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A Minimal Critical Region of the 8p22–23 Amplicon in Esophageal Adenocarcinomas Defined Using Sequence Tagged Site-Amplification Mapping and Quantitative Polymerase Chain Reaction Includes the GATA-4 Gene¹

Department of Surgery, Section of General Thoracic Surgery [L. L., S. A., M. B. O., D. G. B.] and Departments of Human Genetics [T. W. G.] and Pediatrics [T. W. G., S. H.], University of Michigan Medical School, Ann Arbor, Michigan 48109

	ABSTRACT

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The incidence of esophageal adenocarcinomas has increased greatly over the past 20 years. The genetic alterations associated with this disease, however, remain largely unknown. We identified recently a novel amplicon at 8p22–23 in esophageal adenocarcinomas using the restriction landmark genomic scanning two-dimensional gel technique. Four known genes within or near this amplicon were initially characterized. The cathepsin B (CTSB) gene was found to be amplified in 13% of esophageal tumors. CTSB was shown previously to be overexpressed without amplification in many other human cancers. An approach termed sequence tagged site-amplification mapping has been implemented in the present study, allowing the 8p22–23 amplicon to be narrowed from 12 cM to a <2-cM minimal amplified area located between markers D8S552 and D8S1759. The CTSB gene maps within this region. To identify other cancer-related candidate genes in this region, a positional candidate gene approach was subsequently applied to characterize this minimal critical region. An expressed sequence tag (EST), which was included in the minimal critical region, demonstrated both amplification and overexpression. This EST and the extended sequence from the EST were determined to be a novel sequence in the 3' untranslated region of the human GATA-4 gene. GATA-4, a member of a zinc finger transcription factor family, was confirmed to be amplified and overexpressed in esophageal adenocarcinomas and was localized within <0.5 kb from CTSB. Furthermore, amplification of 8p22–23 was detected in one of eight gastric cardia adenocarcinomas but was not observed in either human lung adenocarcinomas (n = 39) or in esophageal squamous cell carcinomas (n = 24). The relatively high frequency of the 8p22–23 amplification in esophageal (13.6%) and gastric cardia (12.5%) adenocarcinomas may indicate a specificity of this amplicon for tumors of gastroesophageal origin.

	INTRODUCTION

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Esophageal adenocarcinoma has been a source of significant and increasing morbidity and mortality in recent decades. The incidence of these tumors has increased >350% in the past three decades, and 5-year survival remains low at <10% (1 , 2) . The precursor lesion for esophageal adenocarcinoma is Barrett’s esophagus, metaplastic columnar epithelia that replace the normal squamous mucosa in the lower esophagus in patients with chronic gastroesophageal reflux (3 , 4) . The presence of Barrett’s metaplasia is the major risk factor for development of esophageal adenocarcinoma. Smoking and familial background also appear to contribute to the development of these tumors, which occur predominantly in males (5, 6, 7) . Compared with many other human tumors, relatively little is known about the genetic alterations that occur in esophageal adenocarcinomas.

Gene amplification is one mechanism of oncogene activation leading to tumorigenesis. Barrett’s esophagus and esophageal adenocarcinoma provide an ideal tumor progression model to examine gene amplification because a well-defined metaplasia-dysplasia-carcinoma sequence is observed (8) . Several proto-oncogenes demonstrate gene amplification in esophageal adenocarcinomas, including the erbB2 gene which is amplified in 22%, the EGFR gene in 13%, and the K-ras gene in 10% (9) .³ Additional chromosomal amplicons have been identified using CGH⁴ as well as other techniques including the amplicon at 8p22–23 (10) . Candidate genes for these amplicons, however, remain largely unknown because narrowing the minimal critical amplified region using CGH alone still leaves relatively large chromosomal intervals involving many uncharacterized genes (reviewed in Ref. 11 ).

