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Departments of Surgery, Section of General Thoracic Surgery [L. L., M. S. P., Z. Z., P. S., S. Y. C., M. B. O., D. G. B.], Human Genetics [T. W. G.], and Pediatrics [T. W. G., R. D. K., S. M. H.], University of Michigan Medical School, Ann Arbor, Michigan 48109
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The genetic alterations underlying development and/or progression of esophageal adenocarcinoma remain poorly understood. Gene amplification and overexpression of the oncogenes erbB2, EGFR, and K-ras have been reported in this type of tumor3 (5 , 6) . A recent study using CGH4 by van Dekken et al. (7) reported amplification at 8q23–24.1, 15q25, 17q12–21, and 19q13.1 in a series of 28 Barrett’s adenocarcinomas. The candidate genes for these amplicons were suggested as myc for 8q23–24.1; IGF1R for 15q25; erbB2 for 17q12–21; and TGFß1, BCL3, and AKT2 for 19q13.1. Another CGH study, analyzing 58 primary gastric cancers, also demonstrated genomic amplification at multiple chromosomal locations including 6p21, 7q31, 8p22–23, 8q23–24, 11q13, 12p12–13, 17q21, 19q12–13, and 20q13 (8) . We have recently identified and characterized the 8p22–23 amplicon that was detected in 12.1% of 66 esophageal adenocarcinomas (9 , 10) . Two genes, the lysosomal protease cathepsin B and the zinc finger transcription factor GATA-4, were localized within the minimal critical region and are likely candidate genes for the 8p22–23 amplicon in esophageal adenocarcinomas (9 , 10) .
In the present study, a potentially amplified NotI/DpnII DNA fragment was identified and cloned from an esophageal adenocarcinoma using the two-dimensional gel RLGS technique. The amplification was confirmed and then localized to 19q12. STS-amplification mapping was applied to characterize the amplicon using the QG-PCR assay with 11 STS markers neighboring the location of the two-dimensional fragment and spanning >8 cM in 19q12. Genomic DNA from 65 normal-esophageal adenocarcinoma pairs and 11 normal-gastric cardia carcinoma pairs were analyzed. The core amplified region was determined to be localized between D19S919 and D19S882, a region of <300 kb that includes cyclin E. Cyclin E is a G1 cyclin that regulates cell entry into the S phase (11 , 12) . Amplification and overexpression of cyclin E have been observed in ovarian and gastric carcinomas (13, 14, 15, 16) . These results strongly support the theory that cyclin E is the likely target gene selected by the 19q12 amplicon.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Two-Dimensional Gel Electrophoresis for RLGS.
High- molecular-weight DNA from normal and tumor samples were subjected
to two-dimensional gel electrophoresis as described previously
(18)
. In brief, DNA samples were digested using the
restriction enzyme NotI (New England BioLabs Inc., Beverly,
MA) followed by labeling NotI ends with
[
-32P]dCTP and
[-32P]dGTP (NEN Life Science Products,
Boston, MA). A second enzyme, EcoRV, was used to digest the
DNA fragments prior to electrophoresis in a 0.9% disc-agarose gel
(one-dimensional). After first-dimensional separation,
additional digestion was carried out within the gel using
HinfI enzyme (or DpnII for cloning purposes). The
disc-agarose gel was then placed on the top of 5.25% polyacrylamide
gel and subjected to electrophoresis. The resulting gels were dried and
autoradiographed using PhosphorImage screens (Molecular Dynamics,
Sunnyvale, CA). Computer analysis of potential amplification events was
performed using previously developed software (18)
. For
cloning, preparative gels were run, and the selected amplified DNA
fragments were cut out for subsequent cloning as described previously
(9)
.
Isolation and Cloning of the Two-Dimensional DNA Fragments.
The gel pieces containing the amplified DNA fragments were
isolated, and the gel was electrophoresed onto a DEAE membrane
(Schleicher & Schuell, Keene, NH). The DNA was then eluted out and
purified as described previously (9)
and was dissolved in
7 µl of distilled water. A pBC vector (Stratagene, La Jolla, CA)
was cleaved with the enzymes NotI and
BamHI (DpnII compatible end), and was
gel-purified using SeaKem GTG agarose (FMC BioProducts, Rockland, ME).
