[雙語翻譯]--外文翻譯--對食管癌19q12擴增子的鑒定和描述顯示cyclin e是這個擴增子的最佳候選基因（原文）

1、[CANCER RESEARCH 60, 7021–7027, December 15, 2000]Identification and Characterization of a 19q12 Amplicon in EsophagealAdenocarcinomas Reveals Cyclin E as the Best Candidate Gene forthis Amplicon1Lin Lin, Michael S. Pres

2、cott, Zhouqin Zhu, Puja Singh, Sang Y. Chun, Rork D. Kuick, Samir M. Hanash, Mark B. Orringer, Thomas W. Glover, and David G. Beer2Departments of Surgery, Section of General Thoracic Surgery [L. L., M. S. P., Z. Z., P. S

3、., 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 48109ABSTRACTGenomic DNA amplification in tumors is f

4、requently associated with an increased gene copy number of oncogenes or other cancer-related genes. We have used a two-dimensional whole-genome scanning technique to identify gene amplification events in esophageal adeno

5、carcinomas. A multicopy genomic fragment from a tumor two-dimensional gel was cloned, and genomic amplification encompassing this fragment was confirmed by South- ern blot analysis. The corresponding DNA sequence was mat

6、ched by BLAST to a BAC contig, which allowed the use of electronic-PCR to localize this amplicon to 19q12. Sequence tagged site-amplification mapping, an approach recently implemented in our laboratory (Lin, L. et al., C

7、ancer Res., 60: 1341–1347, 2000), was used to characterize the amplicon. Genomic DNA from 65 esophageal and 11 gastric cardia adenocarcinomas were investigated for 19q12 amplification using quantitative PCR at 11 sequenc

8、e tagged site mark- ers neighboring the cloned fragment. The amplicon was narrowed from >8 cM to a minimal critical region spanning <0.8 cM, between D19S919 and D19S882. This region includes the cyclin E gene. Four

9、teen expressed sequence tags (ESTs) covering the minimal region were then assayed for potential gene overexpression using quantitative reverse transcription-PCR. Seven of the selected ESTs were found to be both amplified

10、 and overexpressed. Among these seven ESTs, cyclin E showed the highest frequency of gene amplification and overexpression in the tumors examined, which allowed us to finalize the core-amplified region to <300 kb. The

11、se results indicate that cyclin E is the likely target gene selected by the amplification event at 19q12. The fact that cyclin E overexpression was found only in the amplified tumors examined indicates that gene amplific

12、ation underlies the cyclin E gene overexpression. Our study represents the first extensive analysis of the 19q12 amplicon, and is the first to physically map the core-amplified domain to a region of <300 kb that inclu

13、des cyclin E. Amplification of 19q12 was found neither in the 28 esophageal squamous cancers nor in the 39 lung adenocarcinomas examined but was observed in 13.8% of esophageal and 9.1% of gastric cardia adeno- carcinoma

14、s.INTRODUCTIONA striking increase in the incidence of esophageal adenocarcinoma has been observed in the United States over the past 2 decades (1), yet the 5-year survival rate remains unchanged and ?10% (2). Chronic gas

15、troesophageal reflux is the major risk factor for esophageal ade- nocarcinoma, which can result in the replacement of the normal squamous mucosa with a metaplastic, intestinal-like columnar epithe- lia, designated Barret

16、t’s esophagus (3). Esophageal adenocarcinomas are reported to occur at a higher rate among patients with preexisting Barrett’s esophagus as compared with those without, and the malig- nancy is often associated with Barre

17、tt’s epithelia (4).The genetic alterations underlying development and/or progression of esophageal adenocarcinoma remain poorly understood. Gene am- plification and overexpression of the oncogenes erbB2, EGFR, and K-ras

18、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 cand

19、idate genes for these amplicons were suggested as myc for 8q23–24.1; IGF1R for 15q25; erbB2 for 17q12– 21; and TGF?1, BCL3, and AKT2 for 19q13.1. Another CGH study, analyzing 58 primary gastric cancers, also demonstrated

20、 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 wa

21、s detected in 12.1% of 66 esophageal adenocarcino- mas (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 lik

22、ely 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 adenocarci- noma usin

23、g the two-dimensional gel RLGS technique. The amplifi- cation 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 neigh

24、boring 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

25、 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 overexpressio

26、n of cyclin E have been observed in ovarian and gastric carcinomas (13–16). These results strongly support the theory that cyclin E is the likely target gene selected by the 19q12 amplicon.MATERIALS AND METHODSTumor Tiss

27、ue Collection and DNA/RNA Isolations. Sixty-nine esopha- geal adenocarcinomas and the corresponding normal esophageal or gastric mucosa, 20 Barrett’s metaplasia specimens, as well as the esophageal squa- mous cell carcin

28、omas and lung adenocarcinomas were obtained after informed consent from patients undergoing esophagectomy and pulmonary resection for cancer at the University of Michigan Medical Center from 1992 to 1999. Patients receiv

29、ing treatment with chemotherapy and/or radiotherapy prior to surgery were excluded from the present study. A small portion of each tissueReceived 4/4/00; accepted 10/19/00. The costs of publication of this article were d

30、efrayed 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 National Cancer Institute

31、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 Sch

32、ool, 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, re- strict

33、ion landmark genomic scanning; STS, sequence tagged site; EST, expressed se- quence tag; QG-PCR, quantitative genomic-PCR; E-PCR, electronic PCR; RT-PCR, reverse transcription-PCR; Ts/c and Ns/c, the intensity ratio of t

