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[Cancer Research 62, 5867-5873, October 15, 2002]
© 2002 American Association for Cancer Research

Molecular Biology and Genetics

Comparative Gene Expression Profile Analysis of GLI and c-MYC in an Epithelial Model of Malignant Transformation1 ,, 2

Iúri D. Louro, Evans C. Bailey, Xingnan Li, Lindsey S. South, Peggy R. McKie-Bell, Bradley K. Yoder, Conway C. Huang, Martin R. Johnson, Aubrey E. Hill, Ronald L. Johnson3 and J. Michael Ruppert4

Department of Medicine, Division of Hematology/Oncology [I. D. L., L. S. S., P. R. M-B., A. E. H., J. M. R.], Department of Cell Biology [E. C. B., B. K. Y., R. L. J.], Department of Biochemistry and Molecular Genetics [X. L.], Department of Dermatology [C. C. H.], and Department of Pharmacology [M. R. J.], University of Alabama at Birmingham, Birmingham, Alabama 35294


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Transcription factor oncogenes such as GLI and c-MYC are central to the pathogenesis of human tumors. GLI encodes a zinc finger protein that is activated by Sonic Hedgehog signaling. Mutations in this pathway induce GLI expression in basal cell carcinoma, and expression of GLI in mice is sufficient to induce these skin tumors. We used microarrays to identify transcripts regulated by GLI or c-MYC after retroviral transduction and short-term culture of epithelial RK3E cells. Although each of these oncogenes induces malignant transformation of RK3E, two distinct sets of genes were identified. Of ~17,500 transcripts represented on the microarrays, GLI up-regulated the expression of 158 and repressed the expression of 52. In contrast, transcripts regulated by c-MYC were mainly repressed (424 of 682 regulated transcripts). Transcripts induced by the GLI transgene are likewise expressed in association with endogenous GLI in Ptch-deficient murine fibroblasts or in human skin tumors, but are not up-regulated in RK3E cells transformed by c-MYC, KLF4, or HRAS1. Unlike these other oncogenes, GLI induced the expression of mesenchymal cell markers including Snail, a zinc finger protein implicated in epithelial-mesenchymal transition in development and during tumor progression. A novel GLI-estrogen receptor fusion protein rapidly induced Snail mRNA expression in a manner like Ptch, a known direct transcriptional target gene. Induction of Snail expression and epithelial-mesenchymal transition by GLI may account for certain histopathological features of basal cell carcinoma, such as the absence of a well-defined, intraepithelial precursor lesion. In addition, consistent expression of the newly identified GLI-induced transcripts within GLI-expressing tumors in vivo indicates that oncogene-specific transcriptional profiles may be useful diagnostic tools for analysis of human tumors.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Gene expression profiles of human tumors offer great potential to understand cancer pathogenesis and improve treatment (1, 2, 3) . Determining how specific molecules such as transforming oncogenes influence the overall profile has been limited by the paucity of robust and reliable in vitro models. Several factors account for this problem. Although the early genetic alterations that give rise to carcinomas occur in epithelium, few epithelial cell lines have been available for functional analysis of oncogenes. Secondly, oncogenes can have distinct effects in different cell types, inducing malignant transformation, apoptosis, growth arrest, or even differentiation. Thus, expression of an oncogene in a specific cell line may result in gene expression profiles that bear little resemblance to that resulting in vivo. Finally, oncogenes induce a cascade of direct and indirect transcriptional alterations, such that genes altered by a direct mechanism may be difficult to identify.

RK3E epithelial cells provide an excellent in vitro model for analyzing early events in tumor progression (4, 5, 6) . These diploid, immortalized cells were cloned from rat kidney cells by transfection of primary cultures with adenovirus E1a (4) . In response to a select set of oncogenes important in carcinoma, including GLI, c-MYC, HRAS1, KLF4/GKLF, and others, the cells exhibit loss of contact inhibition and malignant transformation in vitro. RK3E cells and transformed derivatives proliferate at similar rates at subconfluence, thus allowing the process of transformation to be analyzed in a setting where nontumorigenic control cells and malignant cells exhibit similar cell cycle parameters (5 , 7) .

