Budesonide Exerts Its Chemopreventive Efficacy During Mouse Lung Tumorigenesis by Modulating Gene Expressions

Budesonide exerts its chemopreventive efﬁcacy during mouse lung tumorigenesis by modulating gene expressions

Ruisheng Yao1, Yian Wang1, William J Lemon1, Ronald A Lubet2 and Ming You*,1

1Department of Surgery and The Alvin J Siteman Cancer Center, Washington University School of Medicine, 660 S Euclid Avenue, St Louis, MO 63110, USA; 2Chemoprevention Branch, National Cancer Institute, Bethesda, MD 20892, USA

Budesonide, a glucocorticoid, was proven to be a highly Introduction effective agent in preventing the development of lung tumors in A/J mice. In a lung tumor bioassay, budesonide Lung cancer is the most common malignancy in both produced 70% inhibition of tumor multiplicity and 94% men and women in the world. In 2003 there will be reduction of total tumor load compared to benzopyrene about 172 000 new cases of lung cancer in the United (B[a]P) treated mice. Gene expression array analysis was States and about 157 000 people will die of this disease performed on mouse lung tumors from this bioassay using (American Cancer Society, 2003). Lung cancer, like Affymetrix U74Av2 GeneChips to determine gene ex- other types of cancer, develops as a multistage process pression changes associated with budesonide treatment. involving the accumulation of genetic alterations that We found 363 genes that were changed between lung affect key proto-oncogenes and tumor suppressor genes tumors induced by treatment with B[a]P and similar (You et al., 1989; Herzog et al., 1997). There are a wide tumors treated with budesonide. Among them, 243 genes variety of genes whose expressions are altered in lung were overexpressed and 120genes were underexpressed tumors (McDoniels-Silvers et al., 2002; Yao et al., 2002, after budesonide treatment. In addition, 108 genes 2003). Many of these genes are probably altered at the differentially expressed during mouse lung tumorigenesis transcription level, and may play important roles in lung (50genes overexpressed and 58 genes underexpressed) tumor development. The A/J mouse has proved to be an were modulated back to normal levels after budesonide extremely valuable animal model for studying lung treatment when compared with the controls group. These adenoma and adenocarcinoma formation as well as lung genes are involved in a broad range of different pathways cancer chemoprevention. In addition to histologic including control of cell cycle, signal transduction, and similarity between adenomas/adenocarcinomas com- apoptosis and may play a role in the observed preventive monly seen in mice and human lung tumors, genetic effect. Our results suggest that budesonide exerts its changes found in mouse lung tumors also resemble those effects of chemoprevention through growth arrest via in humans (Malkinson, 1992; Herzog et al., 1997). Mad2/3 and through apoptosis via Bim/Blk and, by Glucocorticoids and their synthetic analogs have been inference, caspase-8/9. Using the pathway visualization shown to have cancer chemopreventive efficacy in tool GenMapp, G protein pathway and MAPK cascade animal models of lung cancer (Wattenberg and were also regulated by budesonide. Thus, we have Estensen, 1996, 1997; Wang et al., 2003). In fact, it determined, for the first time, the expression profiles of was initially observed that steroid hormones inhibited genes modulated by budesonide during murine lung croton oil-promoted mouse skin tumorigenesis almost tumorigenesis. Our results indicate that the chemopreven- 35 years ago by Belman (Belman and Troll, 1972). tive effects of budesonide in the mouse lung tumorigenesis Recent studies by Wattenberg et al. have demonstrated assay involved increase and decrease expression of a wide that the synthetic glucocorticoid budesonide, has strong variety of genes in multiple signaling pathways. preventive activity in the B[a]P-induced pulmonary Oncogene (2004) 23, 7746–7752. doi:10.1038/sj.onc.1207985 adenoma formation in female A/J mouse model. This ONCOGENOMICS Published online 13 September 2004 inhibitory effect was achieved with budesonide administrated either by diet or aerosol (Wattenberg and Keywords: budesonide; lung cancer; chemoprevention; Estensen, 1996; Wattenberg et al., 2000). An inhibitory A/J mice; expression profile effect of budesonide was observed even at a very low dose level when administrated by aerosol (Wattenberg et al., 2000). Budesonide is one of a few compounds with lung tumor inhibitory effects even when given after carcinogen initiation (Wattenberg and Estensen, 1997). *Correspondence: M You, Department of Surgery and The Alvin J Recently, we have shown that budesonide has both Siteman Cancer Center, Campus Box 8109, Washington University chemopreventive and chemotherapy effects on lung School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA; E-mail: [email protected] tumors in mice with different germline mutations and Received 10 March 2004; revised 10 June 2004; accepted 10 June 2004; that the degree of efficacy depends on genotypes of mice published online 13 September 2004 (Wang et al., 2003). Expression profile of mouse lung tumors treated with budesonide RYaoet al 7747 The mechanisms for chemopreventive effect of (Figure 1). When comparing these genes with the paired glucocorticoid have not been elucidated. Evidence normal tissues, 108 genes that changed during lung indicates that the possible mechanisms include: (1) tumorigenesis induced by B[a]P were modulated back to directly altering gene transcription by regulating the normal levels by budesonide treatment. Clustering glucocorticoid response element (GRE) in the target reveals two major gene expression patterns: in Pattern gene DNA sequence; (2) indirectly altering gene I, 50 genes were found upregulated in B[a]P-induced transcription by interacting with other transcript tumors; however, expression of these genes were factors, such as AP-1, NF-kB, and CREB; (3) altering post-transcriptional events and translation of proteins (Greenstein et al., 2002). Through these possible mechanisms, GCs induce apoptosis via activation of death-inducing genes or inhibition of growth/survival genes. The microarray techniques allow rapid and simulta- neous detection of the expression of thousands of genes. In this study, we employed the Affymetrix oligonucleo- tide arrays representing over 12 000 genes and ESTs for the identification of differentially expressed genes in mouse lung tumors and for analysis of possible modes of action for budesonide in cancer chemoprevention and/ or chemotherapy.

