Quantitative relationship of dioxin-responsive gene expression to dioxin response element in Hep3B and HepG2 human hepatocarcinoma cell lines

Toxicology Letters 165 (2006) 174–181

Quantitative relationship of dioxin-responsive gene expression to dioxin response element in Hep3B and HepG2 human hepatocarcinoma cell lines Won Kon Kim a , Yu-Jung In a , Jin-Ho Kim a , Hyun-Ju Cho a , Ji-Hye Kim a , Sunghyun Kang a , Chul Young Lee b , Sang Chul Lee a,∗ a

Systemic Proteomics Research Center, Korea Research Institute of Bioscience and BioTechnology (KRIBB), Daejeon, South Korea b Regional Animal Industry Research Center, Jinju National University, Jinju 660-758, South Korea Received 25 August 2005; received in revised form 3 March 2006; accepted 10 March 2006 Available online 18 April 2006

Abstract Dioxin response element (DRE) is a cis-acting DNA sequence mediating the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced gene expression. The present study was undertaken to elucidate TCDD-responsive gene expression profiles and their relationships to the number of DREs in liver cancer cells. Hep3B and HepG2 human hepatocarcinoma cells were exposed to 50-nM TCDD for 0, 1, 2 and 4 h in culture, after which gene expression profiles were analyzed by the microarray hybridization using a chip containing 24,000 cDNAs prepared from the human liver. The TCDD-responsive expression levels in each gene were calculated by dividing the densitometric values of the hybridization signal for h1, h2 and h4 by that of h0, followed by transformation of the resulting data into a log scale with the base of 2. Up- and down-regulated gene expressions were defined as >0.585 and <−0.585 by the log scale (>1.5 and <1/1.5 arithmetically), respectively, exhibited at any time after h0. Hep3B and HepG2 cells had 27 and 58 TCDD-responsive, up-regulated genes, respectively, of which 78% (21/27) and 62% (36/58) had one or more DREs. Of these 85, 80 genes were up-regulated exclusively in one of the two lines, with CYP1A1 and PPP1R15A being so regulated in both lines. Expression levels of the up-regulated genes at h1, h2 and h4 were correlated with each other (P < 0.01) and the mean of these regressed to the number of DRE(s) in both lines (P < 0.01). However, expression of a total of 93 TCDD-responsive, down-regulated genes, of which 46% contained DRE(s), had no relation to the number of DRE(s). In conclusion, results suggest that DREs may cooperatively mediate the expression of TCDD-responsive genes in liver cancer cells. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Dioxin; Response element; Liver; Microarray; Gene expression; Gene regulation

1. Introduction Recent completion of the human genome project and the accumulation of an increasing number of gene anno-

∗ Corresponding author. Tel.: +82 42 860 4142; fax: +82 42 860 4598. E-mail address: [email protected] (S.C. Lee).

tations have allowed bioinformaticians to develop new approaches that can help solve various biological problems (Jin et al., 2004; Liu and Zhao, 2004). Moreover, novel computational approaches have been developed to annotate genomic sequences pertaining to structure, function and control processes involving combinatorial regulatory elements (Bork et al., 1998; Qiu, 2003). These also complement genome-wide analysis of gene expression and its regulatory networks.