In human BCC,5 GLI expression is thought to be up-regulated by loss-of-function mutations in the tumor suppressor PTCH or through activating mutations in SMO (8, 9, 10) . Several direct transcriptional targets of GLI have been identified, including PTCH, HNF3ß, PDGFR{alpha}, and MYF5. These genes contain elements that are similar to the GLI binding site, GACCACCCA (11) . However, GLI-regulated transcripts have not been extensively characterized, and it remains unclear how this oncogene induces malignant transformation.

In this study we observed that early passage cells transduced by GLI or c-MYC exhibit gene expression profiles that are oncogene-specific. ISH of human BCCs revealed coexpression of GLI and GLI-induced transcripts. In addition, Snail mRNA is induced by a GLI-ER fusion protein in the absence of protein translation, consistent with a direct interaction between GLI and regulatory elements of the Snail gene (12 , 13) . The GLI-induced profile included other markers of mesenchymal cells, suggesting that GLI and Snail may induce certain of the mesenchymal features observed in BCC in vivo.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid Constructs and Cell Culture.
The human GLI cDNA was modified at the NH2 terminus with a hemagglutinin epitope and cloned into the Moloney murine leukemia virus vector pLJD (obtained from Louise T. Chow, University of Alabama at Birmingham). The c-MYC expression vector pCTV3K-SQC5, cell culture medium, and retroviral transduction of RK3E were described previously (5) . Cells transduced with pLJDgli or pGLI-ER were selected in 400 µg/ml G418 or 0.4 µg/ml puromycin, respectively. Cells transduced with pCTV3K or pCTV3K-SQC5 were selected in 80 µg/ml hygromycin. Total RNA was isolated from RK3E cells at 80% confluence and from Ptch -/- and +/- embryonic fibroblasts at 100% confluence (14) .

Microarray Analysis and SSH.
Retroviral transduction of RK3E, drug selection, RNA preparation, probe preparation, array hybridization, scanning, and data analysis were all performed twice in sequential, independent experiments termed experiments 1 and 2. U34A and U34B arrays were analyzed using GeneChip 3.3 software (Affymetrix) and dCHIP (15 , 16) . Alignment of accession numbers from different data sets was performed using FileMaker Pro version 5.5, and scatter plots were generated using Excel (Microsoft). Gene nomenclature was according to Blake et al. (17) .6 As a measure of quality of experimental technique, 40–52% of transcripts represented on the arrays were scored as present by GeneChip. For selection of GLI target genes for Northern blot analysis we used GeneChip analysis of microarray experiment 1, the more sensitive of the two experiments.

SSH was performed using the PCR-Select cDNA Subtraction kit (Clontech). SSH adaptors were as described (18) . A detailed protocol is available from the authors on request. One-hundred-twenty clones, named SSH1-120, were screened by Northern blot analysis of total RNA from GLI cells and control cells.7

RT-PCR and Northern Blot Analysis.
Primers corresponding to candidate GLI target genes were used to amplify cDNA fragments for use as Northern blot probes (Table 1)Citation . Reverse transcription reactions were performed using total RNA from GLI-transduced RK3E cells, oligodeoxythymidylic acid primer (Invitrogen) at 50 µg/ml, and SuperScript II reverse transcriptase (Invitrogen). PCR conditions, primer annealing conditions, RNA isolation, and Northern blot conditions were as described previously (5) .


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  		Table 1 PCR primers used in this study

 
ISH.
Fresh-frozen samples were obtained through the Tissue Procurement Facility of the University of Alabama at Birmingham. Human cDNAs corresponding to induced transcripts were isolated by RT-PCR analysis of RNA from microdissected BCCs (Table 1)Citation . A T7 RNA polymerase recognition site was incorporated into the Reverse primer. Digoxigenin-labeled transcripts were purified on an RNeasy column (Qiagen). Frozen sections of BCC were analyzed by ISH using antisense transcripts at 150 ng/ml and then developed using the alkaline phosphatase substrate Fast Red (19) .