Results

Bioassay of the chemopreventive efficacy of budesonide on B[a]P-induced mouse lung tumorigenesis showed that in B[a]P-treated group, mice developed lung tumors in all animals with an average of 12 tumors per mouse and a total tumor volume of B24 mm3 per mouse. In contrast, lung tumor multiplicity in the budesonide group was 3–4 per mouse with a total tumor volume of B1.5 mm3 per mouse. Thus, dietary budesonide significantly inhibited lung tumor formation with a 70% inhibition on multiplicity and a 94% inhibition on total tumor volume. Differences in multiplicity and tumor volume for budesonide-treated mice were highly sig- nificant (Po 0.01) (Table 1). The experimental design of microarray includes the use of normal lung tissues and tumors from B[a]P control mice and tumors from mice treated with both Figure 1 Hierarchical clustering of 363 genes and ESTs found to B[a]P and budesonide. When comparing the tumors of be differentially expressed between B[a]P tumors and budesonide mice treated with B[a]P only with the tumors of mice treated tumors (Po0.05, fold change 42). Expression levels for treated with both B[a]P and budesonide, 363 genes each gene were transformed across samples using a normal (0,1) transformation. Green indicates an expression below the mean were found regulated by budesonide of which 243 genes value for the gene, black near the mean, and red above the mean. were overexpressed and 120 genes were underexpressed More than 240 genes were overexpressed and 120 genes were in mouse lung tumors after budesonide treatment underexpressed in mouse lung tumors after budesonide treatment

Table 1 Chemopreventive effects of budesonide on B[a]P-induced lung tumorigenesis in A/J micea Carcinogen Chemo Tumor Tumor no./ Decrease in Total tumor Decrease preventive incidence mouse multiplicity vol./mouse in vol. agent (%) (mm3) (%)