0378-4274/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2006.03.007

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2,3,7,8-Tetrachlorodibenxo-p-dioxin (TCDD) is an environmental pollutant which causes various toxic damages resulting in birth defects, teratogenesis, immunosuppression, tumor promotion and neuroendocrine disturbances, etc. (Nebert et al., 1993; Parzefall, 2002). Biological effects of TCDD are mediated through aryl hydrocarbon receptor (AhR) which is a liganddependent DNA-binding transcription factor (Swanson and Bradfield, 1993; Hankinson, 1995; Hahn, 1998). The AhR, after binding its ligand, associates with AhR nuclear translocator (ARNT) to form a ligand:AhR:ARNT complex which binds to a specific genomic sequence (5 -TNGCGTG-3 ) known as dioxin response element (DRE) to modulate the expression of responsive genes (Reyes et al., 1992; Swanson et al., 1995; Mimura and Fujii-Kuriyama, 2003). Although it is generally believed that DRE is a canonical cis-acting element in TCDD-induced expression of many genes including cytochrome P450 CYP1A1 (Rushmore et al., 1990; Nebert et al., 1993; Mimura and Fujii-Kuriyama, 2003), it is not clear whether this regulatory sequence is a strict requirement for TCDD action. Similarly, it is not known to what extent the expression of TCDD-responsive genes is related to the number of DREs. The present study was therefore undertaken to investigate TCDD-responsive gene expression profiles and also to assess a quantitative relationship of these to the number of DREs in Hep3B and HepG2 human hepatocarcinoma cell lines. 2. Materials and methods 2.1. Computational screening of the DRE Promoter sequences, which span a region between −3000 and +2000 bp relative to the transcription start site, were obtained from UCSC Genome Browser for the RefSeq genes (http://www.genome.ucsc.edu; hg 17). The DRE sequence on both positive (5 -TNGCGTG-3 ) and negative (5 -CACGCNA3 ) strands in each promoter was identified by virtue of the DNA database TFExplorer (Kim et al., 2005; http://mars.kribb. re.kr:8080/tfExplorer). 2.2. Cell culture The Hep3B and HepG2 human hepatocarcinoma cell lines were obtained from American Type Culture Collection (Rockville, MD). Cells were cultured in Dulbeco’s modified Eagle’s medium (Gibco-Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (Sigma Chemical Co., Saint Louis, MO) and 100 ␮g/ml penicillin–streptomycin (GibcoInvitrogen) following addition of 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD; AccuStansndard Int., New Havan, CT) to 50 nM at 37 ◦ C in a humidified atmosphere containing 5% CO2 .

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2.3. Microarray analysis of TCDD-mediated gene expression At 0, 1, 2, and 4 h after the start of cell culture, total cellular RNA was isolated using RNeasy Midi kit (Qigen, Valencia, CA) according to the manufacturer’s instruction. Only RNA preparations of good quality, which exhibited >1.7 of the ratio of the spectrophotometric absorbance at 260 nm to that at 280 nm and also the integrity of the 28S and 18S RNA bands following 1% agarose/formaldehyde gel electrophoresis, were used in all the experiments including the microassay hybridization. Each RNA preparation was labeled with either Cy3 or Cy5 dye using a 3DNA array kit (Genisphere Inc., Hatfield, PA) according to manufacturer’s instruction. The microarray hybridization of the labeled RNA was performed on a chip spotted with 24,000 human cDNAs selected from KUGI Unigenes (http://kugi.kribb.re.kr), followed by densitometric scanning of the hybridization signal using GenePix Pro 5.0 (Axon Instruments, Foster City, CA) on ScanArray 5000 (Perkin-Elmer Life Science Inc., Boston, MA). Expressed genes were identified by one-class significant analysis of microarray analysis (SAM; Sherlock, 2001), with the q-value and false discovery rate of <0.05% and <0.15%, respectively. Genes which had exhibited up- or down-regulated expression in response to TCDD were classified according to their biological functions following the gene ontology (GO) charts of database for annotation, visualization and integrated discovery (DAVID, http://apps1.niaid.nih.gov/david/) (Dennis et al., 2003). 2.4. Reverse transcription-polymerase chain reaction (RT-PCR) Two micrograms of total cellular RNA were denatured at 65 ◦ C for 5 min, after which first-strand cDNA was synthesized using M-MLV reverse transcriptase at 37 ◦ C for 60 min in the presence of 0.5-␮g oligo(dT), 10-mM each dNTP and 0.1-M DTT (all from Invitrogen) in a total volume of 40 ␮l. After terminating the RT reaction by heating at 75 ◦ C for 15 min, double-strand cDNA fragments of several selected TCDD-responsive genes and others encoding ␤-actin and the trans-acting elements of TCDD-induced gene expression were amplified by PCR from the RT product. The PCR products were separated by agarose gel electrophoresis and stained with ethidium bromide, followed by densitometric scanning using a 1D analysis software (Quantity One; Bio-Rad, Hercules, IL) and normalization of the resulting data to that of ␤-actin. 2.5. Statistical analysis Densitometric values of the microarray hybridization for h1, h2 and h4 of the time-course in culture were divided by that of h0 and the resulting data were transformed into a log scale with the base of 2. TCDD-responsive, up- and downregulated genes were defined as those which exhibited >0.585 and <−0.585 of the log2 -transformed expression levels (>1.5

