OBJECTIVETo study the expression of the cyclooxygenase-2 (COX-2) gene in breast cancer in contrast to that of normal breast tissues or benign breast tumors and its significance in the carcinogenesis and development of breast cancer.

METHODSWith reference to the expression of the beta-actin gene, the expression of COX-2 mRNA was examined in cancerous tissues and adjacent normal breast tissue from 30 patients and benign breast tumors from 15 patients by reverse transcription-polymerase chain reaction (RT-PCR). Quantitation of relative band densities was performed using densitometry-scanning software. Estrogen receptors of 30 breast cancers were investigated by immunohistochemistry.

RESULTSEnhanced expression of COX-2 was observed in ninety percent of cancers tissue with a range of 0.05 - 0.91 (median 0.53). Rare cases showed significant COX-2 expression in normal breast tissues with a range of 0 - 0.09 (median 0). In part of benign breast tumors, COX-2 expressions were obviously elevated with a range of 0 - 0.68 (median 0.07). The difference of expression of COX-2 mRNA among breast cancers, normal breast tissues, mastopathy or fibroadenomas was significant (rank-sum test, P < 0.05) and the difference of that between estrogen receptor negative and positive was also observed (rank-sum test, P < 0.01).

CONCLUSIONThe level of expression of COX-2 mRNA is obviously higher in breast cancer tissue than in normal breast tissue, mastopathy or fibroadenomas. The expression of COX-2 in hormone-dependent breast cancer is higher than that in hormone-independent breast cancer. The overexpression of COX-2 may play a crucial role in the carcinogenesis and development of cancer in patients with breast carcinoma.

Breast ; enzymology ; Breast Neoplasms ; enzymology ; Cyclooxygenase 2 ; Female ; Humans ; Isoenzymes ; genetics ; Membrane Proteins ; Middle Aged ; Prostaglandin-Endoperoxide Synthases ; genetics ; RNA, Messenger ; analysis ; Receptors, Estrogen ; analysis

OBJECTIVETo investigate the relationship of telomerase genes and the malignant transformation of atypical mammary ductal hyperplasia.

METHODSTelomerase genes hTR and hTRT in 50 cases of mammary hyperplasia (the cases included 6 benign hyperplasia, 9 mild atypical hyperplasia, 12 medium atypical hyperplasia, 23 severe atypical hyperplasia) and 26 cases of breast carcinoma were detected by in situ hybridization.

RESULTSThe expression of hTR and hTRT mRNA were weak or negative in benign hyperplasia (1/6, 0), weaker in mild-moderate atypical hyperplasia (2/9, 1/9, 4/12, and 3/12), strong in severe atypical hyperplasia (14/23, 60.9% and 12/23, 52.1%), while very strong expression (23/26, 88.5% and 21/25, 80.8%) in carcinoma of the breast. The difference between mild-moderate atypical hyperplasia, invasive ductal carcinoma and severe atypical hyperplasia was significant (P < 0.05) and the difference between severe atypital hyperplasia and intraductal carcinoma was not significant (P > 0.05).

CONCLUSIONSTelmerase genes (hTR, hTRT) expression is closely related to the malignant transformation of atypical hyperplasia. The reactivated telomerase may play a crucial role in the development of breast cancer.

Breast Neoplasms ; enzymology ; pathology ; Carcinoma, Intraductal, Noninfiltrating ; enzymology ; pathology ; DNA-Binding Proteins ; Female ; Gene Expression ; Humans ; RNA, Messenger ; Telomerase ; genetics

OBJECTIVETo assess the association of five types of fucosyltransferase gene (Fuc-T), the important biosynthesis gene of sialylated carbohydrate antigens (Sialyl Lewis A, Sialyl Lewis X), with metastasis and prognosis of breast cancer.

METHODSThe real-time quantitative PCR of five-type Fuc-T III, IV, V, VI, VII genes was performed in 80 patients with breast cancer.

RESULTSThe result showed that Fuc-TVII gene had higher gene copy compared to other type of Fuc-T in breast cancer. The grading of Fuc-TVII gene was related to lymph node metastasis and poor disease-free survival. Statistically difference was significant (P < 0.01). Fuc-T III, IV, V, VI gene were not significant relation to the metastasis and prognosis in 80 cases.

CONCLUSIONSThese findings suggest that Fuc-TVII gene is a prognostic indicator of breast cancer, and it may be play an important role in the biosynthesis of sialylated carbohydrate antigens in breast cancer.

