[雙語(yǔ)翻譯]--醫(yī)學(xué)外文翻譯--epha2efna1在人類(lèi)胃癌中的表達(dá)（原文）

1、42–47 | Cancer Sci | January 2005 | vol. 96 | no. 1 © Japanese Cancer Association doi: 10.1111/j.1349-7006.2005.00007.xBlackwell Publishing, Ltd. EPHA2/EFNA1 expression in human gastric cancerRitsuko Nakamura,1 Hide

2、ki Kataoka,2,3 Naomi Sato,4 Masao Kanamori,5 Megumi Ihara,1 Hisaki Igarashi,1 Sanjar Ravshanov,1 You-Jie Wang,6 Zhong-You Li,7 Takahiro Shimamura,8 Toshihiko Kobayashi,8 Hiroyuki Konno,9 Kazuya Shinmura,1 Masamitsu Tan

3、aka10 and Haruhiko Sugimura1,111First Department of Pathology, 2First Department of Medicine, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu 43-3192; 3Department of Gastroenterology, Hamamatsu Me

4、dical Center, 328 Tomitsuka, Hamamatsu 432-8580; 4Department of Nursing; 5Department of Lifelong Sport, Biwako Seikei Sport College, 1204 Shigachou, Shigagun, Shiga 520-0503; 6School of Public Health, Tongi Medical Col

5、lege, #13 Hong Kong Road, Wuhan 430030, China; 7Department of Cancer Genetics, Roswell Park Cancer Institute, Elm and Carlton Str. Buffalo, NY 14263, USA; 8First Department of Surgery, and 9Second Department of Surgery,

6、 Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu 431-3192; and 10Growth Factor Division, National Cancer Center, 5-1-1 Tsukiji, Cyuo-Ku, Tokyo 104-0045(Received September 17, 2004/Revised November

7、15, 2004/Accepted November 16, 2004/Online publication January 19, 2005)The erythropoietin-producing hepatocellular (EPH)A2 receptor, tyrosine kinase, is overexpressed and phosphorylated in several types of human tumors

8、and has been associated with malignant transformation. A recent report, however, indicated that stimulation of the EPHA2 receptor ligand, ephrinA1 (EFNA1), inhibits the growth of EPHA2-expressing breast cancer. The autho

9、rs examined the expression of EPHA2 and EFNA1 using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) in four gastric cancer cell lines and 49 primary gastric cancer samples, as well as in normal

10、gastric tissue. EPHA2 was more highly expressed in tumor tissue than in normal tissue in 27 cases (55%). EFNA1 was overexpressed in tumor tissue in 28 cases (57%). No significant correlation was detected between the expr

11、ession levels and histologic features such as tumor size, age, vessel invasion, or lymph node involvement. However, EPHA2 overexpression was more prominent in macroscopic type 3 and 4 tumors than in type 1 or 2 advanced

12、gastric cancer. The authors observed EPHA2 expression in three of the four gastric cancer cell lines (AGS, KATO3, and MKN74) that were examined. In one cell line, TMK1, EPHA2 expression was barely detectable using northe

13、rn blotting, RT-PCR, and western blotting. In contrast, EFNA1 was detected in all cell lines. In the gastric cancer cell lines that endogenously expressed EPHA2, stimulation with ephrinA1-Fc led to decreased EPHA2 protei

14、n expres- sion and increased EPHA2 phosphorylation. Finally, the growth of EPHA2-expressing cells was inhibited by repetitive stimulation with soluble ephrinA1-Fc. Taken together, these findings suggest that EPHA2 and EF

15、NA1 expression may influence the behavior of human gastric cancer. (Cancer Sci 2005; 96: 42–47) T he erythropoietin-producing hepatocellular (EPH) receptors represent the largest known family of receptor tyrosine kinases

16、 and are activated by interaction with the cell-surface ligands, ephrins (EFN). There is evidence to suggest that some members of the EPH family and their EFN ligands are involved in angiogenesis and oncogenesis through

17、cell adhesion, morpho- genesis, capillary sprouting, and chemoattraction.(1?5) EPH receptors have been classified into two subfamilies, EPHA and EPHB. EPHA receptors bind mainly to glycosylphosphatidylinositol- anchored

