Adult ; Aged ; Carcinoma, Hepatocellular ; metabolism ; Female ; Humans ; Liver Neoplasms ; metabolism ; Male ; Middle Aged ; RNA, Messenger ; biosynthesis ; genetics ; Receptor, ErbB-2 ; biosynthesis ; genetics ; Receptor, ErbB-3 ; biosynthesis ; genetics

In order to explore a potential indicator of predicting the occurrence and development of gestational trophoblastic tumor, the expression of c-erbB2 oncogene in human normal placenta, hydatidiform mole and choriocarcinoma was investigated. The expression of c-erbB2 was detected immunohistochemically by monoclonal antibody against the gene on the formalin-fixed paraffin sections of 21 hydatidiform moles, 21 invasive moles, 20 choriocarcinomas and 30 normal placentas. Results showed that the expression level of c-erbB2 was significantly higher in gestational trophoblastic tumor than in hydatidiform mole and normal placenta of midterm and term pregnancy (P < 0.05), while there was no significant difference between patients with gestational trophoblastic tumor of stage III, IV and those of stage I, II. It was demonstrated that overexpression of c-erbB2 may closely associated with malignant transformation of hydatidiform mole, not only providing important insight into pathogenesis of gestational trophoblastic tumor, but also having an important significance for the early diagnosis and early treatment of gestational trophoblastic tumor.
Biomarkers, Tumor ; Choriocarcinoma ; metabolism ; Female ; Genes, erbB-2 ; Humans ; Hydatidiform Mole ; metabolism ; Hydatidiform Mole, Invasive ; metabolism ; Placenta ; metabolism ; Pregnancy ; Receptor, ErbB-2 ; biosynthesis ; genetics ; Uterine Neoplasms ; metabolism

To purify the extracellular domain of HER2 in vitro and improve its prokaryotic expression abundance, the cDNA fragment encoding extracellular domain of HER2 was obtained by PCR and cloned into the expression vector pGEX-6P-1. After transforming it into Escherichia coli BL21, we instituted an investigation of different inducing conditions to try out the optimal condition for expressing soluble fusion protein. As for insoluble inclusion bodies, they were dissolved in 8 M Urea and refolded in refolding buffer. The soluble protein and the refolded protein were purified with Glutathione Sepharose 4B, respectively. The results showed that both the soluble and insoluble protein existed in Escherichia coli, but the majority was insoluble. It is beneficial to the expression of soluble fusion protein by induction at lower temperature (30 degrees C) and higher optical density (A600= 1.8) with the use of certain additive in medium. By purification of the supernatant of the lysate and refolded protein, the yield of the fusion protein was about 1.23 mg per liter culture. As a result, we have obtained the maximum soluble extracellular domain of HER2 protein, and thus have laid a foundation for further work on functional study and antibody preparation for HER2.
Cloning, Molecular ; Escherichia coli ; genetics ; Genes, erbB-2 ; genetics ; Humans ; Prokaryotic Cells ; metabolism ; Protein Folding ; Receptor, ErbB-2 ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

Breast Neoplasms/genetics* ; Female ; Gene Amplification ; Humans ; In Situ Hybridization, Fluorescence ; Receptor, ErbB-2/metabolism*

OBJECTIVETo evaluate the application of the immunohistochemistry (IHC) and the fluorescence in situ hybridization (FISH) in detecting the amplification and the expression of HER-2 gene in the breast cancer patients.

METHODSSixty-six cases of paraffin-embeded breast cancer samples with overexpression, low or no expression of HER-2 gene as detected by IHC were analyzed for HER-2 gene amplification using FISH.

RESULTSAmong the 42 samples with HER-2 gene overexpression (3+/2+) detected by IHC, 31 showed positive HER-2 gene amplification and 11 showed negative HER-2 gene amplification in FISH. In the 24 samples with low or no HER-2 gene expression (1+/-) detected by IHC, no HER-2 gene amplification was detected by FISH. The results of the two testing methods showed a good consistency with the kappa coefficient of 0.672 (P<0.001). We also found that the 17 chromosome polysomy in 42% of the samples and the incidence of 17 polysomy was significantly higher in the HER-2 gene overexpression (3+/2+) group than in low or no HER-2 gene expression (1+/-) group (chi(2)=4.688, P=0.03).