Vehicle Vehicle 5/16 (31.3%) 0.3170.47 1.2473.27 B[a]P Vehicle 12/12 (100%) 11.6373.20 24.09715.7 B[a]P Bud 17/19 (89.5%) 3.4772.26b 69.8 1.5171.58b 93.8 aApproximately equal numbers of males and females were used, with no signiﬁcant difference in tumor multiplicity between the sexes. At 6 weeks of age, mice in groups 2 and 3 were given single i.p. injection of B[a]P (100 mg/kg in tricaprylin). Mice in group 3 were fed with budesonide (1.5 mg/kg) diet 2 weeks prior the B[a]P treatment and continue for the entire experiment. bPo0.01, tumor multiplicity or volume in budesonide group was signiﬁcantly different from that from B[a]P treatment group. Bud, budesonide

Oncogene Expression profile of mouse lung tumors treated with budesonide RYaoet al 7748 modulated back to normal levels with budesonide three major regulatory pathways were significantly treatment. In Pattern II, 58 genes were found down- altered by budesonide during lung tumorigenesis. regulated in B[a]P-induced tumors compared with Figures 4 and 5 represent the G protein signaling normal lungs and were modulated back to the level of pathway and MAPK cascade, respectively. normal lung tissues after budesonide treatment (Figure 2). A broad range of functions is associated with these genes, including signal transduction, cell cycle, apoptosis, and transcription (Table 2). These Discussion genes may play important roles in mediating the inhibitory effect of budesonide in mouse lung tumor- In the present study, we have successfully examined igenesis. From the genes found differentially expressed, chemoprevention effects of budesonide against lung nine of 11 genes tested were confirmed by RT–PCR; the tumor induction in A/J mice and its effects on gene confirmation rate is about B80% at the cutoff of two- expression. Continual budesonide administration in the fold change and Po0.05 (Figure 3). diet exhibited a potent protective effect against B[a]P- GenMAPP is a novel tool for visualizing expression induced lung tumorigenesis in mice at 40 weeks. We data in the context of biological pathways (Dahlquist found a strong decrease in tumor multiplicity (70%) and et al., 2002). We imported our data set into the program a profound decrease in total tumor volume (94%) in A/J and used it to convert the expression data into mice. Although glucocorticoids have been used for illustrations demonstrating the implications of signifi- cancer prevention and treatment in animals for a long cant expression changes. We found that genes in at least time, the precise mechanism of action has not been elucidated. Proposed mechanisms include that glucocorticoids: (1) initiation of the apoptotic cascade by inducing transcription of death-specific genes; (2) induce apoptosis through cell cycle arrest; and (3) initiate apoptosis by inhibiting transcription of cytokines (Greenstein et al., 2002; Schmidt et al., 2004). Using Affymetrix GeneChips that contain about 12 000 genes and ESTs, we identified differentially expressed genes between untreated tumors and tumors treated with budesonide which may lead to the identification of possible mechanisms and pathways through which budesonide inhibits lung tumorigenesis. Our results support at least two of the three models of budesonide action. As described in more detail below, budesonide induced expression of a number of genes considered to regulate apoptosis and budesonide induced expression of genes involved in cell cycle arrest. Table 2 lists the most prominent genes. Results suggest that budesonide-treated tumors follow a different apoptotic mechanism than do the untreated tumors. In budesonide-treated tumors, Bim, Blk and ALG-2 are all expressed at higher levels than in untreated tumors, suggesting that the Bcl-2-regulated mechanism of apoptosis is activated. Regulation of this pathway has been recently reviewed and showed it to be conserved across many distant species (Puthalakath and Strasser, 2002). In untreated tumors, Raidd/Cradd is expressed at higher levels than in budesonide tumors, suggesting that the Caspase-2 apoptotic pathway is activated in untreated tumors. Differences in apoptotic mechanism could explain, at least in part, the effectiveness of budesonide. Results also show budesonide-treated tumors having higher expression of Mad3 and Mad2, which play roles in mitotic checkpoint function. Mad2 and Mad3 are Figure 2 Hierarchical clustering of 108 genes and ESTs found differentially expressed between B[a]P induced tumors and part of a larger complex that interacts with the budesonide treated tumors (Po0.05, fold change 42) and these anaphase-promoting complex (APC) and blocks its 108 genes and ESTs were modulated back to/near normal levels by ligase activity (Yu, 2002). Cdc28 is the main CDK budesonide treatment. Pattern 1 contains 50 genes upregulated in driving the cell cycle in budding yeast. Genetic interac- tumors and modulated back to normal levels after budesonide treatment. Pattern 2 contains 58 genes downregulated in tumors tions with checkpoint and APC mutants suggest Cdc28 and modulated back to normal levels by budesonide may regulate checkpoint arrest downstream of the