We have identified recently a novel amplicon at 8p22–23 in esophageal adenocarcinomas using the RLGS 2D-gel technique (10) . The CTSB gene was proposed as a potential candidate gene for the 8p22–23 amplicon, based on the fact that it was found to be amplified in 13% and overexpressed in >25% of esophageal adenocarcinomas examined, and that CTSB is overexpressed in many other human cancers (12, 13, 14) . The 8p22–23 amplicon, however, spans 12 cM and includes many genes in this region. To better characterize the minimal critical region of this amplicon, in the present study, we implemented a novel application, which we have termed STS-amplification mapping. This approach uses integrated physical maps and databases to search highly resolved microsatellite markers (STS) mapped within the chromosomal region of the amplicon. The selected STS markers are then applied to screen a large number of normal-tumor pairs of genomic DNA by a QG-PCR assay. The minimal critical area is determined by mapping the minimal overlapping amplified region between tumors. This application allowed us to construct an STS-amplification map and narrow the 12-cM amplicon to a <5-cM minimal amplified region in the present series of 66 esophageal adenocarcinomas. A positional candidate approach was then applied to this region, and the minimal critical region was further reduced to <2 cM. A YAC clone (725-c-12), which contained a 420-kb insert including CTSB, was analyzed previously using FISH to determine its chromosomal location (10) . This YAC was localized within the minimal amplified region in the present study. The GATA-4 gene was also verified to be included within YAC 725-c-12. GATA-4 was observed to demonstrate gene amplification as well as mRNA and protein overexpression in the esophageal tumors in the present study, as was the case for CTSB in the previous study (10) . GATA-4 is a member of zinc finger transcription factor family that play developmental roles including gut endodermal differentiation (15) . The function of the GATA-4 gene in adult tissue, however, remains unknown. Furthermore, by using QG-PCR assay, we were able to demonstrate genomic amplification of 8p22–23 in both esophageal and gastric cardia adenocarcinomas but not in esophageal squamous cell carcinomas or lung adenocarcinomas.

	MATERIALS AND METHODS

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Tumor Tissue Collection and DNA/RNA Isolations.
Sixty-six esophageal adenocarcinomas and the corresponding normal esophageal or gastric mucosa, as well as 20 Barrett’s metaplasia specimens, were obtained after informed consent from the patients undergoing esophagectomy at the University of Michigan Medical Center from 1992 to 1997. A small portion of each tissue specimen was embedded in OCT compound (Miles Scientific, Naperville, IL), whereas the remainder of the tissue samples was immediately frozen in liquid nitrogen. All samples were stored at -70°C. High molecular weight DNA was isolated as described previously (16) . DNA was dissolved in TE buffer and stored at -20°C. Total RNA was isolated by using Trizol reagent (Life Technologies, Inc., Gaithersburg, MD). Agarose gel electrophoresis and the A260:280 nm ratio were used to assess RNA quality. The RNA samples were stored at -70°C until use.

STS-Amplification Mapping Using QG-PCR.
Two BAC clones (393H12 and 538D6), representing two individual NotI-HinfI two-dimensional gel spots found to be amplified in the esophageal adenocarcinomas and mapped by FISH (10) , were used as anchors to localize the telomeric and centromeric sites of the 8p22–23 amplicon. By STS database analyses,⁵ DNA microsatellite (STS) markers in the 8p22–23 amplicon were selected. PCR primers for each selected DNA fragment were carefully designed (slightly modified from the primers chosen using DNASTAR software) to ensure that the melting temperature (Tm) of the STS PCR fragments matched the Tm of the internal control (GAPDH), which was coamplified in each PCR reaction. Genomic DNA from tumor and normal tissues was quantified by fluorometry (TKO100; Hoefer Scientific Instruments, San Francisco, CA). The forward primers of the control and test fragments were end-labeled with [-³²P[ATP (NEN Life Science Products, Boston, MA) using T4 polynucleotide kinase (New England Biolabs, Beverly, MA). PCR was conducted with incorporation of a 50-ng template in 25 µl of total reaction volume using Taq polymerase (Promega Corp., Madison, WI). The template was amplified for 18–22 cycles to maintain linearity of the PCR products. The PCR conditions included an initial denaturing step at 94°C for 2 min and 18–25 cycles each consisted of 95°C for 40 s, 58–63°C (depending on the Tm of individual primers of the tested STS markers) for 40 s, and 70–72°C for 1 min using the PTC100 thermal cycler (M & J Research, Watertown, MA). The PCR products were then resolved on 8% denaturing polyacrylamide gels. Vacuum-dried gels were exposed to PhosphorImager screens (Molecular Dynamics, Sunnyvale, CA). The signal ratios (Ts/c:Ns/c) for both the tumor (Ts/c, tumor STS fragment/tumor GAPDH fragment) and normal DNA samples (Ns/c, normal STS fragment/normal GAPDH fragment) were determined after quantitation using ImageQuant software (Molecular Dynamics). Esophageal tumors showing recurrent increased DNA copy number were further analyzed, and the minimal amplified region was determined.