CONCLUSIONIHC can be used as a screening method for detecting HER-2 gene amplification, and FISH should be performed in cases of HER-2 gene overexpression (3+/2+) as detected by IHC.

Breast Neoplasms ; genetics ; metabolism ; Carcinoma, Ductal, Breast ; genetics ; metabolism ; Female ; Humans ; Immunohistochemistry ; In Situ Hybridization, Fluorescence ; Receptor, ErbB-2 ; analysis ; genetics

OBJECTIVETo evaluate the clinical value of HER2 overexpression in breast cancer and its prognostic implication in patients with lymph node negative breast carcinoma.

METHODSThe following electronic database were extracted using appropriate inclusive and exclusive standards: Cochrane library, PUBMED, Embase (1984 - 2003), OVID, CMCC and CNKI. Excel and RevMan 4.2 were used for statistical analysis.

RESULTSFifty-six articles were extracted to calculate the positive rate of HER2 overexpression. The pooled positive rate was 23.14% [19.54%, 26.73%], with positive immunohistochemistry (IHC) rate of 23.13% [19.49%, 26.77%] and positive FISH rate of 20.90% [15.54%, 26.25%]. Seven articles were used to evaluate prognostic predication of HER2 expression. It was concluded that in patients with lymph node negative breast carcinoma, HER2 overexpression (both IHC and FISH) independently predicted a poor prognosis based on disease-free survival (DFS) and overall survival (OS) with a P < 0.05. For DFS, the pooled RR was 1.38 [1.07, 1.80] with 1.16 [1.02, 1.31] for IHC and 1.98 [1.56, 2.52] for FISH. For OS, the pooled RR was 1.58 [1.16, 2.14] with 1.37 [1.14 to 1.64] for IHC and 2.33 [1.45 to 3.75] for FISH. HER2 overexpression effectively predicted DFS/OS of patients without adjuvant therapy and OS of patients with the therapy, but not for DFS, with the pooled RR of 1.46 [1.02, 2.09] and 1.11 [0.95, 1.31] for DFS, respectively and the pooled RR of 1.93 [1.44 to 2.58] and 1.25 [1.01, 1.56] for OS, respectively.

CONCLUSIONSIn patients with lymph node negative breast carcinoma, the positive rate of HER2 overexpression is 23.14%. HER2 overexpression indicates a poor prognosis and adjuvant therapy after surgery should be recommended.

Breast Neoplasms ; genetics ; metabolism ; pathology ; therapy ; Chemotherapy, Adjuvant ; Disease-Free Survival ; Female ; Genes, erbB-2 ; Humans ; Lymph Nodes ; pathology ; Mastectomy ; Prognosis ; Receptor, ErbB-2 ; metabolism ; Survival Rate

Breast Neoplasms ; diagnosis ; genetics ; metabolism ; Female ; Gene Amplification ; genetics ; physiology ; Humans ; Middle Aged ; Receptor, ErbB-2 ; metabolism

OBJECTIVETo explore the clinical significance of protein expression and gene amplification of HER2 in gastric cancer.

METHODSImmunohistochemistry (IHC) and chromogenic in situ hybridization (CISH) method were used to detect protein expression and gene amplification of HER2 in 80 specimens of gastric cancer patients.

RESULTSProtein expression of HER2 was negative in 51 cases, (+) in 12 cases, (++) in 12 cases, (+++) in 5 cases, and the positive expression rate was 21.3% (17/80). Seven (8.8%) cases had gene amplification of HER2, including gene critical amplification in 3 (3.8%) cases. The result of IHC was positively correlated with CISH (P<0.05), and the coincidence rate was 85.0% (68/80). HER2 positive expression rate was higher in the gastroesophageal junction carcinoma, poorly differentiated and stage III-IIII gastric cancer (all P<0.05).