Construction of pGLI-ER.
The GLI protein-coding region was amplified from pLJDgli using Pfu polymerase (Stratagene) and primers incorporating BglII restriction sites. The product was cloned into the EcoRV site of pZero-2 (Invitrogen), digested with BglII, and inserted into the BamHI sites of pBpuro c-myc ERTM (20) . This strategy fuses the GLI protein-coding region to an OHT-inducible fragment of the ER.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Gene Expression Profile Analysis.
To better understand how transcription factor oncogenes alter the gene expression profile of transformed cells, RNA was prepared from RK3E cells 10 days after retroviral transduction of GLI (GLI cells), c-MYC (MYC cells), or vector controls. These cells exhibit the distinctive morphologies reported previously for GLI- or c-MYC-transformed RK3E (5 , 7) . Microarray analysis was performed after two independent transduction experiments (Table 2Citation ; Supplemental Data2).


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  		Table 2 Identification of GLI-induced transcripts by use of microarrays and the SSH assay

 
Of the 17,598 transcripts represented on the microarrays, GLI induced 158 (0.86%) and repressed 52 (0.30%) by 3-fold or more relative to the vector control (Fig. 1ACitation ; Table 2Citation ; and Supplemental Data,2 experiment 1). In contrast, c-MYC acted mainly as a repressor, inducing 258 (1.47%) and repressing 424 (2.41%) by 3-fold or more relative to the vector control.



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  		Fig. 1. A, oncogene-induced gene expression profiles in RK3E cells. Cells were stably transduced with GLI or c-MYC expression vectors, and mRNA was analyzed using Affymetrix microarrays. Transcripts are grouped according to fold-induction or fold-repression. Cells transduced with empty vectors served as the control. B, transcripts altered by 3-fold or more relative to the control are ordered on the X axis, and the corresponding fold-change induced by the other oncogene is plotted on the Y axis. Panels on the left include all data points, whereas a magnified view centered at the origin is shown on the right. Linear regression (blue lines) indicates that similar results were obtained for GLI in two independent experiments (bottom row; R2 = 0.865), but there was little correlation between the GLI- and c-MYC-induced profiles (top row, R2 = 0.003; middle row, R2 = 0.014).

 
We used scatter plots to compare the fold-regulation of each transcript by GLI or c-MYC (Fig. 1BCitation ; Supplemental Data2). Transcripts regulated by c-MYC were largely unaltered by GLI expression and clustered near the Y-axis origin (Fig. 1BCitation , top panels). Similarly, there was little regulation of GLI targets by c-MYC (Fig. 1BCitation , middle panels). In contrast, a plot of GLI-regulated transcripts from independent experiments indicated a strong correlation (Fig. 1BCitation , bottom panels).

We also used the SSH assay to identify GLI-induced transcripts (Table 2)Citation . This approach identified several transcripts that were also identified by microarray analysis (i.e., Crlf1, Cdh11, and Snail). In addition, we identified the known genes Ptch, Clu, Wif1, and Fus2, several molecules corresponding to ESTs (Ssh14, AA543886; Ssh20, AA387505), and transcripts not represented in the database (Ssh31, Ssh41, Ssh78, Ssh80, and Ssh85).

Several GLI targets that we identified, including Bcl2, Couptf2, Hnf3b, Igf2, Ptch, Timp3, and Wnt2b were shown previously to be Shh-regulated or were induced in human BCC (21, 22, 23, 24, 25, 26, 27, 28) . As for GLI, known c-MYC-dependent transcripts were identified, including the glucose transporter Slc2a1, nucleolin, RCL, Slc16a1, thrombospondin, MHC class I, tropomysosin 1{alpha}, fibronectin 1, and the cyclin-dependent kinase regulator and tumor suppressor p15ink4b (Table 3Citation ; Refs. 29, 30, 31 ). Interestingly, c-MYC-repressed transcripts included at least two tumor suppressors, p15ink4b and the retinoblastoma suppressor Rb-1. A role for transcriptional repression as a potential mechanism of transformation by c-MYC is well recognized (31, 32, 33) .


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  		Table 3 Transcripts regulated by c-MYC in RK3E cells

 
Northern Blot Analysis.
cDNA probes were prepared for 27 transcripts predicted to be induced by GLI according to GeneChip analysis of data from experiment 1. Each of these transcripts were up-regulated in GLI cells (Fig. 2ACitation , Lanes 1–2; Table 2Citation ). Subsequent comparison of GeneChip and dCHIP outputs for experiments 1 and 2 revealed that dCHIP predicted more consistent fold-changes between experiments, whereas GeneChip correctly identified multiple true positive alterations that were not called by dCHIP (data not shown).



