center for Comparative Respiratory and , Department of Internal Medicine, University of California at Davis, Davis, California.

Abstract. Recent advances in high-density DNA microarray mdm2 that are involved in the regulation of apoptosis, technique allow the possibility to analyze thousands of genes and the mitogen-activated kinase phosphatase 1 that simultaneously for their differential expression patterns in functions as a regulator for various mitogen-activated protein various biologic processes. Through clustering analysis and kinase activities. The second phase, usually 5 h later, includes pattern recognition, the significance of these differentially ex- the induction of various stress and ubiquitin, which are pressed genes can be recognized and correlated with the bio- important in providing the chaperone mechanism and the turn- logic events that may take place inside the cell and tissue. over of damaged macromolecules. The third phase, which is 5 High-density DNA microarray nylon membranes were used to to 10 h later, includes the induction of genes that seem to be explore and regulation associated with smoke- involved in reducing oxidative stress by metabolizing the cel- and hydrogen peroxide–induced injury and repair in differen- lular level of reactive oxygen species. In this phase, enzymes tiated human bronchial epithelial cells in vitro. At least three associated with tissue and cell remodeling are also elevated. phases of change in gene expression could be recognized. The These results demonstrated a complex gene expression array first phase seems to be an immediate event in response to by bronchial epithelial cells in response to a single insult of oxidant injury. This phase includes the induction of bcl-2 and oxidants that are relevant to environmental pollutants.

Conducting airway is one of the primary targeted tissues that among different individuals, different airway regions, and the are constantly exposed to various air pollutants, including chemical nature of the exposures. The difference in the respon- those of environmental oxidant pollutants, tobacco smoke, and siveness of these targeted epithelial cells to environmental ozone. To exert the first line of pulmonary defense, airway oxidants is likely a reflection of differential gene expression by epithelium is responsible for the maintenance of mucociliary these cells. A functional genomic approach at the expression clearance that traps and removes various inhaled air particulate level allows the examination of the relationship between the and infectious agents (1–3). However, this function is fre- change in gene expression pattern and the process of injury and quently impaired in airways after an exposure to these envi- response. Such a study will provide advanced knowledge at the ronmental oxidant pollutants. This impairment may lead to molecular level on how to contest various respiratory diseases more damage on airway epithelium by environmental air pol- that seemed to be caused by environmental oxidant pollutants. lutants and the development of various pulmonary diseases Large-scale study of thousands of gene expressions is a hall- (4–6). The effects of these oxidant pollutants on airway cells mark of the transition from “structural” to “functional” genomics, are not completely understood. Morphologic assessments of in which knowing the complete DNA sequence of the genome is the injury on experimental animals reveal a wide range of cell only the first step in understanding how specific genes function. injury and dysfunction of the epithelium, inflammation, and The next step, probably more challenging, is to sort out the various tissue remodeling evidences (7). In addition, there is biologic function of these genes, and the manner of these genes strong evidence to support the notion that the response by that are expressed is critical to various biologic processes. Central airway epithelial cells to these insults is complicated and varied to the advancement of “functional” genomics is the development of high-density DNA microarray technology (8–16) that is able to profile simultaneously thousands of gene expressions and is also Correspondence to Dr. Reen Wu, Center for Comparative Respiratory Biology able to sort out which pattern of differential gene expression is and Medicine, Surge 1 Annex, Room 1121, University of California at Davis, One Shields Avenue, Davis, CA 95616; Phone: 530-752-2648; Fax: 530-752- associated with a specific biologic event and what cellular process 8623; E-mail: [email protected] is carried out by a certain set of genes. 1046-6673/1408-0284 Several DNA microarray systems are available, which in- Journal of the American Society of Nephrology clude the arrays based on the “gene chip” Copyright © 2003 by the American Society of Nephrology concept (14,17,18) and developed by Affymetrix Incorp, and DOI: 10.1097/01.ASN.0000078023.30954.05 high-density DNA arrays on slide (8–11) or on nylon J Am Soc Nephrol 14: S284–S289, 2003 Gene Expression Pattern Analyzed by DNA Microarray S285 membrane (16). All of these systems take the advantage of Materials and Methods the high-density concept that hybridization ki- Creating a High-Density DNA Microarray in Nylon netics can be efficiently carried out. With computer-assisted Membrane software programs, images of the hybridization intensity on The procedure to generate target DNA inserts and array spotting on thousands of DNA dots on these gene chips can be quanti- nylon membrane has been described (16). A similar procedure is used fied and clustered into various gene expression patterns for DNA spotting on a glass slide surface. Most target DNA are either (19–21). We previously developed a DNA microarray sys- from expression sequence tag (EST) clones of the IMAGE Consor- tem based on spotting DNA on “positively charged nylon tium human cDNA libraries or from cDNA clones derived from a membrane” (16). The reason for choosing nylon membrane cDNA library of a well-differentiated human tracheobronchial epithe- over the glass surface is that the amount of DNA spotted on lial . Currently, there are approximately 45,000 UniEST clones with sequence verification available from Research . nylon membrane is much higher than what a glass surface We used 9600 EST clones from this pool for this initial study. These can receive. Thus, the hybridization kinetics is not limited amplified target DNA are then spotted high-density onto a nylon by the amount of target DNA on the membrane (22). Using membrane with a robotic spotting machine (Arrayer). The one we this system, we initiate the study to profiling the gene used is the Arrayer-02 or -03 from the WITTECH Inc. (Taipei, expression patterns associated with smoke- and hydrogen Taiwan), which is capable of spotting DNA on an area smaller than 75 peroxide (H2O2)-induced injury and repair on an immortal- ␮m diameter and with 100 to 150 ␮m apart from each spot. The size ized human bronchial epithelial cell line, HBE1 (23), and of the DNA microarray membrane is 1.8 ϫ 2.7 cm for 9600 DNA primary human bronchial epithelial cells. spots (cf. Figure 2).