Positional Candidate Analysis Using Quantitative RT-PCR.
Several databases⁶ were searched to select available ESTs and known genes within the defined minimal amplified region of the 8p22–23 amplicon. Total RNAs from all samples were treated with DNase I (Promega) prior to performing reverse transcription. Two µg of total RNA were reverse transcribed using reverse transcriptase (Life Technologies) and primed by both (dT)₁₈ and random hexamers in a total 40 µl of reaction volume. Two µl of the cDNA were then subjected to RT-PCR with GAPDH coamplified as the internal control. The PCR products were resolved on 8% PAGE gels, and gel data analyses were performed using ImageQuant software as in QG-PCR analysis.

cDNA Library Screening.
Human testis cDNA library (Clontech, Palo Alto, CA) was plated onto 150-mm LB agar plates at a high cell titer. The individual clone DNAs were lifted onto nylon membranes and then hybridized to EST stSG1554, according to the supplied protocol from the manufacturer (NEN Life Science Products, Boston, MA). The hybridization probe used was a 296-bp RT-PCR fragment of stSG1554 labeled with [-³²P]dCTP by direct incorporation. A Sephadex G-50 column (Boehringer Mannheim, Indianapolis, IN) was used to remove unincorporated [-³²P]dCTP. After hybridization and washing, the membranes were subjected to autoradiography using Hyperfilm (Amersham). The positive clones were selected for secondary screening, and the isolated clones were then picked. Mini-preps of isolating clone DNA were conducted by the alkaline method as described by Sambrook et al. (17) .

Northern Blot Analysis.
Ten µg of total RNA from tumor samples, Barrett’s mucosa, and surrounding normal esophagus were size-fractionated in 1.2% formaldehyde/formamide agarose gels and vacuum-transferred to nylon membranes. The same 296-bp RT-PCR fragment of EST stSG1554 used for cDNA library screening was [-³²P]dCTP labeled and hybridized to the RNA blotting membranes following the conditions described previously (18) . Hybridization signals and analysis were obtained after PhosphorImager scanning.

Sequencing of Cloned DNA and RT-PCR Products.
The sequences of the cloned DNA were obtained manually according to the protocol of fmol DNA Cycle Sequencing System (Promega) and through the University of Michigan DNA Sequencing Core using an ABI PRISM Model 377XL DNA Sequencer (PE Applied Biosystems, Foster City, CA). A 0.7-kb RT-PCR fragment was amplified from an esophageal tumor that contained the 8p22–23 amplicon. One primer of the RT-PCR products overlapped the 3' end of the published GATA-4 gene sequence (GATA4-s2, 5'-CTGCTGCCGGCCTTTGCTC-3'), and another primer was located within the cloned DNA sequence (SG554-rf1, 5'-TCCAGCATCAGGGGCAGAAAC-3'). The purified RT-PCR products were sequenced through the University of Michigan DNA Sequencing Core from both the 5' and 3' ends, respectively, primed with the sense primer GATA4-s2 and an antisense primer that overlaps the 5' end of the cloned DNA sequence (SG554–75', 5'-ACAGACAGCAGGTGGGCCAGC-3').