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and <1/1.5 by the arithmetic scale), respectively, at any time after h0. Quantitative relationship between DRE and gene expression was determined by regression analysis using SAS (SAS Institute Inc., Cary, NC), preceded by analysis of correlations among expression levels at h1, h2 and h4. In this analysis, the independent and dependent variables were the number of DRE and the mean of h1, h2 and h4 values when the latter variables were correlated. When the dependent variables were not correlated, the regression analysis was separately performed for each of h1, h2 and h4.

3. Results 3.1. Identification of TCDD-responsive and DRE-containing genes The Hep3B and HepG2 cells had 985 and 312 expressed genes, respectively, during the entire time-

course of culture including time zero (h0) after the present microarray analysis and filtering procedure. Of those expressed genes in the Hep3B and HepG2 cells, 27 and 58 genes in the respective cell lines exhibited the up-regulated expression in response to TCDD which was defined as >1.5-fold expression (or >0.585 after the log2 transformation of microarray data) relative to that at h0 at any time after h0 (Table 1). Moreover, 78% (21/27) and 62% (36/58) of the up-regulated genes in the respective cell lines contained one or more DREs. When the TCDDresponsive, up-regulated genes were classified according to the biological function, they mostly fell in the category of metabolism, cellular physiological process, response to stimulus, or cell communication (Table 1). The Hep3B and HepG2 cells, following an exposure to TCDD, also exhibited 64 and 29 down-regulated genes, respectively, which had <1/1.5-fold expression

Table 1 Classification of the genes which exhibited up- or down-regulated expression following an exposure to TCDD in Hep3B and HepG2 hepatocarcinoma cell lines Classification of regulated genes

Up-regulated genesa

Down-regulated genesb

Hep3B

HepG2

Hep3B

HepG2

21 6 27

36 22 58

34 30 64

9 20 29

10 10 4 2 5 2 6 2 1

23 23 14 8 3 3 13 6 1

25 14 5 3 3 3 8 1 2 1

14 4 1 1 1 1

DREc

By the presence/absence of Genes containing ≥1 DRE Genes containing no DRE Total

By biological functionc Metabolism Cellular physiological process Response to stimulus Organismal physiological process Morphogenesis Regulation of cellular process Cell communication Death Cell differentiation Coagulation Regulation of enzyme activity Embryonic development Aging Pathogenesis Viral infectious cycle Membrane fusion Reproduction Membrane fusion Reproduction Learning and/or memory Regulation of physiological process Unclassified a

1

1 1 1 1 1 1 1 1 1

8

18

28

1 1 14

Data denote numbers of genes. Defined as >1.5-fold (or >0.585 after the log2 transformation) expression relative to that at time zero at any time after an exposure to TCDD. b Data denote numbers of genes. Defined as <1/1.5-fold (or <−0.585 after the log transformation) expression relative to that at time zero at any 2 time after an exposure to TCDD. c Raw data are available upon request (e-mail: [email protected]) as well as online [http://sprc.kribb.re.kr/DA/leesc.html].