After the incubation of 7 µl of two-dimensional DNA, 1 µl of vector
(1 ng/µl), and 1 µl of 10x ligation buffer (Boehringer
Mannheim, Indianapolis, IN) at room temperature for 10 min and at
65°C for 7 min, 1 µl T4 ligase (5 u/µl; Boehringer Mannheim,
Indianapolis, IN) was added, and the ligation reaction was carried at
16°C for 25 h. The ligation mixture was purified using a
standard phenol-chloroform extraction protocol (19)
. The
XL1 Blue competent cells were then transformed with the purified vector
two-dimensional DNA via electroporation using E Coli Pulser
(Bio-Rad Laboratories, Hercules, CA). Individual colonies were used for
DNA isolation (mini-preps) via the alkaline lysis method as described
by Sambrook et al. (19)
.
E-PCR.
The cloned fragment was sequenced by University of Michigan Sequencing
Core facility. The sequences were analyzed by BLAST for
similarity matches in the databases. Resulting sequences can be then
used as templates to perform the E-PCR (on the NCBI web site) analysis.
Chromosomal location of the template was determined based on matches to
a previously analyzed group of sequences in the databases.
Southern Blot Analysis.
DNA from three normal-tumor tissue pairs, including the tumor from
which the two-dimensional fragment was isolated, was cleaved by
EcoRI and size-fractionated following the standard protocol
for Southern blot analysis (19)
. The cloned DNA sequence
was used as the probe and hybridized to the membrane containing the
three pairs of DNA samples, using the hybridization and washing
conditions provided by the manufacturer (NEN Life Science Products).
STS-Amplification Mapping Using QG-PCR.
The amplified fragment was used as the anchor to select neighboring STS
markers by searching STS databases as described previously
(10)
. Briefly, PCR primers for each STS fragment
were designed 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 to have equity of starting materials. The forward primers of
the control and test fragments were end-labeled with
[32P-
]ATP (NEN Life Science Products) using
T4 polynucleotide kinase (New England BioLabs). PCR was conducted with
a 50-ng template in 25 µl of total reaction volume using Taqpolymerase (Promega, Madison, WI). The PCR products were then
resolved on 8% denaturing polyacrylamide gels. 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 using
ImageQuant software (Molecular Dynamics).
Positional Candidate Analysis Using Quantitative RT-PCR.
Several databases (NCBI,5
WICGR,6
SHGC7
) were searched to
select available ESTs or known genes within the defined minimal
critical region of the 19q12 amplicon. All of the total RNA samples
used 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.
Immunohistochemical Analysis.
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
three 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 cyclin E protein was detected using the anti-cyclin E antibody
(Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:500 dilution in
PBS-1% BSA. A section of each tissue was also incubated without the
primary antibody as a negative control. Immunoreactivity was detected
using the Vectastain avidin/biotin complex kit (Vector Laboratories,
Burlingame, CA) with 3,3'-diaminobenzadine as a substrate. The slides
were lightly counterstained with Harris-modified hematoxylin and
permanently mounted as described previously (20)
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-32P]dCTP and
[-32P]dGTP and served as the landmarks to
scan the whole genome of tumor samples and associated normal tissues.
Approximately 3,000 restriction fragments were resolved in each
two-dimensional gel (21)
. Comparison of normal and tumor
two-dimensional patterns revealed a NotI/HinfI
fragment that was 3100 bp in size in the first-dimension separation and
400 bp in the second-dimension, which shows higher intensity in
two tumors (F12 and B81) relative to normal tissue controls (Fig. 1, A and B)
.
Two-dimensional gels were digested with
NotI/DpnII restriction enzymes for cloning
purposes (Fig. 1C)
. Most of the very intensive spots visible
in the two-dimensional gels represent ribosomal DNA, which are present
in multiple copies. Changes in the patterns of both of these
two-dimensional fragments and other CpG island-containing fragments,
attributable to alterations of DNA methylation, are often observed
during tumor development (22
, 23)
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-32P]dCTP as the probe and was hybridized
to a membrane containing the paired normal-tumor DNAs. As shown in Fig. 2
, increased DNA copy number of the
two-dimensional fragment was detected in tumor F12 but not in another
two pairs of normal-tumor DNA, F93 and W11.