Adult ; Aged ; Breast Neoplasms ; enzymology ; genetics ; pathology ; Female ; Fucosyltransferases ; genetics ; Genotype ; Humans ; Middle Aged ; Polymerase Chain Reaction ; Prognosis

Centrosomes maintain genomic stability by establishing the bipolar spindles during cell division and, execute accurate segregation of chromosomes during mitosis. In this study, we have demonstrated that there are three forms of STK-15 gene in breast cancer cell lines. Alternative splice positions are located in 5'-untranslated region of STK15 gene. The results of in vitro translation experiments revealed that the alternative splicing in the 5'-untranslated region of STK15 had no effect on protein translation. The differential expression patterns of these alternatively spliced STK15 in breast cell lines and primary tumors therefore suggest that STK15 gene transcription may be differentially regulated or stabilized in these cells.
*5' Untranslated Regions ; *Alternative Splicing ; Base Sequence ; Breast Neoplasms/enzymology/*genetics ; Centrosome/*enzymology ; Female ; Human ; Molecular Sequence Data ; Protein-Serine-Threonine Kinases/*genetics ; Tumor Cells, Cultured

Antigens, Neoplasm ; genetics ; metabolism ; Breast Neoplasms ; enzymology ; genetics ; metabolism ; DNA Topoisomerases, Type II ; genetics ; metabolism ; DNA-Binding Proteins ; genetics ; metabolism ; Female ; Gene Expression Regulation, Neoplastic ; physiology ; Humans

OBJECTIVETo explore the HSVtk gene expression mediated by the retroviral vector and to obtain high titer recombinant retroviral virus.

METHODSThe recombinant vector pRevTRE/HSVtk was constructed by inserting HSVtk gene into pRevTRE. The recombinant retrovirus, which was produced from cloned PA317 cells screened by hygromycin B after "micro-pingpong" technique transferring with pRevTRE/HSVtk plasmids DNA by using modified calcium phosphate precipitation method. HSVtk gene expression was performed on target cells and virus titers were detected in different cultured temper, time and sodium butyrate concentration.

RESULTSThe recombinant retroviral vector pRevTRE/HSVtk was constructed and HSVtk gene expression was detected on target cells after they were infected with the recombinant retrovirus.

CONCLUSIONHigh titer of retroviruses could be obtained in the culture medium of PA317 cell line through "micro-pingpong" technique at 30 hours and 10 mmol/L sodium butyrate concentration followed by frozen ultrafiltration.

Animals ; Breast Neoplasms ; enzymology ; pathology ; virology ; Cell Line ; Cell Line, Tumor ; Female ; Gene Expression Regulation, Enzymologic ; Genetic Vectors ; Humans ; Mice ; NIH 3T3 Cells ; Recombination, Genetic ; Retroviridae ; genetics ; Simplexvirus ; enzymology ; genetics ; Thymidine Kinase ; biosynthesis ; genetics ; Titrimetry ; Transfection

The present study was aimed to investigate the effect of Janus kinase 3 (JAK3) on the migration of breast cancer cells and the underlying mechanism. The expression of JAK3 in breast cancer MCF-7 cells was silenced by siRNA (siJAK3). The migration ability of MCF-7 cells was detected by scratch test. The activity of store-operated calcium channel (SOCC) was detected by fluorescence calcium imaging. The expression levels of Orai1 and STIM1, key molecules in the process of store-operated calcium entry (SOCE) were detected by Western blot and RT-PCR. The results showed that 2-APB, an inhibitor of SOCC, could inhibit the migration ability of MCF-7 cells. siJAK3 transfection significantly inhibited the migration ability of MCF-7 cells, decreased the activity of SOCC, and down-regulated mRNA and protein expression levels of Orai1 and Stim1. Over-expression of Orai1 or STIM1 in JAK3-silenced cells restored their migration ability. These results suggest that JAK3 facilitates the migration of breast cancer cells by SOCC.
Breast Neoplasms ; enzymology ; Calcium ; metabolism ; Calcium Channels ; metabolism ; Cell Movement ; physiology ; Gene Expression Regulation, Neoplastic ; Humans ; Janus Kinase 3 ; genetics ; metabolism ; MCF-7 Cells ; ORAI1 Protein ; genetics