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Table 2 Representative up-regulated genes in response to an exposure to TCDD in Hep3B and HepG2 hepatocarcinoma cell lines RefSeq

Genes

Number of DRE

Cell line

Gene expression (log2 -transformed)a h1

Up-regulated genes in both cell lines NM 000499 CYP1A1

h4

Hep3B HepG2 Hep3B HepG2

1.588 3.450 0.771 0.520

2.350 4.520 0.361 0.412

2.988 4.550 0.444 0.697

Genes up-regulated in Hep3B, but not identified to be expressed in HepG2 NM 000104 CYP1B1 7 Hep3B NM 005746 PBEF1 3 Hep3B NM 002612 PDK4 2 Hep3B NM 001964 EGR1 1 Hep3B NM 002228 JUN 1 Hep3B HES1 1 Hep3B NM 005524

1.879 1.232 0.837 0.872 0.727 1.034

2.102 1.305 0.546 1.144 0.920 0.822

2.055 1.615 0.582 1.215 1.395 0.332

Genes up-regulated in HepG2, but not identified to be expressed in Hep3B NM 003486 SLC7A5 4 HepG2 STC2 3 HepG2 NM 003714 NM 000362 TIMP3 2 HepG2 NM 000596 IGFBP1 1 HepG2 TBX3 1 HepG2 NM 016569 SOS1 1 HepG2 NM 005633 NM 000761 CYP1A2 0 HepG2

1.053 0.748 0.730 1.887 1.520 0.805 2.087

1.369 0.823 0.517 1.927 1.562 2.224 2.478

1.418 1.228 0.892 2.024 1.345 2.414 3.806

1.227 −0.103 0.677 −0.703

0.832 −1.718 0.621 −0.212

0.418 −0.522 0.526 −0.100

NM 014330

PPP1R15A

6

h2

3

Genes which exhibited opposite responses in two cell lines NM 014333 IGSF4 2 NM 018091

ELP3

2

Hep3B HepG2 HepG2 Hep3B

Only representative genes which exhibited up-regulated expression in response to TCDD in either cell line are shown in this table. A complete list of the TCDD-responsive genes is available upon request (e-mail: [email protected]) as well as online [http://sprc.kribb.re.kr/DA/leesc.html]. a Expression levels at the indicated hours after an exposure to TCDD (h1, h2 and h4) were divided by that at h0, followed by transformation of resulting data into a log2 scale. Up- and down-regulations were defined as >0.585 and <−0.585 of the log2 -transformed values, respectively, exhibited at any time after h0.

(or <−0.585 after the log2 transformation of microarray data) relative to that at h0 (Table 1). Percentages of DRE-containing genes in the two cell lines in this group of TCDD-down-regulated genes [53% (34/64) and 31% (9/29) in Hep3B and HepG2 lines, respectively] were less than those of the other group of up-regulated genes. Nevertheless, biological functions of this group of down-regulated genes were similar to those of the other group. Of the 85 up-regulated genes in response to TCDD, only two genes, CYP1A1 and PPP1R15A, were up-regulated in both cell lines (Table 2). All the other TCDD-responsive, up-regulated genes, except for IGSF4 and ELP3, were so regulated only in one of the two cell lines. The IGSF4 and ELP3 genes were upregulated in response to TCDD in Hep3B and HepG2 cell lines, respectively, but in the opposite cell lines, they were down-regulated by TCDD.

3.2. RT-PCR To confirm the fidelity of the present microarray analysis, expression of selected TCDD-up-regulated genes CYP1A1, JUN, EGR1, SOS1 and TBX3 in either cell line was also examined by quantitative RTPCR. As shown in Fig. 1, expression levels of these TCDD-responsive genes determined by the RT-PCR were similar to those of the microarray analysis during the entire 4-h time-course following an exposure to TCDD. By contrast, expression of genes coding for AhR, ARNT, aryl hydrocarbon receptor repressor (AhRR) and aryl hydrocarbon receptor-interacting protein (AIP) barely changed throughout the 4-h culture in either cell line (Fig. 1), implicating that the expression of the TCDD-responsive genes had no relation to the gene expression of these trans-acting elements.

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Table 3 Pearson’s correlation coefficients among expression levels of the up- and down-regulated genes following an exposure to TCDD in Hep3B and HepG2 cell lines Cell line

Hep3B

HepG2

Hour

Up-regulateda

Down-regulateda

h1

h2

h1 h2 h4

– 0.666 (P < 0.001) 0.561 (P = 0.002)

– 0.893 (P < 0.001)

h1 h2 h4

– 0.803 (P < 0.001) 0.784 (P < 0.001)

– 0.772 (P < 0.001)

h4

h1

h2

h4

–

– 0.134 (P = 0.291) −0.011 (P = 0.929)

– 0.580 (P < 0.001)

–

–

– −0.499 (P = 0.006) −0.075 (P = 0.697)

– 0.246 (P = 0.198)

–

a Up- and down-regulations were defined as >0.585 and <−0.585 of the log -transformed ratios of the expression level at any indicated time (h1, 2 h2, or h4) to that at time zero (h0), respectively.