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shows the outline of the six STS markers as well as the amplified
two-dimensional sequence that localized within this BAC clone. This
allowed not only the chromosomal location of the amplicon in question
to be confirmed but the neighboring physical map to be determined as
well. This analysis also revealed that the amplified two-dimensional
NotI/DpnII fragment was actually a part of the 5'
end of the UQCRFS1 gene and was 27 kb away from the STS
marker D19S409.
|
. The 19q12
amplicon spans more than 8 cM in tumor genome and occurred in 13.8% (9
of 65) of esophageal and in 9.1% (1 of 11) of gastric cardia
adenocarcinomas. After quantification, a 6.5-fold increase was observed
in tumor F12 using Southern hybridization analysis (Fig. 2)
, and an
8.6-fold increase was present in tumor B81 using the QG-PCR assay (Fig. 3)
. The minimal critical region was mapped between the markers D19S919
and D19S882, telomeric to the UQCRFS1 gene and was 
0.8 cM
in size based on the physical maps from WIGRC and SHGC (Fig. 4)
. Cyclin E, a
G1 cyclin that regulates cell entry into the S
phase, is located within this region.
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. Quantitative RT-PCR was applied to 7
(of 10 amplified) tumors containing the 19q12 amplicon and 5 tumors
without 19q12 amplification. EST 9 (sts-N21279) showed elevated
expression in tumors P95, B81, F12, and L61, but not in tumors M60 and
S12, relative to their corresponding normal tissue RNA. Tumors P95 and
B81 did not show increased expression at EST 11 (sts-Z41049); however,
tumors M60 and S12 demonstrated a 6- to 20-fold increased expression of
this EST as compared with their normal tissue mRNA (Fig. 5)
. Tumor P15
was not observed to be amplified and was used as a negative control for
the expression assay (Fig. 5)
. The expression levels between normal and
tumor cDNA from patient P15 are thus similar at ESTs 9 and 11. The mRNA
from one Barrett’s tissue, P95, showed overexpression of EST 9 and
cyclin E, but not EST 11 (Fig. 5)
. QG-PCR for assessment of DNA
amplification was then applied to all of the 76 esophageal and gastric
cardia adenocarcinomas using those EST fragments which showed
overexpression in the tumors to determine the frequency of the genomic
amplification of these ESTs. Seven ESTs of the 14 selected within the
minimal region were observed to be both amplified and overexpressed in
the tumors (Fig. 5
; Table 2
). A high
resolution map representing the characteristics of tumor RNA
overexpression and DNA amplification within the minimal critical region
was established, and the core amplified domain of the 19q12 amplicon
was localized between ESTs sts-W74757 (EST 9, 179.90 cR in GB4
RH GeneMap’99) and sts-R63092 (EST 10, 180.67 cR; Fig. 4
).
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100–300 kb
based on the genetic and RH maps, which is in agreement with the
analysis of sequenced contigs of band 19q12 (from the database of
LLNL8
). A BAC clone,
AC008798, containing >137 kb of sequenced DNA (updated as of February
23, 2000), includes the cyclin E sequence but not any of the
other neighboring ESTs analyzed (Table 3)
. The sequences of ESTs 10, 11, 12, and
14 were matched to the clone AC008507, containing >213 kb of 11
ordered pieces of sequences and adjacent telomerically to AC008798.
ESTs 3, 7, 8, and 9 were found to be contained in AC010513, which is
centromeric to AC008798. ESTs 4 and 5 were contained in AC010505 (Table 3)
, centromeric to AC010513. AC007786 which contains the amplified
two-dimensional NotI/DpnII fragment is 2 cM
centromeric to AC010505 (Table 3)
. Dissection or physical mapping of
the minimal critical region with these ESTs in addition to the genomic
amplification mapping data clearly indicates that cyclin Eis located between ESTs 7, 8, 9 and ESTs 10, 11 (Fig. 4
; Table 3
),
rather than between EST 5 and EST 7 as shown in GeneMap’99.