18、EFNA ligands, and EPHB receptors bind to transmembrane EFNB ligands. The expression of EPH family transcripts has been documented in some melanomas and carcinomas.(6,7) Overexpression of EPHA2 is believed to be sufficien

19、t to confer malignant/tumorigenic potential on non- transformed mammary epithelial cells.(8) Esophageal squamous cell carcinomas that overexpress EFNA2 have a poorer prognosis than those that do not.(9)Gastric cancer rem

20、ains a disease with a very poor prognosis, and the role of kinases in gastric cancer cells has been a focus of research. Ogawa et al. identified EFNA1 and EPHA2 expres- sion in a very few cases of gastric cancer in 2000,

21、 but the role of these molecules has remained unclear,(10) despite an extensivesurvey of tyrosine kinases in gastric cancer.(11) Therefore, the authors examined the expression of EPHA2 and EFNA1 in gastric cancer specime

22、ns and gastric cancer cell lines using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), northern blotting, and western blotting. This is the first documented report of EFNA1 and EPHA2 expression

23、 in a series of gastric cancer cases. Furthermore, the authors exam- ined the effects of EFNA1 stimulation on cancer cell lines that endogenously express EPHA2.Materials and MethodsTissues. For RT-PCR, human gastric canc

24、er specimens and corresponding non-tumor tissues were obtained from 49 surgical resections carried out at Hamamatsu University School of Medicine. The clinicopathologic characteristics of these patients are shown in Tabl

25、e 1, and are classified according to the Japanese classification system (JCS).(12) Histologically, these specimens consisted of 22 cases of well-differentiated adenocarcinoma (tubular and papillary types) and 24 cases of

26、 poorly differentiated adenocarcinoma, including the mucinous type, and three other types (two adenosquamous and one neuroendocrine). The samples consisted of six early gastric cancers (the tumor is in the sub- mucosal a

27、nd mucosal layers in the gastric wall) and 43 advanced gastric cancers (the tumor invades through the proper muscle layer of the gastric wall). According to the pathologic TNM classifica- tion, there were 18 cases at sta

28、ges I and II, and 31 cases at stages III and IV. All samples were immediately frozen in liquid nitrogen and stored at ?80°C until RNA preparation was carried out. The study design was approved by the Institutional R

29、eview Board of Hamamatsu University School of Medicine (no. 12–11). Cell cultures. Gastric cancer cell lines (KATO3, MKN74, and TMK1) were cultured in RPMI 1640 (Nissui, Tokyo, Japan) supplemented with 10% fetal bovine s

30、erum (FBS; Gibco Invitrogen, Carlsbad, CA, USA). The AGS cells were cultured in Ham’s F12K medium (ICN Biomedicals, Bryan, OH, USA) supplemented with 10% FBS. The 293T human embryonic kidney cells were cultured in Dulbec

31、co’s modified eagle medium (Nissui) supplemented with 10% FBS. RNA extraction and reverse transcription. Total cellular RNA was extracted from human tissues using the RNA extraction reagent, ISOGEN, (Nippon Gene, Tokyo,

32、Japan) according to the manufacturer’s protocol. Single-stranded cDNA was prepared from total RNA and 1 µg of oligo dT primer (Life Technologies, Rockville, MD, USA) in a total volume of 20 µL containing Molone

33、y murine leukemia virus reverse transcriptase (Life Technologies) and RNase inhibitor (Toyobo, Tokyo, Japan).11To whom correspondence and reprint requests should be addressed. E-mail: hsugimur@hama-med.ac.jp44 © Jap

34、anese Cancer Association doi: 10.1111/j.1349-7006.2005.00007.xnumbers NM_004428 and NM_004431, respectively), have been described previously.(14) Specific probes were constructed from these plasmids using digestion, with

35、 the following appropriate restriction enzymes, EcoRI and BamHI (for EFNA1) or NotI and BamHI (for EPHA2). 18S RNA was used as a control. The probes were labeled with 32P-dCTP using a random primed DNA labeling kit (Taka

36、ra, Otsu, Japan). Hybridization was carried out at 42°C for 10 h. The hybridized membranes were washed three times at 42°C for 10 min and three times at 65°C for 30 min in 0.1% sodium dodecylsulfate (SDS)