Immunohistochemical and Western Blot Analyses.
Frozen specimens were sectioned at 5 µm, placed on 0.1% poly-L-lysine-coated slides, and fixed in 100% acetone at -20°C for 10 min. Endogenous peroxidase activity was quenched with five changes of 1.2% hydrogen peroxide for 30 min each. Nonspecific binding was blocked using a 1:20 dilution of rabbit serum in PBS-1% BSA. The GATA-4 protein was detected by using the anti-GATA-4 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:500 dilution in PBS-1% BSA. The antibody for GATA-4, although demonstrating cross-reactivity with rat, mouse, and human GATA-4, does not recognize other GATA family members (e.g., GATA-1, GATA-2, GATA-3, GATA-5, and GATA-6). Adult mouse heart, which normally expresses the nuclear GATA-4 gene product, was used as positive control. A section of every tissue was incubated without the primary antibody as a negative control. Immunoreactivity was detected by using the Vectastain avidin/biotin complex kit (Vector Laboratories, Burlingame, CA) with 3,3'-diaminobenzidine as a substrate. The slides were lightly counterstained with Harris-modified hematoxylin and permanently mounted as described previously (18) .

For Western blot analysis, tissue protein was extracted in a buffer containing NP40 as described (18) . Ten µg of total protein extract were fractionated by 10% SDS/PAGE and transferred to nylon membranes. The GATA-4 protein was detected with the same anti-GATA-4 polyclonal antibody used in immunohistochemical study.

	RESULTS

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Determination of the Minimal Critical Region in the 8p22–23 Amplicon by STS-Amplification Mapping.
Sixty-six matched normal-tumor DNA pairs were screened using the QG-PCR assay with 11 STS markers spanning 12 cM within the 8p22–23 amplicon. STS markers, spaced 1 cM apart, were carefully chosen from the highly resolved maps integrated from several STS databases, as described in "Materials and Methods." A housekeeping gene, GAPDH, was incorporated as an internal control and coamplified with the genomic target (STS or genes) fragments in each QG-PCR reaction. The primer labeling and reaction cycles were controlled such that neither the internal control nor the test fragment ever reached amplification saturation or reaction plateau, maintaining product linearity. The PCR products were quantified, and the ratio of (Ts/c):(Ns/c) was computed. Fig. 1 shows the results of the PCR-amplified normal-tumor pairs using four different STS or gene markers by the QG-PCR assay. As shown, some tumors demonstrate genomic amplification at only one marker, whereas some tumors show recurrent increased DNA copy number at all markers (Fig. 1) . The data using the QG-PCR assay in the present study were found to be consistent with previous data obtained by Southern blot analysis of genes in the 8p22–23 amplicon in this series of esophageal adenocarcinomas (10) . Fig. 2 shows the tumor STS-amplification map of the 8p22–23 amplicon based on the QG-PCR data by screening the 66 normal-tumor pairs of genomic DNA with 11 STS markers, one EST, and one known gene inside the amplicon. By analyzing the individual tumor patterns for the recurrent increased DNA copy number using these STS and gene markers, the minimal amplified region was mapped to the area between the markers D8S552 and D8S1759, 5 cM. The minimal critical region was further narrowed and determined to be <2 cM, based on the tumor overexpression patterns of the ESTs selected from the RH physical maps within the defined 5-cM area (Table 1A) . YAC 725-c-12, containing 420 kb of the insert and the STS/EST marker WI-8953, was mapped within the minimal amplified region (Fig. 2) . The sequence of WI-8953 is homologous to CTSB and was confirmed in our previous study (10) . EST stSG1554 was analyzed using PCR and confirmed to be located inside this YAC (data not shown), indicating that the distance between the two genes are within 0.5 cM. The map order is based on the regional amplification patterns of our 66 normal-tumor pairs but closely follows the published genetic, YAC contig, and radiation hybrid maps from the NCBI, WICGR, and SHGC databases.