Moreover, an unknown EST, sts-X95406, which is mapped between EST 9 and
EST 10 in GeneMap’99, shows homology to the cyclin E gene.
Furthermore, among these seven amplified and overexpressed ESTs, ESTs
4, 5, and 7 were found to be amplified in 7 of 10 tumors containing the
19q12 amplicon. ESTs 9, 10, and 11 were found to be amplified in 8 of
10 tumors containing the 19q12 amplification. None of the 13 ESTs
(excluding EST 6, which represents cyclin E) were seen to
have increased gene copy number without cyclin Eamplification. Therefore, cyclin E demonstrates the
highest frequency of both gene amplification and mRNA overexpression
(Fig. 4)
.
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, but
not in any tumors without the 19q12 amplification. The increased
staining was also observed in a region of Barrett’s mucosa from
patient P95 that represents either high-grade dysplasia or early
adenocarcinoma in situ (Fig. 6B)
. This was
consistent with the overexpression of cyclin E mRNA observed
in the Barrett’s mucosa from the same patient (Fig. 5)
. To determine
whether increased DNA dosage might underlie the overexpression of
cyclin E in these dysplastic Barrett’s cells, the region of
high cyclin E nuclear staining from the Barrett’s mucosa
was microdissected, and the DNA was isolated. Cyclin Eamplification is observed in the microdissected DNA from this
dysplastic Barrett’s mucosa (Fig. 6E)
.
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Amplification of BCL3,
TGFß1, and the STS Markers Closely
Linked to AKT2 Was Not Detected in This Series of 76
Esophageal and Gastric Adenocarcinomas.
19q13.1 was reported to be amplified in 28 esophageal
adenocarcinomas by a previous study using a CGH approach
(7)
. AKT2, BCL3, and
TGFß1 were proposed as the potential candidate
genes for the 19q13.1 amplicon. To determine whether the 19q13.1
amplicon is an extension from 19q12 or whether two individual amplicons
exist at chromosome 19q, the QG-PCR assay was used to analyze the genes
BCL3 and TGFß1, and two STS
fragments (D19S421 and D19S420) that are closely linked to
AKT2, as well as four other STS DNA markers spanning the
19q13.1 band. Genomic amplification was not detected in these two genes
nor in the STS markers closely linked to AKT2. Because none
of the markers and genes selected from the 19q13.1 region were found to
be amplified (data not shown), this indicates that the 19q12 amplicon
is not an extension of the previously reported 19q13.1 amplicon and is
the only amplicon in the 19q centromeric region in this series of 76
esophageal and gastric cardia adenocarcinomas.
The 19q12 Amplicon Was Not Observed in Esophageal Squamous Cell
Carcinomas nor in Lung Adenocarcinomas.
Cyclin E amplicon was observed in 13.8% of 65 paired
normal-tumors of esophageal adenocarcinoma and in 9.1% of 11 gastric
cardia adenocarcinomas. Using the QG-PCR assay for analyzing genomic
amplification at 19q12, increased DNA copy number, however, was
detected in neither 28 paired normal-esophageal squamous cell
carcinomas nor in 39 paired normal-lung adenocarcinomas. The results
suggest a potential tissue specificity of the 19q12 amplicon in the
development of esophageal adenocarcinoma.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Genomic amplification is an event that may selectively increase the dosage of a gene. Coamplification of neighboring genes may also occur because of the fact that amplification can involve a large chromosomal segment (reviewed in Ref. 24 ). Although programmed gene amplification is observed as a part of a normal developmental progress in lower organisms (25 , 26) , unscheduled gene amplification in mammals is predominantly observed after exposure to cytotoxic drugs (27) and during tumorigenesis (28 , 29) . In tumors, genes considered to be the target of the amplification are frequently dominant-acting oncogenes. Identification of amplicons in a tumor genome, therefore, provides a means to discover dominant-acting oncogenes or tumor-related genes. The two-dimensional RLGS protocol is an effective method to achieve this goal as shown in this and other studies (9 , 10 , 18) . The STS-amplification mapping approach that we have implemented efficiently reduces the size of the minimal critical region, thus narrowing the choice of potential candidate genes (10) .
