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 study the effect of SiRNA-EGFR on the expression of hyaluronidase gene in human breast cancer cells.

METHODSReverse transcription-polymerse chain reaction was used to detect the changes in the expression of EGFR mRNA in human breast cancer cell lines MDA-MB-231, MDA-MB-435S, ZR-75 and ZR-75-30 after transfection by SiRNA-EGFR.

RESULTSAfter transfection with SiRNA-EGFR, the expression levels of EGFR were significantly inhibited in MDA-MB-231, MDA-MB-435S, ZR-75 and ZR-75-30 cells (P<0.05).

CONCLUSIONTransfection by SiRNA-EGFR can inhibit the expression of EGFR mRNA in human breast cancer cells.

Breast Neoplasms ; enzymology ; metabolism ; pathology ; Cell Line, Tumor ; Female ; Humans ; Hyaluronoglucosaminidase ; genetics ; RNA Interference ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; Receptor, Epidermal Growth Factor ; genetics ; metabolism ; Transfection

OBJECTIVETo investigate the effect of membrane type-1 matrix metalloproteinase (MTI-MMP) on the invasive potential of breast cancer cell and analyze its mechanisms.

METHODSAfter treatment of breast cancer MDA-MB-453 cell line with concanavalin A ( ConA, 20 microg/ml) for 24 h, MT1-MMP protein was detected in cancer cells by Western analysis and immunocytochemistry. MDA-MB-453 cells were cultured with exogenous latent proMMP-2 and MMP-2 activity was analyzed by gelatin zymography. The invasive potential of the tumor cells was measured with a membrane invasion culture system. Cancer cells of the cell line were divided into four groups: the control group treated by neither reagent, group ConA was only treated by ConA, group MMP-2 was treated only by MMP-2, and group ConA + MMP-2 was treated by both ConA and MMP-2. RESULTS The expression of MTI-MMP protein could be detected in groups ConA and ConA + MMP-2, but nothing was detected in control and group MMP-2. There was only 72 000 precursor form of MMP-2 in group MMP-2 and there were both 72 000 precursor form and 64 000 active enzyme form of MMP-2 in group ConA + MMP-2, but there was no forms of MMP-2 in the other two groups detected by gelatin zymography. The largest amount of cells penetrated through Matrigel was observed in group ConA + MMP-2 than in the other three groups.

CONCLUSIONMTI-MMP can remarkably promote the invasive potential of breast cancer cells mainly through its ability of activating latent proMMP-2 to degrade

Blotting, Northern ; Blotting, Western ; Breast Neoplasms ; enzymology ; genetics ; pathology ; Cell Line, Tumor ; Cell Movement ; drug effects ; Concanavalin A ; pharmacology ; Female ; Humans ; Immunohistochemistry ; Matrix Metalloproteinase 14 ; genetics ; metabolism ; Matrix Metalloproteinase 2 ; metabolism ; Neoplasm Invasiveness ; RNA, Messenger ; genetics ; metabolism

OBJECTIVETo investigate telomerase activity of MCF-7 mammary cancer cells during apoptosis induced by sodium butyrate (SB) in vitro and its mechanism.

METHODSThe proliferative activity of MCF-7 cells was assessed by morphology and MTT assay. Cell apoptosis was confirmed by DNA fragmentation and phosphatidylserine (PS) externalization. Telomerase activity was examined by TRAP-ELISA. The expression status of telomerase subunits was analyzed by RT-PCR.

RESULTSA time- and dose-dependent inhibition was detected in MCF-7 cells treated with SB. At 72 hr after SB (2.5 mmol/L) treatment, MCF-7 cells were apoptotic with a rate of 84.3% by flow cytometric assay (AnnexinV/PI double staining). Apoptosis was also confirmed by DNA fragmentation. Telomerase activity and expression level of hTERT, the key subunit of telomerase, decreased at 24-hour time point after SB treatment. No significant changes were observed in the expression of hTR and hTP, the other two subunits of telomerase.

CONCLUSIONTelomerase activity decreases in MCF-7 cells during apoptosis induced by sodium butyrate. The underlying mechanism might be related to the down regulation of hTERT transcription.

Apoptosis ; drug effects ; Breast Neoplasms ; enzymology ; pathology ; Butyrates ; administration & dosage ; pharmacology ; Cell Line, Tumor ; DNA-Binding Proteins ; biosynthesis ; genetics ; Dose-Response Relationship, Drug ; Humans ; RNA, Messenger ; biosynthesis ; genetics ; Telomerase ; biosynthesis ; genetics ; metabolism ; Time Factors

OBJECTIVETo discuss the clinical significance of matrix metalloproteinase-2 (MMP-2) mRNA and the relationship of MMP-2 mRNA expression with breast cancer metastasis.

METHODDetect MMP-2 mRNA expression of 30 breast cancer, metastatic lymphnode and 116 clinical breast cancer samples by flurence-quantitative RT-PCR and analysis the relativity of MMP-2 mRNA expression with clinical, pathology factors.