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  		Fig. 2. Expression of GLI-regulated transcripts. A, RK3E cells were cultured in selective medium for 10 days after retroviral transduction of a GLI expression vector (Lane 2) or a control (Lane 1). RNA was analyzed by Northern blot using cDNA probes corresponding to GLI-induced transcripts. Tumorigenic cell lines cloned from transformed foci were similarly analyzed (Lanes 3–7). Parental RK3E cells (not shown) and cells transformed by c-MYC, KLF4, or HRAS1 (Lanes 4–6) expressed low levels of GLI-induced transcripts. Detection of Wnt2b, Fgf10, and Hnf3b required poly(A)+ RNA; total RNA was analyzed for other transcripts. A ß-tubulin probe confirmed equal loading. B, analysis of GLI-regulated genes in Ptch -/- and Ptch +/- mouse fibroblasts. Equal loading of total RNA was confirmed by hybridization to ß-tubulin (not shown).

 
To determine whether candidate GLI target genes were specifically induced in cells transformed by GLI, we examined their regulation in cell lines derived from oncogene-transformed foci of RK3E (Fig. 2ACitation , Lanes 3–7; Table 2Citation ; Refs. 5 , 7 ). Whereas the transcripts were consistently up-regulated in cell lines transformed by GLI (Fig. 2ACitation , Lanes 3 and 7), they were rarely induced in lines transformed by c-MYC, HRAS1, or KLF4 (Fig. 2ACitation , Lanes 4–6).

GLI expression in BCC is thought to result from persistent signaling as a consequence of mutation of PTCH or SMO. To determine whether expression of endogenous Gli is sufficient to induce expression of these transcripts, we examined Ptch-deficient murine fibroblasts (Fig. 2BCitation ; Table 2Citation ; Ref. 14 ). Compared with Ptch +/- cells, the Ptch -/- cells exhibit elevated expression of Gli, of transcripts derived from the modified Ptch locus, and of other known Shh targets (Fig. 2BCitation ; Ref. 34 ). Of the 22 transcripts examined, 8 were up-regulated in the Ptch -/- cells consistent with regulation by Gli. The majority of transcripts tested were expressed at similar levels in the Ptch -/- and Ptch +/- cells (e.g., Snail), perhaps because of cell-type differences between RK3E epithelial cells and fibroblasts. Alternatively, Ptch and Gli may coregulate some transcripts by distinct mechanisms. For example, the proposed Gli target Clu was actually repressed in Ptch -/- cells (Table 2)Citation .

Expression of GLI-induced Transcripts in Tumors.
To determine whether the identified transcripts were expressed in association with GLI in vivo, we analyzed gene expression in human BCCs (25 , 35) . Examination of two nodular BCCs by ISH revealed prominent expression of GLI and of several GLI-induced transcripts (Fig. 3Citation ; Table 2Citation ). In contrast, the transcripts were undetected in many adjacent hair follicles, where GLI is expressed only during the anagen phase of the follicle cycle (25) . ß-TUBULIN, a control for RNA integrity, was similarly expressed in hair follicles and in tumor (Fig. 3F)Citation . Most transcripts were not detected in the surrounding mesenchyme, except for CLU, which was expressed both in the tumor and in the adjacent mesenchyme (Fig. 3D)Citation . Thus, transcripts induced by GLI in RK3E were induced in association with GLI in vivo.



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  		Fig. 3. Expression of GLI-induced transcripts in human BCC. A–F, sections of tumor (bottom left of each panel) and surrounding tissue were hybridized with probes corresponding to GLI or GLI-induced genes and counterstained with hematoxylin. Hair follicles adjacent to the tumor (top right of each panel) were controls and showed either undetectable or reduced levels of expression. ß-TUBULIN was detected in all cells and controls for RNA integrity. Panels are at the same magnification and the scale bar corresponds to 100 µ. G–N, serial sections of a BCC were hybridized with GLI and CDH11 probes and examined at low (G and K) and high (H and L) magnification. GLI is detected in tumor cells, whereas CDH11 is present in the surrounding mesenchyme. For a subset of adjacent hair follicles, GLI is expressed in epithelial cells of the root (I), whereas CDH11 expression is detected in adjacent mesenchymal cells (M). In contrast, cells surrounding GLI-negative hair follicles do not express CDH11 (J and N). Scale bars, 100 µ.