Table 4 Relationship between the expression level of the up- and down-regulated genes after an exposure to TCDD and the number of DRE in Hep3B and HepG2 cell lines: regression analysis Cell line

Hep3B

HepG2

Group of regulated genesa

Dependent variable (gne expression)

Up Down

h1+h2+h4 3

Up Down

h1 h2 h4 h1+h2+h4 3

h1 h2 h4

Regression constantsb Intercept

Slope (P-value)

0.506 −0.696 −0.242 −0.149

0.192 (P < 0.001) 0.004 (P = 0.240) −0.005 (P = 0.531) −0.008 (P = 0.500)

0.774 −0.651 −0.509 −0.607

0.168 (P = 0.008) −0.01 (P = 0.758) −0.046 (P = 0.521) 0.006 (P = 0.923)

a Up- and down-regulations were defined as >0.585 and <−0.585 of the log -transformed ratios of the expression level at any indicated time (h1, 2 h2, or h4) to that at time zero (h0), respectively. b Dependent variable (gene expression) = intercept + slope × number of DRE (independent variable).

3.3. Relationship between the TCDD-responsive gene expression and DRE Quantitative relationship of the TCDD-responsive gene expression to the number of DREs was examined by regression analysis, preceded by analysis of correlations between the expression levels at different times after h0. The h1, h2 and h4 values representing the expression levels at the respective times were highly correlated with each other in the TCDD-up-regulated genes in both cell lines (Table 3), indicating that these variables were not mutually independent. Accordingly, means of the h1, h2 and h4 values were used as dependent variables in

subsequent regression analysis for this group of genes. By contrast, in the TCDD-down-regulated genes, correlations between the h1, h2 and h4 values were not significant except for a single case in each cell line, for which the regression analysis for this group of genes was performed separately for each time-point of the culture in both cell lines. In the group of TCDD-responsive, up-regulated genes, the mean of gene expression levels at h1, h2 and h4 regressed to the number of DREs (P < 0.01) in both cell lines (Table 4). Furthermore, the slope of the linear regression was positive, implicating that expression of this group of genes increased with increasing number of

Fig. 1. Quantitative reverse transcription-polymerase chain reaction (RT-PCR): comparison with microarray data. Panel A: RT-PCR. Following culture of Briefing and HepG2 cells in the presence of TCDD for the indicated time and RT-PCR, DNA products representing the expression of selected TCDD-responsive, up-regulated genes (rows 2–4 from the top) and others coding for the trans-acting elements of TCDD-responsive gene expression (rows 5–8) and ␤-actin gene (row 1) were electrophoresed and stained with ethidium bromide. Panel B: Comparison of the quantitative RTPCR with microarray analysis. Ethidium bromide-stained RT-PCR products following agarose gel glectrophoresis were densitometrically scanned, after which resulting data were normalized to the intensity for ␤-actin of the corresponding time-point of cell culture. Means ± S.E. (n = 3) of the quantitative RT-PCR data at the indicated time-points relative to that at time zero were aligned with corresponding results of the microarray analysis.