Oncogene Expression proﬁle of mouse lung tumors treated with budesonide RYaoet al 7749 Table 2 Genes changed during mouse lung tumorigenesis and modulated by budesonide Gene name GenBankaccession Fold changes Incidence

Bud T vs B[a]P T

Signal transduction MAPKK1 (MEK1) AV332058 7.0* 4/4 MEKK1 U23470 À3.6* 2/4 MEKK2 U43186 2.1 2/4 MKK3 X93150 À5.8** 3/4 MKK6 U39066 6.5*** 2/4 MKK7 AF026216 2.3** 3/4 JNKK1 U18310 1.5 2/4 Jun oncogene X12761 À3.2* 2/4 Phospholipase C, beta 3 (PLC-b) U43144 2.2 2/4 Neuroblastoma ras oncogene X13664 À2.1** 3/4 Rap1, GTPase-activating protein 1 (Rap1GAP) AI853638 À1.7* 4/4 ras homolog gene family, member C (RhoC) X80638 2.0*** 4/4 RalBP1-associated EH domain protein Reps1 AF031939 1.8* 4/4 Braf transforming gene M64429 2.3* 3/4 Fyn proto-oncogene M27266 À2.5* 4/4 Protein kinase A, catalytic, alpha M12303 À2.2 2/4 Protein kinase C, alpha M25811 À4.1** 3/4 Protein kinase C, gamma L28035 À2.2* 4/4 Protein kinase C, mu Z34524 3.1* 2/4 protein kinase C, theta D11091 À3.3* 4/4 G protein, alpha inhibiting 1 U38501 3.8** 4/4 G protein, alpha inhibiting 2 AI841629 À2.5* 4/4 G protein, alpha o AV374213 2.1* 4/4 G protein, alpha q M55412 À3.3** 3/4 G protein, alpha 11 U37413 À2.7 2/4 G protein, alpha 14 M80631 3.1* 3/4 Inositol 1,4,5-triphosphate receptor 1 AV256307 5.4* 3/4 Phosphatase 2B catalytic subunit, gamma isoform M81475 2.2* 3/4

Cell cycle and apoptosis Bcl2-like 11 apoptosis facilitator (Bim) AA796690 2.4* 4/4 Bcl2-interacting killer-like (Blk) AF048838 1.9* 4/4 Ect2 oncogene L11316 6.6* 4/4 Caspase 9 AB019600 2.3* 3/4 Btg3 associated nuclear protein AF091234 2.3* 4/4 Excision repair cross-complementing rodent repair Deﬁciency, complementation group 5 (XPG) U40796 4.0* 4/4 v-maf, protein G (mafG) AB009693 3.7* 4/4 Cell differentiation and embryonic development AF027185 3.3* 4/4 CDC28 protein kinase 1 (sid1334p) AB025409 4.3* 4/4 RAE1 RNA export 1 homolog AI048716 2.0* 4/4 Programmed cell death 6 (ALG-2) U49112 2.7* 4/4 Inhibitor of growth family, member 1 (ING1) AA647507 2.1* 4/4 Death adaptor molecule (Raidd/Cradd) AJ224738 À2.0* 4/4 CDK5, regulatory subunit (p35) U89527 À3.3* 4/4 Histone deacetylase 2 U31758 2.4* 4/4