 
Unlike GLI and the other transcripts examined, the cell-cell adhesion molecule CADHERIN 11 (CDH11) was not expressed in the BCC itself, but in the surrounding mesenchyme (Fig. 3, G, H, K, and L)Citation . Indeed, analysis of serial sections showed that CDH11 expression surrounds GLI-positive hair follicles (Fig. 3, I and M)Citation but not those that are GLI-negative (Fig. 3, J and N)Citation . This nonautonomy suggests that GLI may control the expression of signaling molecules to induce CDH11 in the adjacent dermal cells. In addition, the GLI-induced transcripts SNAIL, TIMP3, OPN, CLU, FUS2, and CRLF1 were also present at reduced levels in a subset of morphologically normal hair follicles adjacent to a tumor (data not shown). Therefore, GLI may regulate these transcripts in normal epithelial cells during the hair follicle cycle.

Characterization of an Inducible GLI-ER Fusion Protein.
To discriminate between indirect effects and direct transcriptional targets, we fused GLI to an OHT-inducible form of the ER (20) . Ptch was rapidly induced by OHT in RK3E cells stably transduced with GLI-ER (Fig. 4ACitation and Fig. 4BCitation , Lanes 5–7). Consistent with a direct interaction of GLI-ER with GLI binding sites in the Ptch promoter (36 , 37) , induction was maintained and even enhanced in the presence of the protein translation inhibitor CHX. Similarly, Snail mRNA was induced by either OHT or OHT + CHX (Fig. 4, A and B)Citation . SNAIL genomic sequence contains four candidate GLI binding sites, each matching eight of nine positions of the consensus (11) . One of these is within the first intron, whereas three others are found upstream, within 12 Kb of the first exon. In these studies, GLI-ER did not induce ß-tubulin, Gapd, or the candidate GLI target Opn, suggesting that Opn activation by GLI is indirect (Fig. 2A)Citation .



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  		Fig. 4. Rapid induction of Ptch and Snail mRNA by a GLI-ER fusion protein. A, RK3E cells were stably transduced with the GLI-ER expression vector, and expression of Ptch and Snail were examined after addition of OHT and CHX to the culture media for 2 h. For comparison, the 10-day sample in the bottom panel represents Snail expression in RK3E cells transduced by wild-type GLI vector. Expression of Gapd and ß-tubulin were unchanged. Similar results were obtained in three independent induction experiments. B, control RK3E cells and GLI-ER cells were treated with OHT and/or CHX as indicated. Gene expression was quantitated by a phosphorimager and normalized to Gapd. The fold-induction relative to the uninduced control (Lanes 1 or 5) was determined.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A major result of this study was the finding that different oncogenes induce distinct expression profiles during transformation of cells in culture. Indeed, cellular transcripts were regulated in an opposite fashion by GLI and c-MYC; whereas most GLI-regulated transcripts were up-regulated, most c-MYC targets were repressed. These results are consistent with previous functional studies identifying activation and repression domains within GLI and c-MYC, respectively, and indicate that c-MYC may transform RK3E cells in part by coordinately repressing expression of tumor suppressor genes (31, 32, 33 , 38) . The very few transcripts that are similarly regulated by both oncogenes largely represent uncharacterized expressed sequence tags or else known genes without a clearcut role in cell cycle regulation or transformation (Supplemental Data).2 Whereas these descriptive studies suggest that GLI and c-MYC induce transformation by distinct mechanisms, the possibility remains that one or more common targets play a critical role.

Oncogene-specific profiles were achieved by use of early passage cells (i.e., 10 days post-transduction of retroviral expression vectors). In initial studies (not shown) we used established cell lines derived from transformed foci as the RNA source for microarray analysis. We identified a prevalent class of transcriptional alterations that were late (i.e., they were not observed in early passage cells), nonspecific (i.e., the alterations were observed in cells transformed by different oncogenes), and inconsistent (i.e., they were not similarly induced in several independent clones of cells transformed by one oncogene). Late alterations involved genes encoding, for example, multiple protein components of the small and large ribosomal subunits. Consistent with these results, a recent microarray study that used mRNA from a late-passage, GLI-transformed RK3E line identified 30 altered transcripts that included only a few of the genes identified by us in early passage cells (e.g., Ptch, Opn; Ref. 39 ). We conclude that transformed cells undergo gradual, nonspecific changes in gene expression that may provide a selective growth advantage and that oncogene-specific profiles can be identified by use of early passage cells for RNA preparations.