34、umor (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; LL

35、NL, Lawrence Livermore National Labora- tory; RH, radiation hybrid; BLAST, Basic Local Alignment Search Tool; BAC, bacterial artificial chromosome; CR, centi-ray.7021?20°C for 10 min. Endogenous peroxidase activity

36、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 an

37、tibody (Santa Cruz Biotechnol- ogy, 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 t

38、he Vectastain avidin/biotin complex kit (Vector Laboratories, Burlingame, CA) with 3,3?-diaminobenzadine as a sub- strate. The slides were lightly counterstained with Harris-modified hematox- ylin and permanently mounted

39、 as described previously (20).RESULTSTwo-Dimensional Gel Analysis of Esophageal Adenocar- cinomas. DNA samples from 44 primary esophageal adenocarcinomas were analyzed using the two-dimensional gel RLGS technique. Cleava

40、ge sites of the rare cutting restriction enzyme NotI were labeled with [?-32P]dCTP and [?-32P]dGTP and served as the landmarks to scan the whole genome of tumor samples and associated normal tissues. Approx- imately 3,00

41、0 restriction fragments were resolved in each two-dimen- sional gel (21). Comparison of normal and tumor two-dimensional pat- terns revealed a NotI/HinfI fragment that was 3100 bp in size in the first-dimension separatio

42、n 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

43、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

44、 fragments and other CpG island-containing fragments, attributable to alterations of DNA methyla- tion, are often observed during tumor development (22, 23). Southern Blot Analysis for Genomic Amplification. High- molecu

45、lar-weight DNA from three normal-tumor pairs of esophageal adenocarcinomas, including patient F12, were digested by restriction enzyme EcoRI prior to Southern blot analysis. The cloned two- dimensional fragment was label

46、ed with [?-32P]dCTP as the probe andwas 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

47、 another two pairs of normal-tumor DNA, F93 and W11. Chromosomal Localization of Amplified Two-Dimensional Fragment. The amplified fragment was sequenced, and a BLAST search revealed that the 396-bp sequence was matched

48、within a BAC clone, AC007786. AC007786 consists of more than 229 kb of completed sequences and was mapped to 19q12 between the STS marker D19S222 and the gene UQCRFS1 (ubiquinol-cytochrome C reductase iron-sulfur subunit

49、), a chromosomal interval of 2–3 cM centromeric to the UQCRFS1 gene. AC007786 was then used as the template and submitted for an E-PCR analysis. Table 1 shows the outline of the six STS markers as well as the amplified t

50、wo-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 ana

51、lysis 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. Characterization of the 19q12 Amplicon and

52、Narrowing of the Minimal Region by STS-Amplification Mapping. To determine the size and frequency of the 19q12 amplicon in esophageal adenocarci- nomas and, most importantly, the minimal critical region, 11 STS markers s

53、panning ?8 cM were selected in proximity to the location of the two-dimensional fragment in 19q12 based on a search of NCBI, WICGR, and SHGC databases. The QG-PCR assay was applied to the DNA from 65 paired normal tissue

54、-esophageal adenocarcinomas and 11 paired normal-gastric cardia adenocarcinomas using these 11 STS markers. The house-keeping gene GAPDH was PCR-coamplified as an internal control. Increased DNA dosage in the tumors cont

55、aining the 19q12 amplicon was observed (Fig. 3). 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.Fig. 2. South

56、ern blot analysis of DNA samples from three paired normal-adenocarci- nomas. The cloned two-dimensional DNA fragment was 32P-labeled and hybridized to the Southern blot membrane, which contained the DNA from normal-tumor

57、 pairs of patients F12, F93, and W11. DNA amplification was confirmed in tumor F12, which demonstrated a 6.5-fold increase as compared with its normal tissue counterpart. The lower panel is a control to show equal DNA lo

58、ading.Fig. 3. The QG-PCR assay was applied to characterize the 19q12 amplicon and to map the minimal critical region in 76 esophageal and gastric cardia adenocarcinomas. The housekeeping gene GAPDH was coamplified in the

59、 PCR reactions along with the STS markers. The STS markers selected were those neighboring the two-dimensional fragment on 19q12 as determined from the physical maps and STS databases. The DNA from the 10 of 76 tumors th

60、at were amplified at 19q12 is presented here. As shown, genomic amplification in tumors B81, M60, and P95 is observed for markers D19S409 and D19S919 but not for the markers D19S882 and D19S225. Amplification is observed

61、 at marker D19S882 in tumors S12, T99, B05, and S32, and also at D19S225 in tumor B05 but not at markers D19S409 or D19S919. Amplification in tumor M55 is observed at all of the loci except D19S409 as shown. D19S409 is a

62、 STS marker tightly linked to the cloned two-dimensional fragment that is 27 kb away (Table 1).Table 1 An outline of the resulting STS DNA fragments and two-dimensional fragment in BAC clone AC007786 retrieved by E-PCR a

63、nalysisName GenBank accession no. Location in contig (bp) Genetic mapSHGC-80721 G54241 19280–19574 NAaSHGC-130179 G59466 31591–31791 NA UTR-9734 G13234 107360–107538 NA F12 two-dimensional fragment Not applied 112467–112

64、870HS269XG5 (D19S409) Z23912 139732–139902 50.0 cM HSA084XF1 (D19S932) Z51953 186844–186987 49.4 cM HSA224WC9 (D19S875) Z52589 206471–206583 49.4 cMa NA, not available.7023CYCLIN E AMPLIFICATION IN ESOPHAGEAL ADENOCARCIN