Figure 1. Microarray analysis of gene expression pattern in airway epithelial cells after H2O2 treatment. Cells were cultured on culture dishes in a serum-free, hormone-supplemented medium (32). At 80% confluence, cultures were exposed to 0.1 mM H2O2. Total RNA were isolated from these cultures at 0 (A), 1 (B), 3 (C), and 48 (D) h later. These RNA were used to produce digoxigenin-cDNA probes as described previously (16). Hybridization on microarray membranes and data imaging and analysis were the same as described before (16). Genes whose expressions were elevated by H2O2 were circled as shown. S286 Journal of the American Society of Nephrology J Am Soc Nephrol 14: S284–S289, 2003

DNA Microarray Imaging and Data Processing cyan, magenta, and yellow colors. Based on the way the human eye The process of hybridization and detection of genes on filter perceives color, a color can be described by three components: hue, membrane is a standard technique in Northern and saturation, and brightness. A slight change in any of these three Southern blot hybridization analyses. For the DNA microarray mem- components results in a perceivable difference. By using true-color brane, a colorimetric detection method has been developed (16). This signals, the digitized image can be reasonably quantified (16). For method is based on the well-established quantification method tradi- quantifying the expression levels of known genes in a cell, six plant tionally used in quantitative protein measurements, such as enzyme gene mRNA with different amounts are included in mammalian immunoassays or ELISA (24–26). cDNA probes labeled by digoxi- mRNA mixture in cDNA probe preparation during reverse transcrip- genin- or -dUTP are hybridized with the membrane. The hy- tion. Hybridization intensities on these plant gene DNA spots in the bridization results are then developed with a single or dual color microarray DNA membrane served as internal references that are then through the enzyme/substrate reaction of color-forming enzymes. We used to quantify the level of the gene expression. Based on this estimated that each 75-␮m-diameter DNA spot on nylon membrane approach, we demonstrated a sensitivity at one to two copies per cell contains more than 10 ng of DNA, which corresponds to approxi- level (at a hybridization condition with mRNA from 1 million cells) mately 109 molecules per spot (assuming 1000 bp DNA insert). This quantified by this colorimetry detection approach. This sensitivity is amount of target DNA is sufficient to carry out first-order kinetics to comparable with those probes used on glass slide surface hybridize cDNA probes generated from mRNA templates, which is (24). approximately 106 molecules per reaction per template. After the color development, the image on each DNA dot is Cell Culture Condition digitized by a scan on a high-resolution flat-bed scanner (Umax In this study, primary cells derived from human bronchial epithelial MagicScan at 3000 dpi). These digitized images are separated into tissues and an immortalized human bronchial epithelial cell line,

Figure 2. Dual-color microarray analysis of smoke-induced gene expression. HBE1 cells were cultured in a condition as described in Figure 1. Fifteen hours after smoke exposure, total RNA were isolated from these cultures treated or untreated with smoke. These RNA were further purified for mRNA preparation. Approximately 1 ␮g of mRNA from each smoke-treated and -untreated culture was used to generate digoxigenin- and biotin-labeled cDNA probes, respectively. Hybridizations were carried out on a membrane of 1.8 ϫ 2.7 cm size containing 9600 Uni-EST DNA spots. Hybridization condition and the color development were the same as those described previously (16). J Am Soc Nephrol 14: S284–S289, 2003 Gene Expression Pattern Analyzed by DNA Microarray S287

HBE1 (23), were cultured under an air-liquid interface culture con- dition in a defined serum-free hormone-supplemented medium as described before (24). Primary human bronchial tissues were obtained from the local hospitals at UC Davis Medical Center with consent. HBE1 cell line is a clonal, papillomavirus-immortalized human bron- chial epithelial cell line (23). These cultured cells at days 14 to 21 under the described culture condition expressed various mucociliary functions. It was under such a differentiated condition that these cells ␮ were exposed to H2O2 (20 to 200 M) and smoke. At various times after these treatments, cultures were harvested for RNA isolation (24).