View larger version (89K): [in this window] [in a new window]   Fig. 1. The QG-PCR assay was applied to map the minimal critical region of the 8p22–23 amplicon in esophageal adenocarcinomas. The housekeeping gene GAPDH was coamplified in the PCR reactions with highly resolved microsatellite (STS) markers that were selected by integrating physical maps and STS databases released recently. As shown, genomic amplification in tumor B95 is observed for marker stSG1554 (D) but is negative for the markers shown in A–C. Amplification of tumor H70, however, is observed at markers D8S1695 (C) and EST stSG1554 (D) but not at markers D8S520 (A) and D8S550 (B). Genomic amplification of esophageal tumors, e.g., tumors L86, B29, R93, D06, B42, and S88, is observed in all four loci as shown. Tumor B05, however, did not demonstrate genomic amplification in any of the markers shown.

View larger version (25K): [in this window] [in a new window]   Fig. 2. Diagrammatic representation of the minimal critical region of the 8p22–23 amplicon in esophageal adenocarcinomas as determined using the data from QG-PCR analysis shown in Fig. 1 as well as the data from quantitative RT-PCR data (Fig. 3 and Table 1 ). •, observed genomic amplification in the individual tumors at the markers tested. , the DNA amplification is not detected at the loci applied. The map order is based on the DNA amplification analysis of 66 normal-tumor pairs but closely follows the published genetic, YAC contigs, and radiation hybrid maps from the NCBI, WICGR, and SHGC databases. As shown in the map, the minimal amplified area is confined to an approximate 5 cM region between markers D8S1759 and D8S552. The minimal amplified region was further limited to a <2-cM region, including the YAC 725-c-12, by analyzing the ESTs selected that spanned 3–5 cR in the GeneMap’99 physical map. This YAC was confirmed to contain CTSB and was hybridized to metaphase chromosomes to determine the chromosomal location using FISH analysis (10) . EST and stSG1554 were mapped to this YAC in the present study, indicating that the distance between these two genes is <0.5 cM.

Nine available ESTs within the minimal amplified region⁷ and four potential cancer-related genes in the 8p22–23 amplicon⁸ were selected for quantitative RT-PCR analysis (Table 1) . Quantitative RT-PCR of normal-tumor pairs was directly chosen as the testing method, rather than testing DNA amplification first, because of the lack of genomic information for these ESTs. Theoretically, all ESTs within the minimal amplified area should show amplification in tumors bearing the amplicon. Moreover, the ultimate goal of the study is to identify overexpressed gene(s) within this amplified genomic DNA segment. All total RNAs were treated with DNase prior to performing reverse transcription to avoid any DNA contamination. Quantitative RT-PCR was performed with the normal-tumor pairs containing genomic amplification as well as with pairs not demonstrating increased DNA copy numbers. Among the nine ESTs and four known genes examined in the present study, EST stSG1554 was the only one demonstrating RNA overexpression in the amplified tumors relative to their corresponding normal tissues. Table 1 lists the expression status of all 12 ESTs within the minimal amplified region and four known genes examined from the present study as well as from our previous study (10) . The four selected known genes are cytogenetically close to the minimal amplified region; however, their precise positions in terms of location within the genetic map are not currently available.