OBJECTIVE: To determine the effect and molecular mechanism of DNA damage caused by suicide gene therapy system HSV-TK/GCV under Tet-On regulation in human breast cancer cell line MCF-7 infected by recombinant adeno-associated virus (rAAV). METHODS: We used comet assay to detect the effect of HSV-TK/GCV suicide gene regulation system on MCF-7 DNA damage, and analyzed the expression change of relative DNA damage response active genes and proteins with RT-PCR and Western blot. RESULTS: Compared with other control groups, the comet assay showed that MCF-7 cells with HSV-TK/GCV treatment had obvious comet tails, and the expression level of DNA damage response active genes and proteins changed obviously in the HSV-TK/GCV treatment group,such as ATM, p53 and p27,but CyclinE and CDK2 did not change. CONCLUSION DNA damage on MCF-7 cells is resulted from HSV-TK/GCV in suicide gene therapy system through a p53-dependent signal pathway, causing cell cycle arrest and cell death.
Breast Neoplasms ; genetics ; pathology ; therapy ; DNA Damage ; Dependovirus ; genetics ; Female ; Ganciclovir ; metabolism ; pharmacology ; Gene Expression Regulation, Neoplastic ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; Humans ; MCF-7 Cells ; Recombinant Fusion Proteins ; genetics ; metabolism ; Simplexvirus ; enzymology ; Thymidine Kinase ; genetics

OBJECTIVETo construct a RNA interference expression vector targeting human telomerase reverse transcriptase gene (hTERT) gene and investigate its effects on telomerase activity and proliferation in breast cancer cells in vitro.

METHODSThe shRNA sequences targeting hTERT gene were designed and recombined into pSuper-retro-puro vector. The breast cancer cell lines MCF-7 and MDA-MB231 were transfected with the recombined vector, and the telomerase activity of the cells was tested by telomerase repeat sequence amplification-enzyme linked immunosorbent assay (TRAP-ELISA). The proliferation of the transfected cells was assessed using MTT and soft-agar clone formation assays.

RESULTSThe recombinant plasmids pSuper-retro-puro-TERT RNAi#1 and #2 were successfully constructed as confirmed by enzymatic digestion and DNA sequencing. The telomerase activity in the transfected breast cancer cells were down-regulated significantly as compared with that in negative control cells (Plt;0.005). The transfection resulted in significant inhibition of the proliferation of both MCF-7 and MDA-MB231 cells as detected by MTT assay (Plt;0.05) and soft agar clone formation assay (Plt;0.001).

CONCLUSIONTransfection with the recombinant plasmid containing the shRNA targeting hTERT gene can down-regulate telomerase activity and inhibit proliferation of breast cancer cells in vitro, suggesting the potential of gene therapy targeting telomerase in the treatment of breast cancer.

Breast Neoplasms ; enzymology ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Female ; Genetic Therapy ; Genetic Vectors ; genetics ; Humans ; RNA Interference ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics ; metabolism ; Telomerase ; biosynthesis ; genetics ; metabolism ; Transfection

To investigate the effects of estrogen (E2) on telomerase activity and its mechanism in human breast cancer cells, estrogen receptor positive MCF-7 cells were treated with different concentrations of E2. Telomerase activity was measured by using TRAP-ELISA method, the cell cycle phases analyzed by using flow cytometry, and the expression of Cyclin D1 detected by using immunohistochemistry method. The results showed that telomerase activity levels were increased in MCF-7 cells treated with 10(-8) mol/L E2 during the observed period (P < 0.05), and E2 increased telomerase activity levels in a dose-dependent manner(10(-10)-10(-8) mol/L); Simultaneously, the cell cycle phases of MCF-7 cells treated with 10(-8) mol/L E2 were changed significantly: G0/G1 phase decreased from 60.52% to 50.93%. S phase increased from 29.03% to 30.83%; However, the expression of Cyclin D1 was decreased. It was concluded that estrogen can upregulate telomerase activity of MCF-7 cells, and the effect can be blocked by antiestrogen tamoxifen. Its mechanism may be closely associated with modulation of cell cycle phases.
Breast Neoplasms ; chemistry ; enzymology ; pathology ; Cell Cycle ; Cyclin D1 ; analysis ; Estrogens ; pharmacology ; Female ; Humans ; Receptors, Estrogen ; analysis ; Telomerase ; genetics ; metabolism ; Tumor Cells, Cultured