Functional genomics of UV radiation responses in human cells

Mutation Research 549 (2004) 65–78

Functional genomics of UV radiation responses in human cells Christine A. Koch-Paiz a , Sally A. Amundson a,1 , Michael L. Bittner b,2 , Paul S. Meltzer b , Albert J. Fornace Jr. a,∗ a

Gene Response Section, Center for Cancer Research, NCI, Bethesda, MD, USA b Cancer Genetics Branch, NHGRI, Bethesda, MD, USA

Received 4 January 2004; received in revised form 26 January 2004; accepted 26 January 2004

Abstract 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. © 2004 Elsevier B.V. All rights reserved. Keywords: UV irradiation; cDNA microarray hybridization; Cell signaling; Suramin

1. Introduction

∗ Corresponding author. Tel.: +1-301-402-0745; fax: +1-301-480-1946. E-mail address: [email protected] (A.J. Fornace Jr.). 1 Present address: Center For Radiologic Research, Columbia University, NY, USA. 2 Present address: Translational Genomics Research Institute (TGen), Phoenix, AZ, USA.

Genotoxic stress responses are complex, and can involve a substantial portion of the eukaryotic genome. Early studies in yeast indicated that numerous genes, likely more than 1% of the organism’s genome [1], were regulated in response to genotoxic stress. With the advent of functional genomics approaches to survey large portions of the genome, estimates of the fraction of responsive genes have increased even further;

0027-5107/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2004.01.010

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e.g., up to 20% of the 6200 genes in yeast may actually be responsive to genotoxic stresses as determined by microarray hybridization [1]. These functional genomic approaches are being applied further to define stress-specific signatures or “fingerprints” for different classes of toxicants [2–4]. For example, gene expression profiles were found to distinguish between treatment with the genotoxic agent cisplatin and non-genotoxic agents [5], and the response to ionizing radiation (IR) was distinguished from responses to DNA base damaging agents, such as cisplatin and UV radiation [6]. In addition to the variations in gene expression profiles found in response to different types of damaging agents, there can also be substantial variation in gene responses depending on the cell type [7]. As discussed recently [8], the patterns of stress gene responses to IR often show substantial differences in various human cell lines, implying variation among the responses of individuals. The large number of potentially stress-responsive genes and the emerging complexity of the signaling networks involved, have inspired the use of functional genomics approaches in toxicological studies with both chemical agents and physical agents, such as UV radiation and IR [3–5,9]. In the specific case of photobiology, the toxicogenomic research can be expected to provide insight into the injury and protective responses of cells exposed to radiation of various UV wavelengths, as well as the inter-individual variability in such responses. Different wavelengths of UV radiation produce a wide range of lesions in DNA and other cellular targets, triggering complex patterns of stress responses. The UV spectrum is divided into three general classes on the basis of wavelength: UVA (>320 nm), UVB radiation (280–320 nm), and UVC (<280 nm). Probably, the most important of these for cytotoxicity are the middle wavelengths of UVB radiation [10]. Highly energetic UVC radiation is not relevant from a public health standpoint, since essentially all of it is absorbed by atmospheric ozone. However, because UVC radiation studies can be carried out with inexpensive germicidal lights that emit primarily at 254 nm, near the maximum for DNA absorption, there is vast literature using this type of UV radiation. The cytotoxicity of UVC radiation is due primarily to well-defined DNA lesions, such as pyrimidine dimers and pyrimidine–pyrimidone (6–4) photoprod-

ucts, which are repaired by nucleotide excision repair mechanisms [10]. While the same lesions are generated by UVB radiation, they here account for only about half the cytotoxicity, with most of the remainder due to oxidative damage to DNA and other cellular targets. Both UVB and UVA radiation generate appreciable levels of reactive oxygen species, such as various peroxides, singlet oxygen, and transient hydroxyl radicals. Damage by the less energetic UVA radiation is primarily due to oxidative damage with production of only negligible levels of photoproducts, such as pyrimidine dimers [10,11]. The overall frequency of DNA lesions, induced by UVB or UVC radiation compared to equitoxic doses of IR, is more than two-orders of magnitude greater, and thus, UVB and UVC radiation probably trigger some responses more strongly than IR. As discussed in more detail elsewhere [7], UV radiation can trigger a variety of signaling pathways, either through direct DNA damage or via reactive oxygen species in the nucleus, plasma membrane, cytoplasm, and mitochondria. As with exposure to many genotoxic agents, p53-signaling plays an important role in mediating UV-induced DNA damage responses. However, UV radiation also triggers prominent membrane-mediated signaling, such as rapid activation of receptor tyrosine kinases including growth factor and cytokine receptors [12,13]. These receptor pathways lead to rapid activation of MAP kinase signaling presumably by UV radiation damage to membrane targets, although there is also evidence for a DNA damage component to MAP kinase signaling [14]. This UV radiation responsiveness of the MAP kinase pathway has been conserved from yeast to mammalian cells [15], highlighting its importance. Interestingly, UV irradiation led to activation of NF␬B signaling and MAP kinase signaling even in enucleated cells, which strongly argues for direct membrane targets [16]. Clustering of growth factor receptors in the absence of ligand has also been observed soon after UV irradiation [13]. UV radiation signaling can also be blocked using general growth factor receptor inhibitors, such as suramin. For example, suramin blocked UV-induction of GADD45A and GADD153, but had no effect on signaling after exposure to IR or methylmethane sulfonate [17], consistent with the stronger activation of the MAP kinase pathway by UV radiation than by IR [8,18].

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In the current study, we have used suramin to selectively block the membrane receptor-mediated signaling after UV radiation. cDNA microarray hybridization was carried out in a responsive p53 wild-type (wt) human cell line after UVB irradiation and after equitoxic doses of UVC and UVA radiation. A large number of responsive genes were detected, and suramin attenuated the responses in nearly all the cases. Inhibition by suramin was greater for UVC compared to UVB irradiation, which probably reflects more prominent signaling by reactive oxygen species in the latter.

2. Materials and methods 2.1. Cell culture and treatment The human breast cancer line MCF-7 was grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Biowhittaker, Walkersville, MD) as described previously [19]. Stock cultures were maintained without antibiotics, and were monitored periodically to ensure they remained free of mycoplasma contamination. One hundred units per milliliter of penicillin and 100 ␮g/ml streptomycin were added to the medium of experimental cultures prior to irradiation. Clonogenic cell survival studies were carried out to determine the dose producing 37% survival for MCF-7 cells irradiated with UVA, UVB, and UVC wavelengths. Cells were irradiated in RPMI saline, a phosphate-buffered saline formula (0.1 M NaCl, 11 mM glucose, 5.6 mM Na2 HPO4 , 5.4 mM KCl, 0.4 mM Ca(NO3 )2 , 0.4 mM MgSO4 , pH 7.0) [20], and then plated at different densities in duplicate for 7–10 days. Colony survival of replicate samples was carried out as described previously [19]. Based on clonogenic survival studies, cells were irradiated in 150 mm culture dishes, with 50 kJ m−2 UVA radiation using a Honle UVA system with H1 filter (315–400 nm) (Honle UV America Inc., Marlboro, MA) with a dose rate of 510 J m−2 s−1 ; at 125 J m−2 UVB radiation using four Westinghouse FS20 SunLamp bulbs (270–385 nm emission spectrum with peak at 313 nm) with a dose rate of 1.19 J m−2 s−1 ; and at 12 J m−2 UVC radiation using four General Electric 15 W G15T8 Germicidal (254 nm) bulbs with a dose rate of 0.402 J m−2 s−1 . The energy emitted by the lamps was measured with a model PMA2100 me-

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ter (Solar Light Co., Philadelphia, PA). After irradiation, saline was replaced with complete medium. For UVA irradiation, cells were exposed in 150 mm culture plates under temperature-controlled conditions to avoid overheating of cells during irradiation. For all experiments, mock-irradiated parallel control cultures were handled identically with the experimental cultures, except they were not exposed to UV radiation. A stock solution of 30 mM suramin sodium salt (Calbiochem, La Jolla, CA; CAS number: 129-46-4) in water was diluted 100-fold for cell treatment. MCF-7 cells were grown in 150 mm tissue culture dishes, and RPMI 1640 medium was changed the day before treatment. On the day of treatment, 0.3 mM suramin was added to cells 45 min prior to irradiation or mock-irradiation for controls. For irradiation, the suramin-containing medium was removed and retained; the cells were rinsed once with phosphate-buffered saline and were UV-irradiated in 5 ml of RPMI saline containing 0.3 mM suramin at the same doses of UV radiation as described above. Control cultures were treated in an identical manner except for UV radiation exposure. After irradiation the saline was removed and the suramin-containing medium was returned to the cells. Cells were incubated at 37 ◦ C for the indicated times prior to harvest. 2.2. Measurement of relative gene expression Following incubation after treatment, RNA was extracted using the guanidine thiocyanate method [21]. Serial dilutions of RNA were immobilized on nylon membranes, hybridized with cDNA probes at 52 ◦ C in a buffer containing 50% formamide (Hybrisol I, Oncor, Gaithersburg, MD), and washed under standard conditions. Hybridization was quantitated on a phosphorimager (Molecular Dynamics, Piscataway, NJ) and relative signal levels, normalized to the poly(A) content of each sample, were determined using the RNA-Think program. Results represent the average of at least four determinations. Using this approach, the values for relative RNA levels are directly proportional to RNA abundance, and differences of 1.5-fold or more can be reliably measured [22]. Unless otherwise specified, RNA for these experiments was isolated from cells on different days than that used for microarray analyses.

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2.3. Microarray analysis One hundred to one hundred and twenty micrograms of whole-cell RNA were labeled and hybridized to a 7684 element microarray [23] as described previously [24]. Probes were prepared by PCR amplification of IMAGE consortium clones and arrayed on poly-l-lysine coated glass slides. In addition, 96 additional cDNA clones of relevant stress response genes, such as WAF1 (CIP1/WAF1, Cdkna1) were included. Fluorescent labeled cDNA was prepared from mock-irradiated control and UVA, UVB, and UVC irradiated MCF-7 whole-cell RNA by a single round of reverse transcription (Superscript II, Invitrogen, Carlsbad, CA) in the presence of fluorescent dUTP (Cy3 dUTP or Cy5 dUTP, Perkin-Elmer, Wellesley, MA). Experiments (summarized in Fig. 3) were carried out separately on different days. To highlight reproducibility, RNA samples from cells UVB-irradiated on three different days (and their accompanying mock-irradiated controls) were analyzed by microarray hybridization in replicate experiments, which were all carried out on different days. Probes and targets were hybridized together for 16–18 h in 3× SSC at 65 ◦ C in the presence of blocking agents, human CoT1 DNA, yeast tRNA, and polydeoxyadenine. Following hybridization, slides were washed at room temperature twice in 0.5× SSC, 0.01% SDS for 5 min and once in 0.06× SSC for 5 min. Cy3 and Cy5 fluorescence were scanned using a laser confocal scanner (Agilent Technologies, Palo Alto, CA) and images were analyzed using the ArraySuite 2.1 Extensions (Y. Chen, NHGRI) on the IPLab program (Scanalytics Inc., Fairfax, VA) to calibrate relative ratios and develop confidence intervals for their significance [25]. The ArraySuite Extensions analysis also incorporates four separate metrics of hybridization quality for each target, taking into account the intensity, background, and other characteristics of each spot for both fluorochromes. An overall quality assessment is then produced for each target on the array [26], and is used as a parameter for data filtering. The ratios were normalized to a set of 88 internal controls [26] with a theoretical ratio of 1.0. The variance in the housekeeping set was used to determine the significance of expression changes following UV irradiation. Up- or down-regulated genes were called at the level of 99% confidence for the current analysis.

Gene selection and cluster analysis of the microarray data was performed using the NHGRI Online microarray analysis tools (www.arrayanalysis.nih.gov) developed jointly with CIT, NIH. For identification of genes discriminating between different treatment conditions, the maximum pairwise t-statistic, distance-based method, Wilcoxon–Kruskal–Wallis statistic, and class-correlation methods were used. Genes identified as significantly discriminating the groups, and which occurred less than once in 10,000 iterations randomizing the groups, were compared for each of the methods. The final gene set included genes identified as significantly discriminating by at least three of the four selection methods, in order to produce a fairly stringent selection of the best possible discriminators for each comparison.

3. Results 3.1. Cellular responses to UV radiation The human cell line MCF-7 was chosen for several properties including functional wt p53, proficient stress-induced cell cycle checkpoint activation, well-characterized genotoxic stress responses, and proficient DNA repair [27,28]. A moderately toxic dose of UVB radiation was chosen which also produced appreciable changes in gene expression. As shown in Fig. 1, a dose of 125 J m−2 reduced clonogenic survival to 37% (D37 ). D37 doses for UVC and UVA radiation were determined to be 12 J m−2 and 50 ± 10 kJ m−2 , respectively. In order to keep the radiation conditions as constant as possible, cells were irradiated in buffered saline, and this probably increased the apparent incident dose required to reduce survival to 37% after UVC radiation. In addition, MCF-7 cells were relatively resistant to UV radiation; e.g., the D37 doses for MCF-7 cells reduced clonogenic survival in RKO cells (another p53 wt human line [24]) to 10–8% after UVB and UVC radiation, respectively (data not shown). 3.2. Time course of transcriptional responses to UV radiation Quantitative single-probe hybridization studies were carried out to choose optimal times for cDNA

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Fig. 1. Cytotoxicity of different wavelengths of UV radiation. Clonogenic survival was carried out after: (A) UVB; (B) UVC; or (C) UVA irradiation of MCF-7 cells in saline as described in Section 2.

microarray hybridization. Three well-characterized stress response genes (e.g., see [29]), WAF1 (CIP1/ WAF1, Cdkna1), DDB2, and GADD45A, were studied and relative changes in their transcript levels are shown in Fig. 2A–C. All genes showed appreciable induction 12 h after UVB irradiation and 6 h after UVC and UVA irradiation. Induction usually persisted for at least 8 h after irradiation; so these time points, 12 h for UVB and 6 h for UVC and UVA irradiation, were chosen. Particularly in the case of UVA radiation, a second gene induction peak frequently occurred later at 24 h after irradiation; because of this and since the overall responses were weaker after UVA radiation, an additional time point at 24 h was also included for microarray analysis. 3.3. Identification of UV-responsive genes by cDNA microarray hybridization A cDNA set containing nearly all named human genes was used to identify UVB-responsive genes, and then to compare these with responses to the other UV wavelengths. The set of 7684 genes on the array was filtered to include only those exceeding established thresholds for hybridization intensity and quality (see Section 2). From this filtered gene set, a set of 310 UVB-responsive genes significantly induced or repressed in one or more of the UVB experiments was identified. Determination of significant ratio change is

based on the 99% confidence interval developed from the range for the housekeeping gene set in each microarray hybridization, as described in Section 2. With a large microarray, it is likely that some of these genes may not be consistently responsive. A set of 172 genes was next identified that was reproducibly responsive in at least two-thirds of the UVB experiments. From these genes, a subset of 155 genes that showed an average induction or repression of greater than or equal to 2-fold was developed. This subset is summarized in Fig. 3A, where induction and repression are designated by red and green shadings, respectively. Interestingly, there were more repressed genes (114) than induced genes (41). The magnitude of responsiveness varied widely with a maximum for average induction of 27-fold for WAF1 and the greatest average repression for catenin, 16% of the level in un-irradiated cells. Results for UVA and UVC irradiated cells are included for comparison, and there are many cases where responsiveness appeared to be UVB-specific. 3.4. Validation of cDNA microarray hybridization results Considering that microarray hybridization utilizes complex probes containing thousands of different sequences, quantitation is challenging even with advanced data analysis [30]. To directly determine the accuracy of our microarray approach, array results

70 C.A. Koch-Paiz et al. / Mutation Research 549 (2004) 65–78 Fig. 2. Relative mRNA levels after UV irradiation. Cells were treated with 125 J m−2 UVB (A and D), 12 J m−2 UVC (B and E), or 50 kJ m−2 UVA (C) radiation and harvested at the indicated times for quantitative single-probe hybridization analysis. Values represent the relative expression of irradiated samples normalized to un-irradiated controls. A value of 1 indicates no responsiveness and is shown at the zero time point in (C) and (F); for clarity, the zero time points have been omitted from the other panels. Results designated by dashed lines refer to the right y-axes of (A) for WAF1, (B) for WAF1, (D) for cytokine inducible factor ATF3 and BTG2, and (E) for BTG2 and EGR1. (D) Shows repression of polo-like kinase (Image ID: 744047) by UVB and UVC irradiation.

C.A. Koch-Paiz et al. / Mutation Research 549 (2004) 65–78 Table 1 Comparison of hybridizationa Probesb

WAF1 DDB2 BTG2 Cytokine inducible kinase Killer/DR5 Polo-like kinase

microarray Image ID

753447 213136 825080

744047

to

quantitative

UVBc

single-probe

induction at 24 h after UVC radiation. The cause for this biphasic response is uncertain, but this similarity may well reflect common regulatory mechanisms.

UVCd

Arraye

Blotf

Arraye

Blotf

26.7 4.8 6.6 4.0

34.6 9.7 15.0 10.7

24.0 2.3 13.8 7.9

26.7 3.9 16.1 15.9

4.1 0.31

71

3.8 0.22

2.2 0.22

4.6 0.21

a Representative results are shown for relative gene expression by cDNA microarray compared to quantitative single-probe hybridization (see Section 2). b cDNA probes for the indicated Image ID sequences were used for quantitative single-probe hybridization. The CIP1/WAF1 (CDKN1A) and Killer/DR5 are not image clones. c Cells were treated with 125 J m−2 of UVB radiation, and harvested 12 h later. d Cells were treated with 12 J m−2 of UVC radiation, and harvested 6 h later. e Results from cDNA microarray hybridization represent average of different experiments. f The same RNA samples were used to measure relative expression by quantitative single-probe hybridization; results were normalized to untreated controls (Section 2).

were compared to quantitative single-probe hybridization measurements (Table 1). The latter approach has been shown to reliably measure transcript levels relative to a control, which in this case is RNA from mock-irradiated parallel cell cultures. Overall, there was a good concordance between measurements made with the two techniques, the majority showing variation of less than 2-fold. The genes in Table 1, showing induction or repression by microarray, were confirmed with the single-probe hybridization approach. In many samples, there is signal compression with the microarray approach compared to the more quantitative method. Such signal compression is typical of results seen previously [24,31]. Subsequent time course experiments were carried out with selected cDNA probes, and also confirmed that many genes identified by microarray hybridization were consistently UV-responsive (Fig. 2D–F). The general kinetics of induction were similar to those seen for the three genes analyzed prior to microarray analysis in Fig. 2. Like GADD45A and DDB2, many of the genes in Fig. 2E showed a second late peak in

3.5. Differences in transcription responses to different wavelengths of UV radiation The same filtering approach described above was applied to all of the microarray experiments (UVA, UVB, and UVC exposures) to identify a general set of UV-responsive genes. A set of 795 UV-responsive genes with significant expression change in one or more hybridizations was identified; Gene selection algorithms (see Section 2) were then applied to this gene set to delineate gene expression patterns best discriminating between specific radiation conditions. As illustrated by K-means clustering in Fig. 3B, many genes responded to both UVB and UVC irradiation but not to UVA irradiation, which produced generally weak responses. In Fig. 3C, UVB, and UVC radiation responses were compared, and again cluster analysis discerned a variety of responses that were more robust after UVB radiation. As discussed earlier, UVB radiation generates the same DNA photoproducts as UVC radiation, but also induces oxidative damage, which is specific for longer wavelength UVB and to some extent, UVA radiation. Responses to such damage would be consistent with our overall results where UVB radiation triggered responses seen with UVC radiation and additional responses that were not strongly triggered by UVC radiation. 3.6. Major role(s) for membrane growth factor receptors in UV radiation responses In addition to DNA damage and p53-mediated responses, a variety of membrane bound growth factor receptors and their cellular kinase cascades have been shown to contribute to the activation of UV radiation transcriptional responses. Such pathways can be blocked with suramin, which binds to growth factor receptors [17]. To discern the role of growth factor receptor signaling on global UV radiation responses, gene expression studies were carried out in the presence of suramin. Our expectation was that a subset of stress genes would show attenuated UV radiation-responsiveness with suramin. Surprisingly, nearly all the UV radiation induced and repressed

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Table 2 Suppression of UV radiation-responsiveness by suramin Probesa

ATF3 Cytokine inducible kinase BTG2 Killer/DR5 EGR1 GADD45A

Image ID

51448 825080 213136 893245

Suraminb

Suraminb

UVBc

UVBc

No UVd

UVCe

UVCe

No UVd

2.3 18.6 11.9 8.4 8.3 36.5

1.1 7.6 4.8 5.7 1.9 11.1

1.0 1.2 1.9 1.2 0.93 1.6

3.3 18.6 20.6 6.4 3.4 26.6

0.94 3.0 0.85 2.2 1.2 2.7

0.44 0.94 1.6 1.2 0.87 0.86

a

Representative results are shown for relative gene expression by quantitative single-probe hybridization (see Section 2). Suramin was added 45 min prior to irradiation. c cDNA probes and the indicated Image ID sequences were used for quantitative single-probe hybridization. Cells were treated with 125 J m−2 of UVB radiation, and harvested 12 h later. d Cells were treated identically, except mock-irradiated. e Cells were treated with 12 J m−2 of UVC radiation, and harvested 6 h later. b

genes showed substantial decreases in responsiveness with suramin. As shown in Fig. 3D, cluster analysis clearly distinguished the UVB and UVC irradiation experiments from those carried out with suramin. In nearly all cases, responsiveness was markedly reduced in the presence of suramin. Follow-up experiments were carried out with quantitative single-probe hybridization for selected genes in Table 2, and in all the cases, responsiveness was reduced by suramin. In most cases, treatment with suramin alone had only slight effects on gene expression in the absence of irradiation. In the case of many of the genes in Table 2, suramin inhibition was more pronounced for

UVC radiation responses compared to those of UVB radiation. For example, suramin diminished BTG2 induction by UVB radiation to 35% of that without suramin, while totally blocking induction by UVC radiation. In contrast, ATF3 and EGR1 induction by either UVB or UVC radiation was almost totally ablated by suramin. Suramin has previously been shown to inhibit the induction of GADD45A and GADD153 by UV radiation but to be an ineffective inhibitor after IR or alkylating agent treatment [17]. The generally weaker effect of suramin on UVB radiation responsiveness probably reflects the role(s) of oxidative damage in triggering stress responses with this damaging

Fig. 3. Clustered results of gene selection analysis of UV-responsive genes identified by cDNA microarray hybridization. In (A), the genes showing a significant response in two-thirds or more of the UVB hybridizations are ranked from highest induction to greatest repression, based on the average response in six separate experiments with UVB irradiation; results for UVA and UVC irradiation are included. In the case of UVB irradiation, each pair of samples (lanes 7 and 8, lanes 9 and 10, lanes 11 and 12) represents different microarray hybridizations carried out on different days but with the same RNA sample; the RNA for the three pairs of samples was isolated from cells on different days. Each row represents results for the indicated gene, which is designated by the Image ID number. Each column represents results from an independent experiment, and letters above these columns indicate UVB, UVC, or UVA irradiation. Results are only shown for average induction (≥2) or repression (≤0.5). For UVA radiation, results in the first two columns were samples harvested 6 h after irradiation and the others at 24 h. Relative expression is also shown by color-coding; e.g., relative expression of 0.7–1.4 is designated by the lightest yellow color. In (B, C and D), results of K-means cluster analysis are shown for genes that discriminate the indicated data sets (see Section 2). Relative expression is shown graphically using a color-code scale as indicated below these panels. Values outside this range were set at six (red) for induction or one-sixth (green) for repression. Similar responses are clustered together, and cluster branch diagrams have been omitted for clarity. A grey dot in the upper right hand corner of an individual square designates a target with a low-quality factor (<0.4) for hybridization in that particular experiment [26]. In (B), results for UVA irradiation (first six columns) are compared to results for UVB (next six columns) combined with UVC irradiation (last two columns); for UVA radiation, results in the first and third column were samples harvested 6 h after irradiation and the others at 24 h; for UVB and UVC radiation, the cells were harvested 12 and 6 h, respectively, after irradiation. In (C), results for UVC irradiation (first two columns) are compared to results for UVB (next six columns) irradiation. In (D), clustering results are shown for irradiated samples (UVB and UVC radiation) compared to irradiated samples treated with suramin (designated “sur”), as described in Section 2.

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Fig. 4. cDNA microarray results for representative strongly induced and repressed genes are ranked as in Fig. 3D. The average induction or repression for the UVB experiments is shown in the first column. Each column represents an independent experiment; results for individual experiments are shown in the colored boxes. Values are also included for results after UVC irradiation and UVA (6 h or 24 h) irradiation. Relative expression is also shown by color-coding as in Fig. 3A. The “gene ontology” column represents GO annotation categories, as designated below. These were derived from analysis using database for annotation, visualization and integrative discovery (DAVID) software at http://david.niaid.nih.gov [32]: A, cell cycle regulation; B, apoptosis related; C, tumor suppressor; D, cell adhesion or cell adhesion molecule activity; E, histogenesis and organogenesis; F, cell membrane related; G, DNA repair; H, transcription factor activity or cofactor; I, proteolysis, peptidolysis, or ubiquitin-dependent protein catabolism; J, receptor binding or ligand-dependent nuclear receptor activity; K, signal transduction or protein kinase related; L, protein modification; M, response to toxin, oxidative stress related, or xenobiotic metabolism; N, G protein related; O, cytoskeleton or cytokinesis; P, DNA packaging or chromatin related; Q, chromosome segregation; and R, RNA processing.

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agent. In contrast, UVC radiation produces relatively little oxidative damage, and one must conclude that growth factor receptors have a critical role(s) in stress signaling of membrane photoproducts, and perhaps, of pathways triggered by DNA photoproducts. 3.7. UV radiation-responsive genes have a wide range of physiologic roles A variety of genes with wide-ranging functions are induced by UV radiation. As a representative example, results for a sampling of strongly induced and repressed genes are summarized in Fig. 4, using the same formatting as in Fig. 3A. UVB radiation responses were relatively consistent in the six independent experiments. There was substantial overlap with UVC radiation where many of the genes were also responsive, while the UVA radiation responses were less pronounced as discussed previously. Gene ontology (GO) databases are being developed for many named genes, and all of the genes in Fig. 4 have been annotated in this regard. As described in the legend for Fig. 4, GO annotations obtained using the database for annotation, visualization, and integrated discovery (DAVID) suite of tools [32], have been condensed to general categories designated by the letters A–R in this figure, and a remarkable number of different roles can be discerned for even this limited sampling of genes including roles in cell cycle regulation, apoptosis, DNA repair, and proteolysis, among others. A substantial number of genes involved in driving cell cycle progression are repressed, such as polo-like kinase, cyclin A2, and catenin. In Fig. 2F both UVB and UVC radiation caused a similar decrease in polo-like kinase transcript levels within several hours after irradiation. A much greater number of genes with cell cycle related functions can be found in the larger set of repressed genes of Fig. 3A (data not shown).

4. Discussion The aims of our study were to use UVB radiation as a model stress agent for functional genomics analysis, and to compare these responses to other UV wavelengths in the presence and absence of a pharmacological inhibitor. The strategy was to choose time points when robust induction occurred and to focus on early

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rather than later times where cytotoxic responses may be more pronounced. As shown in Fig. 3, a large number of genes were responsive to UVB radiation, and a subset of these genes was also responsive to UVC and to a lesser extent, UVA radiation. Considering that the spectrum of lesions produced by UVB radiation encompasses that produced by UVC as well as UVA radiation, these results are not surprising. Cluster analysis clearly illustrates that the patterns of responses to different wavelengths could be distinguished. Responsiveness to UVA radiation, even at a later (24 h) time point, was weaker than with the other wavelengths. While we employed a dose of UVA, 50 kJ m−2 , which is somewhat lower than that used in other microarray studies; the weaker relative responses may reflect differences in signaling in response to lower energetic radiation. Even studies employing higher doses of UVA have found relatively few changes in gene expression. For instance, in melanocytes only 11 genes were found to be up-regulated by a 200 kJ m−2 dose of UVA radiation [33]. In another study only three UVA responsive genes were identified with an 80 kJ m−2 dose [34]. While the heme oxygenase gene HO-1 is typically UVA radiation inducible in certain cell types [35], we consistently observed no appreciable induction of this gene, which may reflect cell type variability in its regulation. Regarding the reliability of responses, replicate determinations are important because of inter-experiment variability in gene expression as well as the need for statistical confidence in such large datasets. Burczynski et al. emphasized this point in their study to distinguish the response to a genotoxic agent from that to non-DNA damaging agents [5]. In the case of our UVB radiation experiments, 55% of responsive genes showed consistent regulation in four or more independent experiments. With this dataset, patterns of response to other UV radiation wavelengths with or without the inclusion of a pharmacological inhibitor could be easily distinguished. The development of larger replicate datasets should allow for the development and continued refinement of stress-specific signatures. The results with the growth factor receptor inhibitor were somewhat unexpected, and exemplify the power of a functional genomics approach to monitor the scope of a pharmacological effect. Our expectation was that a subset of genes, such as EGR1 and others regulated by receptor signaling, would show

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an effect while others would not. In contrast, nearly all of the UV radiation-responsive genes showed attenuated induction or repression with suramin. For example, suramin treatment reduced induction of the p53-regulated gene GADD45A after UVB and UVC radiation by 71.5 and 93.4%, respectively, compared to that seen without the inhibitor (Table 2). The average induction of the p53-regulated gene WAF1 was likewise reduced 58.4 and 86.7% by suramin, as measured by microarray hybridization. While IR induction of GADD45A and WAF1 is strictly p53-dependent [27] and not affected by suramin [17], both genes show partial p53-independent UV radiation responsiveness [36]. However, ablation of p53 function reduced the UV radiation induction of these two genes to approximately one-third of that seen in MCF-7 cells with intact p53 signaling [37]. These and many other results suggest that the cellular stress network integrates even seemingly independent signaling pathways. Another not mutually exclusive possibility is that early events in growth factor receptor mediated signaling contribute to the activation of other pathways. For example, inhibition of caspase-mediated apoptosis can dampen stress-induced activation of p53 and p53-regulated genes [38]. It is noteworthy that ATF3 and EGR1 induction was nearly entirely blocked by suramin (Table 2). The subset of strongly suramin-inhibited genes includes others such as CSNK1A1, TEBP, ACLY, TARDBP, WTAP, PSPH, TBL1X, VAPA, C1QA, CORO1C, G3BP, YWHAG, RAP1B, CTNND1, USP7, and USP14. Identification of such genes may provide the basis for development of experimental therapeutics that target various receptor-dependent signaling pathways. Many UV radiation-responsive genes with diverse roles in cellular function have been identified with our functional genomics approach, and there are many similarities with stress studies carried out in other eukaryotic cells. For example, genotoxic stress-responsive genes in yeast included many involved in cell cycle control, DNA repair, signaling, transcription, protein degradation, and RNA metabolism [1]. In UVB-irradiated keratinocytes, 249 rapidly responsive genes were identified with the majority (74%) showing stress-induced repression [39]. As in our study (e.g., Fig. 4) as well as in the preceding yeast study, groups of genes with many cellular functions were identified. In maize, 304

UVB radiation-responsive genes have been identified; although, here the majority were induced rather than repressed. Even though plants lack receptor tyrosine kinases, a similarly wide spectrum of genes with diverse cellular functions was identified [40]. In both our study and the preceding studies, it is interesting that a prominent number of responsive genes with roles in protein synthesis and degradation were identified. While cell cycle and DNA repair genes have well-known roles in genotoxic stress responses, another emerging area is translation control responses [41]. Some of our early studies identified stress-responsive genes, such as GADD34/MyD116 and eIF1A121/SUI1 , whose encoded proteins have critical roles in regulating translation and the fidelity of translation after stress (reviewed in [41]). Others have found similar evidence for stress responsiveness for genes involved in protein synthesis such as eukaryotic translation elongation factor 1 (ε1) as well as eukaryotic translation initiation factors 1A, 2␤, 3␯, and 3 (subunit 6) [6]. In addition, we have found UV radiation responsiveness for a variety of other ribosome-related proteins including RPS29, RPS23, RPS27, RPS27, RPS15A, RPL19, RPL21, RPL35, RPL31, RPL34, RPL35A, and LAMR1. Taken together, our study and others highlight the broad effects of genotoxic stresses, such as UV radiation, on the expression of genes with diverse physiologic roles, and should provide leads for future therapeutics and risk assessment in photobiology and toxicology. Our results with suramin demonstrate the power of a functional genomics approach to survey the global effects of a pharmacologic inhibitor on cell signaling, and it is likely that such approaches will have utility in screening for the effects of pharmaceuticals and toxicants on signaling pathways.

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