RESULTSMMP-2 mRNA expression in 30 metastatic lymphnode is lower than in primary breast cancer (t = -3.293, P < 0.05). The mRNA expression of MMP-2 in tumor > 2 cm is lower than in tumor

CONCLUSIONSMMP-2 mRNA expression is up-regulated in early clinic stage, lymphnode metastatic early stage, and down regulated with tumor progressing. MMP-2 mRNA expression is related with the metastasis of breast cancer.

Adult ; Aged ; Breast Neoplasms ; enzymology ; pathology ; Female ; Humans ; Lymph Nodes ; pathology ; Matrix Metalloproteinase 2 ; biosynthesis ; genetics ; Middle Aged ; Neoplasm Metastasis ; RNA, Messenger ; genetics ; Receptors, Estrogen ; biosynthesis ; Receptors, Progesterone ; biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction

Antibiotics, Antineoplastic ; pharmacology ; Breast Neoplasms ; enzymology ; pathology ; Cell Line, Tumor ; Doxorubicin ; pharmacology ; Drug Resistance, Multiple ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Female ; Glucosyltransferases ; biosynthesis ; genetics ; Humans ; Oligodeoxyribonucleotides, Antisense ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Transfection

CYP17 gene is involved in steroidogenesis and steroid metabolism. Epidemiologic results on the association between the CYP17 polymorphism and breast cancer risk have been inconsistent. We examined the association between the MspAI polymorphism at +27 relative to the start of transcription in the 5'-untranslated region of CYP17 gene and breast cancer risk in Korean women. Four hundred and sixty-two incident cases and 337 controls were recruited from three teaching hospitals in Seoul during 1994-2001. Polymorphism of the CYP17 gene was determined by a single base extension assay. Demographic and lifestyle characteristics were identified using structured questionnaire. Age-adjusted (aOR) and multivariate odds ratios (mOR) and 95% confidence intervals (CI) were estimated by unconditional logistic regression. The proportions of A1/A1, A1/A2 and A2/A2 genotypes among controls were 20.8%, 45.1% and 34.1%, respectively. Compared to the A1/A1 genotype, A1/A2 or A2/A2 genotype was not statistically significantly associated with overall breast cancer risk (i.e., mOR=1.01, 95% CI=0.69-1.47 and mOR=0.76, 95% CI=0.51-1.14, respectively). However, a significant association between CYP17 A2/A2 genotype and breast cancer was observed among women aged 50 years or less (mOR=0.58, 95% CI=0.34-0.99, P=0.04) and leaner women (body mass index < 22 kg/m2) (mOR=0.48, 95% CI=0.23-0.97, P=0.04). Our results suggest that genetic polymorphism in 5'-untranslated region of CYP17 might play a role in breast cancer development in Korean women among younger women aged less than 50 or leaner women with body mass index less than 22 kg/m2.
5' Untranslated Regions/*genetics ; Breast/metabolism/pathology ; Breast Neoplasms/enzymology/epidemiology/*genetics ; Case-Control Studies ; Comparative Study ; Demography ; Female ; *Genetic Predisposition to Disease ; Genotype ; Humans ; Korea/epidemiology ; Middle Aged ; Odds Ratio ; Polymorphism, Genetic/*genetics ; Questionnaires ; Research Support, Non-U.S. Gov't ; Risk Factors ; Steroid 17-alpha-Hydroxylase/*genetics

OBJECTIVETo study the expression of thymidine phosphorylase (TP), thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) mRNA in breast cancer and its correlation with prognosis.

METHODSExpression levels of TP, TS and DPD mRNA in 86 micro-selected breast cancer tissues and 9 normal breast tissues were detected by real-time quantitative PCR.

RESULTSThe median expression levels of TP, TS and DPD mRNA in tumor tissue and in normal tissues were 16.54, 0.38, 2.47 and 11.75, 0.25, 8.33, respectively, there were no significant differences (P >0.05). The expression levels of TP, TS and DPD mRNA showed no association with tumor size, lymph node metastasis, pathological grade and clinical stage, except that of DPD showed a negative association with patients' ages. There was no significant difference in disease-free survival or overall survival between the patients with high and low TP or DPD mRNA levels. Disease-free survival tends to be better in the patients with low TS mRNA level than those with high TS mRNA, but the difference was not significant (P=0.069), while the overall survival showed a statistically difference (59.00 month and 70.30 month) (P=0.0496).

CONCLUSIONThe expression level of TS mRNA may serve as a prognostic marker for breast cancer patients.

Adult ; Age Factors ; Aged ; Aged, 80 and over ; Breast ; enzymology ; Breast Neoplasms ; enzymology ; mortality ; pathology ; Dihydrouracil Dehydrogenase (NADP) ; biosynthesis ; genetics ; Disease-Free Survival ; Female ; Follow-Up Studies ; Humans ; Lymphatic Metastasis ; Middle Aged ; Neoplasm Staging ; Prognosis ; RNA, Messenger ; biosynthesis ; genetics ; Survival Rate ; Thymidine Phosphorylase ; biosynthesis ; genetics ; Thymidylate Synthase ; biosynthesis ; 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

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