Transcription factors Max dimerization protein 3 (Mad3) U32394 2.8* 4/4 MAD2-like 1 (Mad2) U83902 2.9* 4/4 C/EBP, gamma AB012273 2.1* 4/4 Transcription factor 21 AF035717 2.8* 4/4 PDZ and LIM domain 1 AF053367 1.7* 4/4 LIM domain binding 1 U69270 À2.5* 4/4 LIM domain binding 2 U89489 À2.0* 4/4 LIM motif-containing protein kinase 2 AB005132 À2.0** 4/4

*Po 0.05; **Po 0.01; ***Po 0.001

MAD2 and BUB2 pathways (Kitazono et al., 2003). Yin Budesonide-treated tumors also show decreased et al. (2001) recently observed increased expression of expression of the proto-oncogene Fyn, which is asso- Mad2 and Mad3 in response to glucocorticoid stimula- ciated with tumor progression in cancer models tion in vitro. Increased expression of these genes including oral squamous cell carcinoma and metastatic therefore suggests a growth arrest pathway for budeso- murine melanoma (Huang et al., 2003; Li et al., nide-treated tumors. 2003). This suggests a progressive phenotype for the

Oncogene Expression profile of mouse lung tumors treated with budesonide RYaoet al 7750 Next, we imported our data set into the program and used GenMAPP to illustrate the pathways modulated by budesonide treatment. As expected, the G protein pathway coupled with ras MAPK pathway is intimately involved in murine lung tumorigenesis since virtually all of these tumors have mutations in the Kras2 gene. Figures 4 and 5 depict the modulation of G protein pathway and MAPK pathway by budesonide, respectively. Gq/11 pathway is activated by calcium-mobiliz- ing hormones and stimulates PLC-b to produce IP3 and DAG. PLC-b can also activate the MAPK pathway. Gai can regulate signals from c-Src to the Rap pathway, activated Rap1GAP can effectively block the small GTPase Ras and inhibit the MAPK pathway (Rozengurt, 1998). Modulation of these genes by budesonide may indicate that budesonide exerts its chemopreventive effects on mouse tumorigenesis by influencing the G protein and MAPK pathways in addition to its effects on apoptosis. The mechanisms of glucocorticoids’ chemopreventive effects on tumorigenesis are not known. A number of theories have been proposed, which suggest that there are multiple signal pathways and mechanisms that account for the chemoprevention effects mediated by the glucocorticoids. However, to date, the major molecular changes associated with their chemopreventive effects have not been identified. In this study, we have presented the unique molecular profile of mouse lung tumors in mice treated with budesonide in contrast to untreated tumors, and identified a broad range of budesonide associated genes. Our results suggest that budesonide exerts its effects of chemoprevention through growth arrest via Mad2/3 and through apoptosis via Bim/Blk and by inference caspase-8/9. Con- tinued study is needed to further define the targets of Figure 3 Semiquantitative RT-PCR confirmation for selected glucocorticoids and to further elucidate the mechanisms genes. (a) RT–PCR confirmation. Lanes 1–4 are mouse normal by which glucocorticoids prevent tumor development in lung tissues, lanes 5–8 are mouse lung tumors induced by B[a]P, the clinical setting, and perhaps to novel modes of and lanes 9–12 are B[a]P-tumors treated with budesonide. (b) chemopreventive and/or chemotherapeutic intervention. Comparison of fold change produced by DNA microarray with relative expression ratio obtained from RT–PCR