Cloning and Sequencing a Novel Sequence Leading to the Identification of Amplification and Overexpression of the GATA-4 Gene in Esophageal Adenocarcinomas.
The EST, stSG1554, was found to show elevated mRNA expression in all tumors containing the 8p22–23 amplicon using quantitative RT-PCR but not in nonamplified tumors (Figs. 1D and Fig. 3, A and B ). Northern blot hybridization using stSG1554 as the probe confirmed the RT-PCR results (Fig. 4) . Overexpression of this EST was observed in primary and metastatic adenocarcinomas but not in Barrett’s metaplasia (Fig. 4) . BLAST search of this EST (407 bp, gi = 689325) indicated >82% sequence homology with the rat GATA-GT2 gene (gb = L22761) in the 3'UTR region. The human GATA-4 gene is the homologue of rat GATA-GT2 gene and has been mapped previously to chromosome 8p22 (19) . The sequence retrieved for human GATA-4 from GenBank (gb = L34357) is 2226 bp in length. The coding sequence of the human GATA-4 mRNA starts at 241 bp and ends at 1569 bp. To further examine the relationship between the EST stSG1554 and the GATA-4 gene, RT-PCR was applied to the coding region of the GATA-4 gene from 890 to 1188 bp (Fig. 3C) . A similar mRNA expression pattern was observed between EST stSG1554 and human GATA-4 gene in esophageal adenocarcinomas (Fig. 3) . To obtain and fill the sequence gap between the EST stSG1554 and human GATA-4 mRNA, a human testis cDNA library was screened using EST stSG1554 as a probe. Two clones were obtained and yielded a 577-bp-read sequence but still did not reach the sequence at the 3' end of the published human GATA-4 sequence. A 0.7-kb RT-PCR fragment was then obtained from tumor cDNA using two primers. One primer overlapped the 3' end of the published GATA-4 sequence, and the other primer was derived from the clone sequence. An 865-bp sequence of the human GATA-4 gene at 3'UTR region was identified, and this novel sequence is now accessible in the GenBank with the accession number of AF180736.

View larger version (76K): [in this window] [in a new window]   Fig. 3. Representation of quantitative RT-PCR analysis applied to all available ESTs within the minimal critical region of the 8p22–23 amplicon mapped in the GeneMap’99. One EST, stSG1554, was demonstrated to be overexpressed in esophageal adenocarcinomas. Analyses of this EST, the sequences of clone DNAs obtained by cDNA library screening, and the database search indicate that stSG1554 and its extended sequence are part of the 3'UTR of the human GATA-4 gene, a zinc finger transcription factor. A, stSG1554 is overexpressed in tumors (T) B95, L86, R93, and T67 containing the 8p22–23 amplicon but is very faint or not detected in the surrounding normal (N) tissues. Tumor M47 (A–C) demonstrated mRNA overexpression of stSG1554; however, its genomic amplification status is unknown because of the unavailability of tumor DNA. GAPDH was coamplified as the internal control. B, RT-PCR reaction of stSG1554 without the internal control was conducted using the same cDNA sources shown in A to confirm the results in the absence of any interference from the primers of GAPDH for PCR amplification. C, RT-PCR of the coding region of GATA-4 results in a similar expression pattern as the EST stSG1554 in the tumors. N, normal; T, tumor.

View larger version (34K): [in this window] [in a new window]   Fig. 4. Northern blot analysis of normal esophagus, Barrett’s metaplasia, and esophageal adenocarcinomas using stSG1554 as a probe. The results are consistent with the RT-PCR analyses shown in Fig. 3 and also indicate overexpression of GATA-4 in a metastatic lymph node (L86-GLN) from the tumor containing the 8p22–23 amplicon. Both Northern blot and RT-PCR analyses provide evidence that GATA-4 shows a very low abundance in normal human esophagus.

View larger version (89K): [in this window] [in a new window]   Fig. 5. Immunohistochemical analysis of GATA-4 protein expression observed in human esophageal adenocarcinomas and control issues. The antibody for GATA-4 cross-reacts with rat, mouse, and human GATA-4 but does not recognize other GATA family members, e.g., GATA-1, GATA-2, GATA-3, GATA-5, and GATA-6. A, an adult mouse heart, normally expressing the nuclear GATA-4 gene product, was used as a positive control. Arrows, positive nuclear staining for the GATA-4 protein. B, GATA-4 protein is found to be highly expressed in the nuclei of an esophageal adenocarcinoma (arrows), L86, which demonstrates gene amplification of GATA-4. C, the nuclei of Barrett’s metaplasia shows very little, if any, GATA-4 protein expression (arrows).

