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<p><b>OBJECTIVE</b>To investigate the expression of NOEY2 gene in breast cancer tissue and its relation to clinicopathological and other molecular parameters.</p><p><b>METHODS</b>The mRNA expression of NOEY2 gene was monitored in benign and malignant breast lesions by RT-PCR and in situ hybridization (ISH) with transcripted antisense RNA probes. The protein expression of estrogen receptor (ER), