Analysis of the ultraviolet B response in primary human keratinocytes using oligonucleotide microarrays Angela Sesto*, Manuel Navarro*†, Frank Burslem‡, and Jose´L. Jorcano* *Department of Molecular and Cell Biology, Centro de Investigaciones Energe´ticas, Medioambientales y Tecnolo´gicas (CIEMAT), Avenida Complutense 22, 28040 Madrid, Spain; and ‡Pfizer Global Research and Development, Sandwich, Kent CT13 9NJ, United Kingdom Communicated by A. Garcia-Bellido, Autonomous University of Madrid, Madrid, Spain, December 17, 2001 (received for review October 19, 2001) UV radiation is the most important environmental skin aggressor, To understand the mechanisms of UV response, we sought to causing cancer and other problems. This paper reports the use of obtain the transcriptional profile of primary keratinocytes after oligonucleotide microarray technology to determine changes in UVB irradiation. Cultured human epidermal keratinocytes were gene expression in human keratinocytes after UVB treatment. treated with three UV doses, and samples were collected at Examination of the effects of different doses at different times different intervals. Oligonucleotide microarrays containing over after irradiation gave a global picture of the keratinocyte response 6,000 genes (HuGeneFL, Affymetrix, Santa Clara, CA) were to this type of insult. Five hundred thirty-nine regulated transcripts used to quantitatively assess changes in gene expression. The were found and organized into nine different clusters depending subset of regulated transcripts was classified into self-organizing on behavior patterns. Classification of these genes into 23 func- maps according to their different behaviors. Biological functions tional categories revealed that several biological processes are were assigned to these genes, which were then correlated with globally affected by UVB. In addition to confirming a majority expression patterns. These approaches provide new insights into up-regulation of the transcripts related to the UV-specific inflam- the genes and biological processes involved in the UV response matory and stress responses, significant increases were seen in the and demonstrate the complexity of the UVB transcriptional GENETICS expression of genes involved in basal transcription, splicing, and profile in keratinocytes, allowing global characterization of translation as well as in the proteasome-mediated degradation UV-regulated genes and pathways. category. On the other hand, those transcripts belonging to the metabolism and adhesion categories were strongly down- Materials and Methods regulated. These results demonstrate the complexity of the tran- Cell Culture. Primary human keratinocytes from foreskin were scriptional profile of the UVB response, describe several cellular cultured on a fibroblast feeder layer (50-Gy-irradiated 3T3 J2 processes previously not known to be affected by UV irradiation, cells) as described by Rheinwald and Green (12). Cells were and serve as a basis for the global characterization of UV-regulated grown to 80% confluence in 100-cm2 plates in 3:1 DMEM͞ genes and pathways. Ham’s-F12 medium (GIBCO͞BRL Life Technologies) supple- mented with 10% FBS (BioWhittaker)͞5 mg/ml of insulin he epidermis is a physiological barrier that protects the (Sigma)͞0.1 nM cholera toxin (Sigma)͞10 ng/ml of epidermal Torganism against pathogens and chemical or physical dam- growth factor (Serono, Geneva)͞0.5 mg/ml of hydrocortisone age. UV radiation is the most important physical carcinogen (Sigma)͞1% antibiotics (GIBCO͞BRL Life Technologies). in the environment, and the skin is its main target. In this tissue, UV induces photochemical changes that may lead to Irradiation. Fresh culture medium was supplied 24 h before acute effects such as erythema or sunburn (1), or chronic treatment. For irradiation, the medium was removed and the effects that include premature skin aging (2, 3) and skin tumors keratinocytes treated with 10, 20, or 40 mJ͞cm2 UVB. The (4, 5). In recent years, the importance of UV radiation has collected medium was then replaced and the cells maintained in increased because of the thinning of the ozone layer (6) and culture for 4 or 24 h. A bank of six filtered UVB Sankyo 615T8E increasing skin cancer rates (7). UV light affects the skin in 15-W lamps, which emit 80% of the energy in the UVB range as different ways depending on its wavelength. UVA (320–400 measured by a UVX radiometer, were used as the radiation nm) effects are primarily oxidative in nature. UVC (200–290 source. nm) hardly ever reaches the surface of the earth. UVB (290–320 nm) is the most important and is considered the RNA Extraction and Preparation for Hybridization. Total RNA was causative agent of many of the effects attributed to UV (8), isolated by using TRIzol (GIBCO͞BRL Life Technologies). giving rise to mutations in DNA and modifying the pattern of RNA was amplified and labeled with biotin as described by gene expression. This transcriptional regulation is part of the Wodicka et al. (13). cellular reaction to UV-induced stress and operates as a defense mechanism. The known UV response comprises, for Array Hybridization and Scanning. Samples for oligonucleotide instance, the rapid activation of genes with reparative, pro- microarray hybridization were prepared as described by Lock- tective, or apoptotic functions and the communication of stress hart et al. (14), except that hybridization was performed in 1ϫ to other cells via secreted signaling molecules (9). Mes buffer (0.1 M Mes, pH 6.7͞1 M NaCl͞0.01% Triton X-100), Although a number of UV-regulated genes and processes have and the chips were washed in 0.1ϫ Mes buffer. The target been described (see refs. 9 and 10 for review), many of the concentration was 15 ␮g of adjusted fragmented cRNA in 300 molecular events involved in the UV response remain unknown. However, many studies on UV lack physiological relevance because they were performed by using UVC and established Abbreviation: cn, cluster n. epithelial cell lines or fibroblasts. Habitually exposed to UV †To whom reprint requests should be addressed. E-mail: [email protected]. radiation, keratinocytes are more resistant to this insult than are The publication costs of this article were defrayed in part by page charge payment. This fibroblasts (11), which probably means they have developed a article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. specialized response. §1734 solely to indicate this fact. www.pnas.org͞cgi͞doi͞10.1073͞pnas.052678999 PNAS ͉ March 5, 2002 ͉ vol. 99 ͉ no. 5 ͉ 2965–2970 Downloaded by guest on September 25, 2021 Fig. 1. Analysis of the experimental variability of the method. The data are represented as log͞log scatter plots of the expression values provided by GENECHIP software. For each graph, only those transcripts present in both samples are considered. The upper and lower boundaries represent a 2-fold change in expression. (A) Control 1 vs. Control 1Ј. Technical variability is provided by the comparison of the expression levels of two different chip analyses performed with the same sample of untreated keratinocytes. The vast majority of the points fall within the Ϯ2-fold limit. (B) Control 1 vs. Control 2. Biological variability is provided by the comparison of two different control samples. A slight increase in the number of data points outside the borders indicates changes in the patterns of gene expression attributed to culture-specific characteristics. (C) Control 1 vs. 40 mJ͞cm2 UVB, 24 h. Changes in gene expression because of the UVB response are represented. Up- and down-regulated genes are represented by the points located outside the Ϯ2-fold lines, although most of the transcripts fall within these limits. ␮m of hybridization solution. Samples were hybridized to Af- Results and Discussion fymetrix GeneChip HuGeneFL arrays. Images were scanned at Experimental System. Primary human epidermal keratinocytes ␮ 3- m resolution by using a GeneArray Scanner made for Af- were grown to subconfluence using culture conditions that fymetrix by Hewlett–Packard. maintain many of the characteristics these cells show in vivo (12). The UVB irradiation doses applied (10, 20, and 40 mJ͞cm2) Analysis. All samples were prepared and hybridized in duplicate. represent low, middle, and high exposures to UV considering GENECHIP ANALYSIS SUITE software (Affymetrix) was used to that, in our experimental system, few changes in expression are analyze image data. Values representing genes not expressed at seen at 10 mJ͞cm2, and nondesired high apoptotic rates are a given experimental point are unreliable and are referred to as observed only at doses over 40 mJ͞cm2 (data not shown). ‘‘absent call’’ entries. To gain a more complete picture, those Irradiation with 40 mJ͞cm2 caused undetectable apoptosis at transcripts that showed at least one ‘‘present call’’ in one 24 h and less than 4% of apoptotic cells at 48 h, as estimated by experimental point in both duplicates were included. When cell viability assays and analysis of DNA content by flow required, the expression levels provided by the ‘‘absent call’’ cytometry (data not shown). These conditions approach the entries were modified to up- or down-regulation values no higher situation in skin during moderate to intense exposure to the sun. than 4-fold. The results of the whole experiment are published Samples were collected at 4 and 24 h after irradiation, time as supporting information on the PNAS web site (www. points for early- and late-regulated genes. The final analysis pnas.org). To classify the temporal profiles of gene expression, therefore comprises the effects of both low and high doses in the cluster analysis was performed by using the GENE CLUSTER 1.1 short and long term. program [http:͞͞www.genome.wi.mit.edu͞MPR͞GeneCluster Microarray experiment results were represented in log͞log (15)]. Genes that, after irradiation, showed differences in their scatter plots to check the consistency of the data. Fig. 1 shows the expression levels of at least a factor of 2.0 in at least one expression pattern of untreated keratinocytes, on the horizontal experimental point in both duplicates were selected for cluster- axis, against its duplicate (Fig.