View larger version (60K): [in this window] [in a new window]   Fig. 6. Analysis of the amplification of a recently published novel gene, MASL1, in esophageal adenocarcinomas. MASL1 amplification was assessed using QG-PCR and its expression by RT-PCR. The results suggest that MASL1 is unlikely to be a candidate gene for the 8p22–23 amplicon of esophageal adenocarcinoma. A, a series of QG-PCR results in different normal-tumor pairs, including those found amplified in the CTSB-GATA-4 minimal region of the 8p22–23 amplicon. Densitometric analysis using ImageQuant software demonstrates that D06 is the only tumor showing MASL1 genomic amplification among the esophageal adenocarcinomas containing the 8p22–23 amplicon. B, RT-PCR results examined the expression of MASL1 in esophageal adenocarcinomas. As shown, the expression of MASL1 is even higher in normal tissues of patients L86, R61, and W12 as well as in the Barrett’s mRNA of patient R16. C, a schematic drawing of genomic amplification status of MASL1 in the esophageal adenocarcinomas at 8p22–23. The amplification of MASL1 was detected in only one tumor, D06, that has the largest amplicon in this series of 66 normal-tumor pairs but not in any other esophageal tumors known containing the same 8p22–23 amplicon.

	DISCUSSION

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Because many known genes, ESTs, and genomic sequences are now accessible using extensive databases, the positional candidate approach used in the present study is an effective way to identify new genes of interest (27 , 28) . Twelve ESTs and four known genes were analyzed as potential candidate genes within the minimal amplified region in the 8p22–23 amplicon. The location of the ESTs were determined using GeneMap’99.⁹ Known genes were selected on their potential relationship to tumor development and progression. All four selected known genes did not demonstrate mRNA overexpression in this series of esophageal adenocarcinomas, suggesting that these genes are unlikely to be critical candidate genes for the 8p22–23 amplicon.

8p22–23 amplification was identified recently as a novel amplicon in esophageal adenocarcinomas using RLGS 2D-gel technique (10) . Genomic amplification of 8p21–22 was also observed in gastric adenocarcinomas by CGH technology (29) . Gene amplification of erbB2 was observed in 21–22% of esophageal adenocarcinomas, EGFR in 13%, and K-ras in 10% (9) .³ Comparable with the frequencies of amplification for these oncogenes, genomic amplification at 8p22–23 is detected in 13.6% in esophageal adenocarcinomas and 12.5% in gastric cardia adenocarcinomas in the present study. These results suggest a nonrandom event favoring tumorigenesis or tumor progression of the cancer types examined and indicate that an oncogenic gene(s) may reside in this region. Interestingly, genomic amplification of 8p22–23 was not observed in lung adenocarcinomas or in esophageal squamous carcinomas examined in the present study, suggesting that the oncogenic event of the 8p22–23 amplicon may be tumor type specific, favoring the development or progression of esophageal and gastric cardia adenocarcinomas.

EST stSG1554 was the only one among 12 ESTs examined that showed elevated mRNA expression in the tumors with 8p22–23 amplification. Further analysis of this EST led to the identification of novel sequences and the determination of these sequences as part of the human GATA-4 gene. The GATA-4 gene has been mapped previously to 8p22 (19) . GATA genes are a family of zinc finger transcription factors, recognizing a consensus WGATAR motif of target genes through a conserved multifunctional DNA-binding domain (30) . Six GATA genes have been identified, among which GATA-1, GATA-2, and GATA-3 function in the hematopoietic cell lineages (31) and GATA-4, GATA-5, and GATA-6 regulate gene expression in developing tissues, including heart and gut (32) . Several target genes in the cardiac system regulated by GATA-4 have been studied, including the brain natriuretic peptide gene (33) , the -myosin heavy-chain gene (34) , and a recently identified gene, FOG2 (35 , 36) . Rat GATA-GT2, the homologue of human GATA-4, was identified in the gastric epithelium and found to bind to the upstream sequence of the H⁺/K⁺-ATPase ß gene containing the GATA motif (37) . The intestinal fatty acid-binding protein was also proposed as the downstream target of GATA-4 (38) . Murakami et al., (39) recently reported that FGF3 oncogene may be a target of the GATA-4 transcription factor in the undifferentiated tumor cells. The function and downstream target gene(s) for GATA-4 in adult tissues, however, are unclear and will require further study.

