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Genomics and Proteomics
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Application of High-Density DNA Microarray 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
JASN August 2003, 14 (suppl 3) S284-S289; DOI: https://doi.org/10.1097/01.ASN.0000078023.30954.05
Ken Yoneda
Center for Comparative Respiratory Biology and Medicine, Department of Internal Medicine, University of California at Davis, Davis, California.
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Mary Mann-Jong Chang
Center for Comparative Respiratory Biology and Medicine, Department of Internal Medicine, University of California at Davis, Davis, California.
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Ken Chmiel
Center for Comparative Respiratory Biology and Medicine, Department of Internal Medicine, University of California at Davis, Davis, California.
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Yin Chen
Center for Comparative Respiratory Biology and Medicine, Department of Internal Medicine, University of California at Davis, Davis, California.
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Reen Wu
Center for Comparative Respiratory Biology and Medicine, Department of Internal Medicine, University of California at Davis, Davis, California.
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Abstract

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

Conducting airway is one of the primary targeted tissues that are constantly exposed to various air pollutants, including those of environmental oxidant pollutants, tobacco smoke, and ozone. To exert the first line of pulmonary defense, airway epithelium is responsible for the maintenance of mucociliary clearance that traps and removes various inhaled air particulate and infectious agents (1–3⇓⇓). However, this function is frequently impaired in airways after an exposure to these environmental oxidant pollutants. This impairment may lead to more damage on airway epithelium by environmental air pollutants and the development of various pulmonary diseases (4–6⇓⇓). The effects of these oxidant pollutants on airway cells are not completely understood. Morphologic assessments of the injury on experimental animals reveal a wide range of cell injury and dysfunction of the epithelium, inflammation, and various tissue remodeling evidences (7). In addition, there is strong evidence to support the notion that the response by airway epithelial cells to these insults is complicated and varied among different individuals, different airway regions, and the chemical nature of the exposures. The difference in the responsiveness of these targeted epithelial cells to environmental oxidants is likely a reflection of differential gene expression by these cells. A functional genomic approach at the expression level allows the examination of the relationship between the change in gene expression pattern and the process of injury and response. Such a study will provide advanced knowledge at the molecular level on how to contest various respiratory diseases that seemed to be caused by environmental oxidant pollutants.

Large-scale study of thousands of gene expressions is a hallmark of the transition from “structural” to “functional” genomics, in which knowing the complete DNA sequence of the genome is only the first step in understanding how specific genes function. The next step, probably more challenging, is to sort out the biologic function of these genes, and the manner of these genes that are expressed is critical to various biologic processes. Central 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 able to sort out which pattern of differential gene expression is associated with a specific biologic event and what cellular process is carried out by a certain set of genes.

Several DNA microarray systems are available, which include the oligonucleotide arrays based on the “gene chip” concept (14,17,18⇓⇓) and developed by Affymetrix Incorp, and high-density DNA arrays on glass slide (8–11⇓⇓⇓) or on nylon membrane (16). All of these systems take the advantage of the high-density concept that nucleotide hybridization kinetics can be efficiently carried out. With computer-assisted software programs, images of the hybridization intensity on thousands of DNA dots on these gene chips can be quantified and clustered into various gene expression patterns (19–21⇓⇓). We previously developed a DNA microarray system based on spotting DNA on “positively charged nylon membrane” (16). The reason for choosing nylon membrane over the glass surface is that the amount of DNA spotted on nylon membrane is much higher than what a glass surface can receive. Thus, the hybridization kinetics is not limited by the amount of target DNA on the membrane (22). Using this system, we initiate the study to profiling the gene expression patterns associated with smoke- and hydrogen peroxide (H2O2)-induced injury and repair on an immortalized human bronchial epithelial cell line, HBE1 (23), and primary human bronchial epithelial cells.

Materials and Methods

Creating a High-Density DNA Microarray in Nylon Membrane

The procedure to generate target DNA inserts and array spotting on nylon membrane has been described (16). A similar procedure is used for DNA spotting on a glass slide surface. Most target DNA are either from expression sequence tag (EST) clones of the IMAGE Consortium human cDNA libraries or from cDNA clones derived from a cDNA library of a well-differentiated human tracheobronchial epithelial cell culture. Currently, there are approximately 45,000 UniEST clones with sequence verification available from Research Genetics. We used 9600 EST clones from this pool for this initial study. These amplified target DNA are then spotted high-density onto a nylon membrane with a robotic spotting machine (Arrayer). The one we used is the Arrayer-02 or -03 from the WITTECH Inc. (Taipei, Taiwan), which is capable of spotting DNA on an area smaller than 75 μm diameter and with 100 to 150 μm apart from each spot. The size of the DNA microarray membrane is 1.8 × 2.7 cm for 9600 DNA spots (cf. Figure 2).