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DREs. However, in the down-regulated genes, the number of DREs had no relation to the gene expression level at any time-point of the culture in either cell line. 4. Discussion Expression of many biological effects of the environmental pollutant TCDD is known to be mediated via a transcriptional regulatory sequence DRE (Lai et al., 1996; Mimura and Fujii-Kuriyama, 2003). The present study was performed to further find out TCDD-responsive genes by the microarray analysis and also to assess a quantitative relationship between the TCDD-responsive gene expression and the number of DRE in Hep3B and HepG2 human hepatocarcinoma cell lines. The DRE sequence in responsive genes was identified by virtue of the TFExplorer which is an integrated DNA database for predicted regulatory elements and hence is very useful as a DNA motif-searching tool in the human, mouse and rat. For instance, a total of 12,567 human genes were identified to contain the DRE sequence by the TFExplorer in a preliminary trial prior to the present study. Moreover, the DRE sequence which was found by Thomsen et al. (2004) at a −48/−42 region of the promoter of HES-1 gene was also found at the same location in the present study. Sun et al. (2004) also found the DRE in 48 genes out of 2437 human–mouse–rat orthologous genes, but this could not be directly compared with the present result because of a narrower promoter region scanned in their study (−1500 to +1500) versus ours (−3000 to +2000). A number of new or previously known TCDDresponsive genes have been identified by the present microarray analysis. Interestingly, of the 85 TCDDresponsive, up-regulated genes identified in the present study, 80 genes were so regulated only in one of the two cell lines. It is thus temping to speculate that like the known organ-specific gene expression in response to TCDD (Yamamoto et al., 2004; Volz et al., 2005), the TCDD-responsive gene expression may also be cell line-specific even within a class of cancer cells derived from a common organ or tissue. Apart from this speculative cell-line specificity, the up-regulated expression of CYP1A1 gene in both cell lines and IGF-binding protein-1 (IGFBP-1) gene in HepG2 cells was consistent with previous reports for the respective genes in nonhepatic and HepG2 hepatocarcinoma cells (Adachi et al., 2004; Marchand et al., 2005). In addition, the TCDDresponsive, up-regulated expression of early growth response-1 (EGR-1) and hairy and enhancer of split homolog-1 (HES1) genes in Hep3B cells also was consistent with previous reports in non-hepatic cells (Liu et

al., 1996; Martinez et al., 2004; Thomsen et al., 2004). It was also noticeable that TBX3 gene, whose responsiveness to dioxin had not been known, exhibited over 2-fold elevated expression following an exposure to TCDD in HepG2 cells. These genes, which en masse are involved in metabolism, apoptosis, cell proliferation and differentiation, have at least one DRE sequence. In contrast to this, CYP1A2 gene containing no DRE also exhibited over 4-fold elevated expression in response to TCDD in HepG2 cells. In this gene, the response to TCDD has been reported to be mediated via a second cis-acting element known as DRE-II (Boutros et al., 2004; Sogawa et al., 2004). The TCDD-responsive, up-regulated gene expression detected in the present study is likely to have been mediated by an action or cooperative interaction of DRE(s), because the mean of gene expression levels after h0 regressed significantly to the number of DREs which were present in the majority of genes of this group in both cell lines. Furthermore, the significant correlations among the gene expression levels at different times in this group of genes indicate that the cells responded to TCDD consistently throughout the culture. On the other hand, in the group of TCDD-responsive, down-regulated genes, the gene expression levels at different times were not correlated with each other and did not regress to the number of DREs at any time-point in either cell line. This suggests that the down-regulated gene expression is not likely to be mediated via DRE, although a significant percentage of this group of genes contained this cis-acting element. It thus seems probable that TCDD-responsive, down-regulated gene expression be mediated via unknown cis-acting element(s) other than DRE. Alternatively, it also seems possible that the down-regulation, defined as <1/1.5-fold expression at any time-point after addition of TCDD, was less selectively detected than the up-regulation (>1.5-fold expression) because of a smaller numerical change in the former versus the latter. In summary, approximately 160 TCDD-responsive, up- or down-regulated genes were identified to be so regulated almost exclusively in one of the Hep3B and HepG2 hepatocarcinoma cell lines. The majority of the up-regulated genes and a significant percentage of down-regulated genes contained one or more DREs. The gene expression levels during a 4-h timecourse were correlated with each other and the mean of these regressed to the number of DREs in the TCDDresponsive, up-regulated genes in both cell lines, but not in the down-regulated genes in either line. Results suggest that expression of a number of TCDD-responsive, up-regulated genes may be mediated by an action or cooperative interaction of DRE(s).

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