Our primary characterization of the 8p22–23 amplicon revealed that CTSB was both amplified and overexpressed in esophageal adenocarcinomas (10) . Overexpression of CTSB has been observed in many other tumor types, including lung, colon, breast, prostate, and gastric (12, 13, 14) . The data from the present study demonstrate that amplification of the GATA-4 gene results in GATA-4 overexpression in the same esophageal tumors that have also been found to have CTSB amplification. STS-amplification mapping and YAC 725-c-12 analyses indicate that CTSB and GATA-4 are located within the minimal amplified region and closely linked to each other (<0.5 Mb). Overexpression of GATA-4 was only observed in the tumors containing the 8p22–23 amplicon. Abundant nuclear staining was also observed in small portions of two additional tumors by immunohistochemical analysis; however, gene amplification analysis of these two tumors was not possible because of lack of tumor DNA. Gene amplification of CTSB was observed in 13% of the tumors examined, but overexpression of the mRNA was seen in 25% of the tumors, including those without gene amplification (10) . Increased CTSB protein staining was detected even in 75% of the tumors examined, suggesting that overexpression of CTSB could result from other mechanisms in addition to gene amplification. It is possible that amplifications of GATA-4 and CTSB are attributable to their cosegregation with an additional cancer-related gene located within the minimal critical region of the 8p22–23 amplicon. Further functional characterization is required to clarify the roles of CTSB, GATA-4, and other unknown genes within the minimal critical region in the development or progression of esophageal and gastric cardia adenocarcinomas.

	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 NCI Grant CA71606.

² To whom requests for reprints should be addressed, at Department of Surgery, Section of General Thoracic Surgery, MSRB II B560, Box 0686, University of Michigan Medical School, Ann Arbor, MI 48109. Phone: (734) 763-0325: Fax: (734) 763-0323; E-mail: dgbeer{at}umich.edu

³ D. G. Beer et al., unpublished data.

⁴ The abbreviations used are: CGH, comparative genomic hybridization; CTSB, cathepsin B; STS, sequence tagged site; QG-PCR, quantitative genomic-PCR; YAC, yeast artificial chromosome; EST, expressed sequence tag; FISH, fluorescence in situ hybridization; RLGS, restriction landmark genomic scanning; Ts/c and Ns/c, the intensity ratio of tumor (Ts/c) or normal (Ns/c) sample versus GAPDH control from QG-PCR; NCBI, National Center of Biotechnology Information; WICGR, Whitehead Institute Center for Genome Research; SHGC, Stanford Human Genome Center; UTR, untranslated region.

⁵ Internet addresses: NCBI, http://www.ncbi.nlm.nih.gov; WICGR, http://www.genome. wi.mit.edu; SHGC, http://www.shgc.stanford.edu.

⁶ NCBI, WICGR, and SHGC, and the Human Genome Research Center (http://www.genethon.fr).

⁷ NCBI, http://www.ncbi.nlm.nih.gov/genome/guide.

⁸ NCBI, http://www.ncbi.nlm.nih.gov/Omim/.

⁹ Internet address: http://www.ncbi. nlm.nih.gov/genemap.

Received 8/27/99. Accepted 1/ 5/00.

	REFERENCES

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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