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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 μg 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 × 2.7 cm size containing 9600 Uni-EST DNA spots. Hybridization condition and the color development were the same as those described previously (16).

DNA Microarray Imaging and Data Processing

The process of hybridization and detection of genes on filter membrane is a standard molecular biology technique in Northern and Southern blot hybridization analyses. For the DNA microarray membrane, a colorimetric detection method has been developed (16). This method is based on the well-established quantification method traditionally used in quantitative protein measurements, such as enzyme immunoassays or ELISA (24–26⇓⇓). cDNA probes labeled by digoxigenin- or biotin-dUTP are hybridized with the membrane. The hybridization results are then developed with a single or dual color through the enzyme/substrate reaction of color-forming enzymes. We estimated that each 75-μm-diameter DNA spot on nylon membrane contains more than 10 ng of DNA, which corresponds to approximately 109 molecules per spot (assuming 1000 bp DNA insert). This amount of target DNA is sufficient to carry out first-order kinetics to hybridize cDNA probes generated from mRNA templates, which is approximately 106 molecules per reaction per template.

After the color development, the image on each DNA dot is digitized by a scan on a high-resolution flat-bed scanner (Umax MagicScan at 3000 dpi). These digitized images are separated into cyan, magenta, and yellow colors. Based on the way the human eye perceives color, a color can be described by three components: hue, saturation, and brightness. A slight change in any of these three components results in a perceivable difference. By using true-color signals, the digitized image can be reasonably quantified (16). For quantifying the expression levels of known genes in a cell, six plant gene mRNA with different amounts are included in mammalian mRNA mixture in cDNA probe preparation during reverse transcription. Hybridization intensities on these plant gene DNA spots in the microarray DNA membrane served as internal references that are then used to quantify the level of the gene expression. Based on this approach, we demonstrated a sensitivity at one to two copies per cell level (at a hybridization condition with mRNA from 1 million cells) quantified by this colorimetry detection approach. This sensitivity is comparable with those fluorescence probes used on glass slide surface (24).

Cell Culture Condition

In this study, primary cells derived from human bronchial epithelial tissues and an immortalized human bronchial epithelial cell line, HBE1 (23), were cultured under an air-liquid interface culture condition 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 bronchial 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 μM) 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 synthesis and secretion, conspicuous mucus-secreting granule formation, and mucin gene message expression (unpublished observations). 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 genes whose expression is significantly elevated after H2O2 treatment. The genes found in this initial study were bcl-2 and nmn2 genes. The expression of these genes was the earliest induced ones by H2O2. The time course study with mRNA samples from cultures after H2O2 treatment further the inducibility of these genes by this treatment.

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Figure 1. Microarray analysis of gene expression pattern in airway epithelial cells after H2O2 treatment. Cells were cultured on plastic 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.

With the use of a dual-color approach, the effects of tobacco smoke on HBE1 cells were investigated on a microarray membrane containing 9600 EST cloned DNA (Figure 2). Both biotin- and digoxigenin-labeled cDNA probes were prepared from mRNA of cells untreated and treated with smoke, respectively. After hybridization and color development, the cDNA molecules labeled with biotin yielded a blue chromogen and the cDNA molecules labeled with digoxigenin appeared red. The majority of spots appeared purple, indicating the level at which the expression of these genes was not affected by smoke. However, some spots exhibited more distinctive colors, more toward the red or the blue, which can be used as an indication of differentially expressed genes after smoke exposure. After the image was digitized and these color spots were quantified, DNA clones with more distinctive color toward red were selected as smoke-induced genes. A similar approach was also carried out in H2O2-treated cells. Twenty-two genes were viewed as inducible genes (27), and 14 of these genes were further confirmed by Northern blot hybridization; all of them were commonly elevated in smoke- and H2O2-exposed cells. A time course Northern blot hybridization study was carried out to elucidate the time course induction by smoke (Figure 3). It seems that three phases of gene expression induction are recognized in this study. The first phase occurs immediately after the smoke and H2O2 treatment. These are genes of bcl-2, mdm-2 (see Figure 1), and mitogen-activated protein kinase phosphatase 1 (Figure 3). The second phase of genes induced by smoke and H2O2 are various stress proteins and ubiquitin genes, which seemed to be induced in cells after 5 h of exposure. The third phase of gene induction is gene products related to oxidant metabolism and cell tissue remodeling. The expression of these genes appeared 5 to 10 h later after smoke exposure. In addition to these 14 genes that are subsequently confirmed by Northern blot hybridization, other induced genes picked in this study required further confirmation at the mRNA level.