Although gene expression profiles may be altered during passaging of transformed cells in culture, alterations that we identified in early passage cells appear to be stably maintained. Transcripts induced by GLI in early passage cells were consistently up-regulated by Northern blot analysis of several independent cell lines that were derived from transformed foci and then passaged >20 times (see "Results"). In addition, these alterations were induced in association with endogenous GLI in embryo fibroblasts deficient in Ptch or in human BCC. Therefore, enforced expression of nonphysiological levels of GLI in RK3E cells did not account for the observed expression profile.

EMT is a normal developmental process in which epithelial cells, attached to the underlying extracellular matrix, lose apical-basal polarity and migrate into the matrix (40 , 41) . EMT occurs to a variable degree during tumor progression in humans, resulting in mesenchymal-like cells that invade and metastasize. The zinc finger protein Snail is implicated as an inducer of EMT in development, and more recent results suggest a role for Snail in EMT during progression of human tumors (12 , 13 , 41) . When expressed in certain epithelial cells, Snail induces fibroblastic conversion, loss of E-cadherin, and malignant transformation. E-CADHERIN expression is reduced in human BCC (42 , 43) , consistent with our demonstration of SNAIL expression and with the locally invasive nature of these tumors (Fig. 3C)Citation . In addition to Snail, our study indicates that GLI up-regulates other mesenchymal markers in RK3E cells (e.g., Cdh11 and Clu). Previous studies have not linked GLI activity to EMT in development, although a role for Shh as an inducer of EMT in developing somites is well established (40) .

GLI-induced transcripts identified in this study may account for some of the known properties of human BCC. BCC does not exhibit an intraepithelial precursor lesion analogous to actinic keratosis, papilloma, or adenoma (44) . By regulating genes implicated in EMT and in multiple other aspects of the malignant phenotype (39 , 45) , GLI may convert normal epithelial cells into invasive tumor cells in a relatively direct fashion, without the need of multiple other genetic changes. Furthermore, induction of potential tumor suppressor molecules such as Timp3 or Wif1 may help to explain the infrequent occurrence of metastasis in patients with this disease. A role for these transcripts in BCC biology can be readily determined by genetic studies that use existing mouse models of BCC in combination with loss-of-function alleles for specific target genes.


   ACKNOWLEDGMENTS
 
We thank Scott Mordecai for assistance in analysis of Affymetrix data, and Brad St. Croix and Ken Kinzler for sharing their mRNA in situ hybridization method.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by NIH Grants RO1CA65686 and P50CA89019 (to J. M. R.) and HD37505 (to R. L. J.), by United States Army Grant PC970408 (to J. M. R.), by a Companha de Aperfeiçoamento de Pessoal de Nível Superior Grant from the Brazilian government (to I. D. L.), and by a gift to the Comprehensive Cancer Center from the Avon Foundation. The University of Alabama at Birmingham Microarray and Oligonucleotide Core Facilities are supported by NIH Grant 5P50 CA13148. Back

2 Supplemental data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org). Back

3 Present address: Human Genome Sciences, 9410 Key West Avenue, Rockville, MD 20850. Back

4 To whom requests for reprints should be addressed, at Department of Medicine, Room 570 WTI, University of Alabama at Birmingham, School of Medicine, Birmingham, AL 35294-3300. Phone: (205) 975-0556; Fax: (205) 934-9511; Email: mruppert{at}uab.edu. Back

5 The abbreviations used are: BCC, basal cell carcinoma; dCHIP, DNA-Chip Analyzer, version 1.1; EMT, epithelial-mesenchymal transition; ISH, mRNA in situ hybridization; SSH, suppression subtractive hybridization assay; ER, estrogen receptor; OHT, 4-hydroxytamoxifen; Shh, sonic hedgehog; CHX, cycloheximide; RT-PCR, reverse transcription-PCR. Back

6 Internet address: http://www.informatics.jax.org. Back

7 Data Deposition: Sequences identified by the SSH assay were submitted to Genbank (accession nos. AF354121-AF354125). Back

Received 4/29/02. Accepted 8/20/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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