Materials and methods

untreated tumors that was ameliorated by budesonide Experimental design and collection of lung tumors and lung treatment. tissues It was recently reported that a combination treatment of dexamethasone and cisplatin on solid tumors of the B[a]P (99% pure), budesonide (499% pure), and tricaprylin were obtained from Sigma Chemical Co. (St Louis, MO, lung and cervix resulted in less apoptosis compared with USA). A/J mice were obtained from the Jackson Laboratory cisplatin alone (Herr et al., 2003). The mechanism for (Bar Harbor, ME, USA). Animals were housed in plastic cages inhibition of apoptosis was shown to involve down- with hardwood bedding and dust covers, in a HEPA-filtered, regulation of CD95-L and TRAIL resulting in a environmentally controlled room (24711C, 12/12 h light/dark reduction of caspase-8/9-mediated apoptosis. In another cycle). B[a]P was prepared immediately before use in animal study, Dorscheid et al. (2001) showed that a number of bioassays by dissolving in tricaprylin. All mice were fed AIN- corticosteroids including both dexamethasone and 76A purified diet during the experiment. In the vehicle group, budesonide elicited apoptosis via a caspase-9 mediated 16 6-week-old A/J mice were given a single i.p. injection of mechanism using epithelial cells. In this study, budeso- tricaprylin (0.1 ml). In the carcinogen control group, 12 6- week-old A/J mice were given a single i.p. injection of B[a]P nide treatment appeared to recruit the caspase-8/9 and (100 mg/kg body weight) in 0.1 ml of tricaprylin. In the to downregulate the caspase-2. It would be interesting to budesonide group, 19 mice were given a single i.p. injection carefully optimize combination treatment of these of B[a]P (100 mg/kg body weight) in 0.1 ml of tricaprylin and tumors since there are so many advantages to using were fed AIN-76A purified diet containing budesonide the glucocorticoid adjuvant. (1.5 mg/kg of diet) beginning 2 weeks prior to B[a]P treatment

Oncogene Expression proﬁle of mouse lung tumors treated with budesonide RYaoet al 7751 G Protein Signaling Pathways

Gs Gi Gq Upregulated by budesonide G alpha s G alpha o 1 Downregulated by budesonide G alpha q G alpha olf G alpha o 2 No criteria met G alpha 11 G alpha z Not found G alpha 14 G alpha i 1 G alpha 15 G alpha i 2 G alpha i 3 Phosopholipase C beta 3

Adenylate Cyclases IP 3 AC1 AC6 GIRK1 K+ Chnl hRAS DAG AC2 AC7 nRAS AC3 AC8 kRAS A IP3 Receptor AC4 AC9 kRAS B AC5 rRAS Ca++ Protein Kinase C cAMP PKC alpha PKC beta Protein Kinase A Pho sphatase 2B PKC gamma PKA 1 alpha reg catalytic alpha Calmodulin PKC delta PKA 1 beta reg catalytic beta PKC epsilon PKA 2 alpha reg catalytic gamma PKC iota PKA 2 beta reg Cell Response PKC eta PKA alpha cat PKC mu PKA beta cat Transcription PKC theta PKA gamma cat PKC zeta

Transcription Cell Response Figure 4 GenMAPP –G protein pathways integrating our expression data. Yellow indicates a higher level of expression in budesonide-treated samples. Blue indicates a lower gene expression by budesonide. Grey indicates that the selection criteria were not met but the gene is represented on the array. White boxes indicate that the gene was not present on the array

MAPK Cascade

Gi Gq β G an d G Upregulated by budesonide RapGAPII PLC-beta3 Downregulated by budesonide GTP No criteria met Not found H-ras P21/ras activ. Activated N-ras Rap1GAP Rap1GAP R-ras K-ras