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Figure 3. Northern hybridization analysis for smoke-induced genes. RNA samples were isolated from these smoke-exposed cultures at 0, 5, 10, and 15 h after smoke exposure. 32P-labeled probes were prepared from these selected genes.

Discussion

We have developed a high-throughput microarray system on nylon membrane that can be used to profile differential gene expression patterns. The sensitivity of the detection is at one to two copies per cell, and the reactivity is linear to the level of the expression. The only limitation of the approach is the inherent nature of the color resolution, which is absorbance based at 1 to 2 order resolution, whereas the resolution for a fluorescence probe is at 3 to 5 orders of magnitude (24). Despite this limitation, the system is useful for a simultaneous quantification of much gene expression by a single hybridization step. The information generated from this approach is superior to that obtained by Northern blot hybridization, which is one gene per hybridization. What needs to be further improved in the system in the future, in addition to the improvement on the quantitative analysis and standardization and the cluster analysis, is the development of bioinformatics tools that can integrate various array data and pattern recognition with various biologic processes. Such a development will fulfill the theme of functional genomics and provide integrated information regarding the various molecular events associated with the cellular process.

Using the microarray membrane generated in our laboratory, we initiated the study to profile different gene expression patterns associated with H2O2- and smoke-induced injury and repair on human bronchial epithelial cell line HBE1. Northern blot and time course study were used to verify the expression pattern of these inducible genes. From this study, three phases of gene induction can be assessed (27). The first one is an immediate phase, in which inducible genes could be seen within 1 h after smoke exposure. These genes include MKP-1, mdm-2, and bcl-2. In contrast to MKP-1, the expression of the rest of the genes is not transient, and their inductions are persistently seen after 24 h after exposure. It is interesting that these genes are involved in the prevention of cell apoptosis. MKP-1 is an important negative regulator of mitogen-activated protein kinase pathways, especially the JNK/SAPK and p38 kinase pathways (28–30⇓⇓). The second phase of gene induction occurs later, and the induction is transient and the expression level is back to normal at 15 h after smoke exposure. The representative genes in this phase are HSP40, HSP70, HSP90a, and ubiquitin p62. Most of the functions of these genes are involved in repairing denatured proteins. Stress proteins are an important chaperone for the proper folding of macromolecules, whereas ubiquitin protein can tag those molecules that are irreparable for protease degradation. The third phase of gene induction occurs 10 h after smoke exposure. These genes are glutaredoxin, light chain of ferritin, dihydrodiol dehydrogenase, MMP-1, and SPRR1B. Except for MMP-1, most of the induced genes in this phase are involved in the metabolism of oxidants. MMP-1 is responsible for a variety tissue remodeling, whereas SPRR1B is a squamous cell differentiation marker (31) under AP-1 transcription factor’s control (32). Thus, an early but transient expression of MKP-1 may block the expression of this differentiated marker. Further study is needed to elucidate the significance of such a complex array in gene expression by bronchial epithelial cells after oxidant injury.

Acknowledgments

The authors acknowledge the grant support from the National Institutes of Health (ES09701, ES06230, HL35635, AI50496, ES04699, and ES05707) and the California Tobacco-Related Disease-Research Program (10RT-026).

  • © 2003 American Society of Nephrology

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Application of High-Density DNA Microarray 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, Reen Wu
JASN Aug 2003, 14 (suppl 3) S284-S289; DOI: 10.1097/01.ASN.0000078023.30954.05

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Application of High-Density DNA Microarray 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, Reen Wu
JASN Aug 2003, 14 (suppl 3) S284-S289; DOI: 10.1097/01.ASN.0000078023.30954.05
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