CONCLUSIONThe gastric cancer tissue has high positive rate of protein expression and gene amplification of HER2, which is closely correlated to the development of gastric cancer.

Adult ; Aged ; Female ; Gene Amplification ; Humans ; Male ; Middle Aged ; Receptor, ErbB-2 ; genetics ; metabolism ; Stomach Neoplasms ; genetics ; metabolism ; pathology

OBJECTIVETo prepare the modified ZHER2V2 affibody with amino-terminal HEHEHE sequence and carboxyl-terminal GGGC sequence by gene recombinant expression,which is the basis for invasive HER2 imaging with affibody.

METHODSThe encoded affibody gene was optimized by codon preference of E. coli with gene designer software. The N-terminal of affibody was fused with HEHEHE sequence,while the C-terminal was fused with GGGC sequence. The synthetic gene was confirmed by Hind 3 endonuclease restriction and gene sequencing. The human epidermal growth factor receptor-2(HER2)affibody gene was sub-cloned into pET22b(+)plasmid and transformed into competent BL21(DE3)bacteria. The expression of modified affibody was induced with isopropyl Β-D-1-thiogalactopyranoside(IPTG)and identified by SDS-PAGE. The affibody was purified by nickel affinity binding and imidazole elution. The purified affibody was labeled with (68)Ga and its affinity was determined by saturation analysis with HER2-positive cells MDA-MB-361.

RESULTSThe affibody gene containing N-terminal HEHEHE and C-terminal GGGC sequences were confirmed by Hind 3 endonuclease restriction and gene sequencing. A newly expressed 8×10(3) protein was expressed from the induced recombinant bacteria identified by SDS-PAGE after sub-cloning HER2 affibody gene into pET22b(+)plasmid,transforming recombinant plasmid into competent BL21(DE3)bacteria and inducing the recombinant bacteria with IPTG. The expressed protein was purified from nickel agarose by 60 mmol/L imidazole eluting. The affinity Kd value of (68)Ga labeled affibody to HER2 positive MDA-MB-361 cells was 1.5 nmol/L.

CONCLUSIONThe affiibody ZHER2V2 containing N-terminal HEHEHE and C-terminal GGGC was successfully prepared by gene optimization,recombinant expression and affinity purification.

Affinity Labels ; isolation & purification ; Escherichia coli ; metabolism ; Gene Expression ; Humans ; Receptor, ErbB-2 ; genetics ; Recombinant Fusion Proteins ; biosynthesis

OBJECTIVE: To explore the molecular mechanism of proliferation inhibition of human breast cancer cell MCF-7 regulated by E1A gene. METHODS: E1A gene was transfected into MCF-7 cells by liposome reagents. RT-PCR and Western blot were used to detect E1A mRNA and protein expression and HER-2 mRNA in MCF-7. The proliferation and colony formation of MCF-7 were measured by 3-(4,5-dinmethylthiahiazo-z-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and soft agar formation assay. The apoptosis of MCF-7 cells regulated by E1A expression was examined by flow cytometry. RESULTS: E1A was not endogenously expressed in MCF-7. E1A expression in MCF-7 could significantly decrease HER-2 mRNA and protein expression. Flow cytometry indicated that the apoptosis of MCF-7 could be induced by E1A. Meanwhile, E1A gene could significantly inhibit MCF-7 proliferation and colony formation in soft agar. CONCLUSION E1A gene can decrease HER-2 expression and induce the apoptosis of human breast cancer cell MCF-7, and inhibit the proliferation and colony formation of MCF-7.
Adenovirus E1A Proteins ; biosynthesis ; genetics ; Apoptosis ; genetics ; Breast Neoplasms ; genetics ; metabolism ; Cell Proliferation ; Female ; Genes, erbB-2 ; genetics ; Humans ; RNA, Messenger ; biosynthesis ; genetics ; Receptor, ErbB-2 ; biosynthesis ; genetics ; Transfection ; Tumor Cells, Cultured