Results These human bronchial epithelial cells grown under an air- liquid interface cell culture system are able to express mucous cell differentiation activity with an expression of mucin syn- thesis and secretion, conspicuous mucus-secreting granule for- mation, and mucin gene message expression (unpublished ob- servations). Figure 1 shows a single-color approach on the

hybridization of cDNA probes of H2O2-treated and untreated control cultures on a microarray membrane. Arrows indicate Figure 3. Northern hybridization analysis for smoke-induced genes. genes whose expression is significantly elevated after H O 2 2 RNA samples were isolated from these smoke-exposed cultures at 0, treatment. The genes found in this initial study were bcl-2 and 5, 10, and 15 h after smoke exposure. 32P-labeled probes were nmn2 genes. The expression of these genes was the earliest prepared from these selected genes. induced ones by H2O2. The time course study with mRNA samples from cultures after H2O2 treatment further the induc- ibility of these genes by this treatment. expression of these genes appeared 5 to 10 h later after smoke With the use of a dual-color approach, the effects of tobacco exposure. In addition to these 14 genes that are subsequently smoke on HBE1 cells were investigated on a microarray mem- confirmed by hybridization, other induced genes brane containing 9600 EST cloned DNA (Figure 2). Both picked in this study required further confirmation at the mRNA biotin- and digoxigenin-labeled cDNA probes were prepared level. from mRNA of cells untreated and treated with smoke, respec- tively. After hybridization and color development, the cDNA Discussion molecules labeled with biotin yielded a blue chromogen and We have developed a high-throughput microarray system on the cDNA molecules labeled with digoxigenin appeared red. nylon membrane that can be used to profile differential gene The majority of spots appeared purple, indicating the level at expression patterns. The sensitivity of the detection is at one to which the expression of these genes was not affected by two copies per cell, and the reactivity is linear to the level of smoke. However, some spots exhibited more distinctive colors, the expression. The only limitation of the approach is the more toward the red or the blue, which can be used as an inherent nature of the color resolution, which is absorbance indication of differentially expressed genes after smoke expo- based at 1 to 2 order resolution, whereas the resolution for a sure. After the image was digitized and these color spots were fluorescence probe is at 3 to 5 orders of magnitude (24). quantified, DNA clones with more distinctive color toward red Despite this limitation, the system is useful for a simultaneous were selected as smoke-induced genes. A similar approach was quantification of much gene expression by a single hybridiza-

also carried out in H2O2-treated cells. Twenty-two genes were tion step. The information generated from this approach is viewed as inducible genes (27), and 14 of these genes were superior to that obtained by Northern blot hybridization, which further confirmed by Northern blot hybridization; all of them is one gene per hybridization. What needs to be further im-

were commonly elevated in smoke- and H2O2-exposed cells. A proved in the system in the future, in addition to the improve- time course Northern blot hybridization study was carried out ment on the quantitative analysis and standardization and the to elucidate the time course induction by smoke (Figure 3). It , is the development of tools that seems that three phases of gene expression induction are rec- can integrate various array data and pattern recognition with ognized in this study. The first phase occurs immediately after various biologic processes. Such a development will fulfill the

the smoke and H2O2 treatment. These are genes of bcl-2, theme of and provide integrated informa- mdm-2 (see Figure 1), and mitogen-activated protein kinase tion regarding the various molecular events associated with the phosphatase 1 (Figure 3). The second phase of genes induced cellular process.

by smoke and H2O2 are various stress proteins and ubiquitin Using the microarray membrane generated in our laboratory, genes, which seemed to be induced in cells after5hof we initiated the study to profile different gene expression

exposure. The third phase of gene induction is gene products patterns associated with H2O2- and smoke-induced injury and related to oxidant and cell tissue remodeling. The repair on human bronchial epithelial cell line HBE1. Northern S288 Journal of the American Society of Nephrology J Am Soc Nephrol 14: S284–S289, 2003 blot and time course study were used to verify the expression 6. Balmes JR, Aris RM, Chen LL, Scannell C, Tager IB, Finkbeiner pattern of these inducible genes. From this study, three phases W, Christian D, Kelley T, Hearne PQ, Ferrando R, Welch B: of gene induction can be assessed (27). The first one is an Effects of ozone on normal and potentially sensitive human immediate phase, in which inducible genes could be seen subjects. Part I: Airway inflammation and responsiveness to within 1 h after smoke exposure. These genes include MKP-1, ozone in normal and asthmatic subjects. Res Rep Health Eff Inst mdm-2, and bcl-2. 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