Cyclin E is a regulatory subunit of the cdk2-E complex, which acts during later G1 phase into the S phase to ensure cells entry into the S phase (11 , 12) . Amplification of the cyclin E gene underlies the observed up-regulated cyclin E mRNA and protein levels in the tumors containing the cyclin E amplicon as shown in the present study. Interestingly, the presence of cyclin E is required for amplification of another gene, chorion, during the Drosophila oogenesis (30) . Cyclin E-cdk2 activity is necessary for the growth anchorage-dependence of nontransformed cells (31) , and the cytoplasmic displacement of the cyclin E-cdk2 complex, along with the cdk inhibitors, is believed to contribute to the growth anchorage-independence of transformed cells (32) . Anchorage-independence of transformed cells is thought to contribute to tumor invasiveness and metastasis (33) . Deregulated cyclin E may also be a factor contributing to genomic instability in tumor genomes (34 , 35) . Cyclin Ecan function like cyclin D1 in the setting of cyclin D1 deficiency (36) , and deregulated cyclin D1 was proposed to act like an oncogene and is found to be amplified and overexpressed in many human tumors (37 , 38 and reviewed in Ref. 39 ). Cyclin Eamplification is observed in ovarian (12.5–21%), colon (9.4%), and gastric (15.6%) carcinomas (13, 14, 15, 16) . Expression of the cyclin E protein was reported in dysplastic Barrett’s mucosa as well as in adenocarcinomas, and increased nuclear staining was associated with regeneration and proliferation of adjacent metaplastic epithelium of erosions or ulcerations in Barrett’s (40) . Overexpression of the cyclin E mRNA and protein was also observed in chemical-induced rat esophageal tumors (41) . Taken together, the previous existing evidence and our present results indicate that cyclin E is the best candidate selected by the 19q12 amplification event.
Genomic amplification in tumors does not usually contain only the core domain but may extend several hundred kilobases to several megabases flanking the selected gene(s) (42 , 43) . One potential hypothesis is that the additional coamplified and cooverexpressed gene(s) could be a factor that influences the tumor phenotype and/or clinical outcome. This may explain why patients with the same amplicon in their tumors demonstrate a different clinical prognosis (44) . In the present study, at least six other ESTs were shown to be coamplified along with cyclin E. Whether the genes coamplified in a given amplicon are just physically linked by sharing the same origin, or whether there is any functional correlation between the coamplified genes in the same amplicon is unclear and will require further study.
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FOOTNOTES |
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1 Supported by National Cancer Institute Grant
CA71606 and by the Roy Weber Endowment. 
2 To whom requests for reprints should be
addressed, at MSRB II B560, Box 0686, Department of Surgery, Section of
General Thoracic Surgery, University of Michigan Medical School, Ann
Arbor, MI 48109. Phone: (734) 763-0325;
Fax: (734) 763-0323; E-mail:
dgbeer@umich.edu.
3 D. G. Beer et al.,
unpublished data.
4 The abbreviations used are: CGH,
comparative genomic hybridization; RLGS, restriction landmark genomic
scanning; STS, sequence tagged site; EST, expressed sequence tag;
QG-PCR, quantitative genomic-PCR; E-PCR, electronic PCR; RT-PCR,
reverse transcription-PCR; Ts/c and Ns/c, the intensity ratio of tumor
(Ts/c) or normal (Ns/c) sample versus GAPDH control from
QG-PCR; NCBI, the National Center for Biotechnology Information; WICGR,
Whitehead Institute Center for Genome Research; SHGC, Stanford Human
Genome Center; LLNL, Lawrence Livermore National Laboratory; RH,
radiation hybrid; BLAST, Basic Local Alignment Search Tool; BAC,
bacterial artificial chromosome; CR, centi-ray.
5 Internet address for NCBI:
http://www.ncbi.nlm.nih.gov.
6 Internet address for WICGR:
http://www-genome.wi.mit.edu.
7 Internet address for SHGC:
http:www-shgc.stanford.edu.
8 Internet address for LLNL:
http://www-bio.llnl.gov.
Received 4/ 4/00. Accepted 10/19/00.
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