37、and 0.1× standard saline citrate. For normalization of signal intensity, the membranes were stripped and then rehybridized with an 18S probe. The autoradiographic densities were measured using a bio-imaging analyzer

38、 (BAS-1000; Fuji Film). Protein extraction from tissue. Proteins were extracted from gastric cancer tissue and from non-tumor gastric tissue, dissected, and identified microscopically. They were powdered and homo- genize

39、d in TXB (10 mM Tris [pH 7.6], 150 mM NaCl, 5 mM ethylenediamine tetra-acetic acid [EDTA], 10% glycerol, 1 mMNa3VO4, 1% TritonX-100, aprotinin [10 µg/mL], leupeptin [10 µg/mL], and phenyl-methyl-sulfonyl-fluo

40、ride [PMSF; 20 µg/mL]). After keeping on ice for 30 min, the proteins were centrifuged twice at 18 000 × g for 30 min. These lysates were then used for western blotting. EPH receptor stimulation, immunoprecipi

41、tation, and western blots. A soluble form of ephrinA1-Fc, which is a mouse ephrinA1- human IgG1 Fc chimeric protein, was purchased from R Upstate Biotechnology, Lake Placid, NY, USA) followed by precleaning and immunopre

42、cipitation with protein G sepharose (Amersham Biosciences). Immunoprecipitated lysates were then separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), transferred to nitrocellulose membran

43、es (Amersham Biosciences), and immunoblotted with antiphosphotyrosine antibody (Upstate Biotechnology). The total lysate was also loaded in parallel for comparison. Immunohistochemistry. The formalin-fixed paraffin-embed

44、ded gastric cancer tissue next to the portions taken for RNA and protein analysis was immunostained with anti-EPHA2 (C-20, dilution 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) according to the manufacturer’s in

45、structions. We gathered nine cases of gastric adenoma and immunostained these in the same manner. Antibody specificity was demonstrated using an immuno- absorption test with 6 µg/mL of EPHA2(C-20) blocking peptide (

46、Santa Cruz Biotechnology). All the immunostains in this study were accompanied by this absorption test. The specificity of this antibody has also been tested by carrying out western blotting with this antibody to the lys

47、ate of 293T cells that were trans- fected with a plasmid that encoded EPHA2. Cell growth assay. A quantity of 1 × 105 cells of MKN74 and TMK1 was seeded into each well of a six-well tissue culture dish (Greiner Bio-

48、one, Kremsmüster, Austria). The cells were replenished with a medium that contained clustered ephrinA1- Fc (4 µg/mL) or clustered Fc every 24 h. After 3 days of incubation at 37°C, cells were harvested in

49、trypsin-EDTA solution (0.05% Trypsin, 0.53 mM EDTA-4 Na; Invitrogen, Carlsbad, CA, USA) and the number of cells was counted using a hemacytometer. The experiment was repeated at least three times. Statistical analysis wa

50、s carried out using Student’s t-test (Microsoft Excel, Seattle, WA, USA), with P < 0.05 defined as significant.ResultsExpression of EPHA2 and EFNA1 in gastric cancer tisseus, paired non-tumor tissues, and gastric canc

51、er cell lines. The authors examined the expression of EPHA2 and EFNA1 RNA in cancerous and non-cancerous gastric tissue using semiquantitative RT-PCR analysis (Fig. 1A). EPHA2 was more highly expressed in tumor tissues t

52、han in normal tissues in 27 of 49 cases (55%). EFNA1 was also overexpressed in substantial subsets of tumor tissues relative to the normal counterparts, specifically in 28 of 49 cases (57%). When classified using histolo

53、gy, EPHA2 overexpressionFig. 1. Semi-quantitative reverse transcription-polymerase chain reaction data of two cases of primary gastric cancer and corresponding non-tumor tissue, and gastric cancer cell lines. (A) Both

54、 ephrinA1 (EFNA1) and EPHA2 were overexpressed in these tumors (cases 1 and 2). ACTB is shown as an internal control. (B) EFNA1 was expressed in all gastric cell lines, particularly in TMK1. EPHA2 was detected in these f