GDP C-raf MEKK1 A-raf MEKK2 B-raf MEKK3

MEK1 MEK2 Stress Activated Pathways P38 MAPK Pathways

MKK7 MKK6 ERK1 ERK2 JNKK 1 p38

mbp JNK 3 MKK3 MAP2 Elk1

Nucleus Growth and Mitogenesis c-jun

Apoptosis Figure 5 GenMAPP –MAPK cascade integrating our expression data. Yellow indicates a higher level of expression in budesonide- treated tumors. Blue indicates a lower level of expression in the budesonide-treated samples. Grey indicates that the selection criteria were not met but the gene is represented on the array. White boxes indicate that the gene was not present on the array

Oncogene Expression profile of mouse lung tumors treated with budesonide RYaoet al 7752 and continuing for an additional 40 weeks. Food and water to the manufacturer’s recommendations. To validate the were available ad libitum. At termination, the lung tumors were microarray results, 11 genes were randomly selected from harvested and frozen in liquid nitrogen until RNA analysis. All the genes detected in the microarray assay for further frozen tumor tissues were microdissected to determine the confirmation by semiquantitative RT–PCR as previously tumor vs normal cell ratio for each specimen. Tumor tissue described (Yao et al., 2002). After normalization to the level sections corresponding to the microscopic sections containing of b-actin mRNA, the student’s t-test was used to determine X80% tumor cells were isolated and stored at À801C for the differences in the signal intensity of phosphor imaging subsequent RNA isolation. Matching normal tissues were also among the normal lungs, lung tumors and budesonide treated microdissected to ensure that specimens consisted of purely lung tumors. normal lung tissue. Lung tumor development was then evaluated by counting tumor number (N), calculating tumor volume (V) and the total tumor load (total tumor V per Cluster and GenMapp mouse). Four independent samples were collected for each group. Array normalization and gene expression estimates were RNA isolation and amplification obtained using Affymetrix Microarray Suite 5.0 software Total RNA from each sample was isolated by Trizol (MAS5). The array mean intensities were scaled to 1500. (Invitrogen, Carlsbad, CA, USA) and purified using the Genes with negative expression values were removed. Differ- RNeasy Mini Kit and RNase-free DNase Set (QIAGEN, ential expression was determined on the combined basis of Valencia, CA, USA) according to the manufacturer’s proto- statistical testing using t-test and expression ratio with cutoff cols. In vitro transcription-based RNA amplification was then of Po0.05 and expression ratio (fold change) 42oro0.5 performed on each sample. cDNA for each sample was being called positive for differential expression. Genes passing synthesized using a Superscript cDNA Synthesis Kit (Invitro- this filter were allocated to four mutually exclusive groups gen) and a T7-(dT)24 primer: 50-GGCCAGTGAATTGTAA- according to their expression pattern including upregulated or TACGACT-CACTATAGGGAGGCGG-(dT)24-30. The downregulated to near normal level. To perform hierarchical cDNA was cleaned using phase-lock gel (Fisher Cat ID clustering, expression for each gene was transformed across E0032005101) phenol/chloroform extraction. Then, the biotin- samples to a standard normal deviate. These values were labeled cRNA was transcribed in vitro from cDNA using a supplied to the Gene Cluster program of Eisen and used BioArray HighYield RNA Transcript Labeling Kit (ENZO without further transformation (Eisen et al., 1998). Signal Biochem, New York, NY, USA) and purified, again using the transduction pathways, metabolic pathways and other func- RNeasy Mini Kit. tional groupings of genes were evaluated for differential regulation using the visualization tool GenMAPP (UCSF, www.genmapp.org; (Dahlquist et al., 2002). We imported our Affymetrix genechip probe array and RT–PCR confirmation data set into the program and used GenMAPP to illustrate The labeled cRNA was applied to the Affymetrix MG_74Av2 pathways containing differentially expressed genes. Differen- GeneChips (Affymetrix, Santa Clara, CA, USA), which tial gene expression was based on budesonide treatment vs contains 412 000 genes and ESTs on one array according nontreatment expression change.

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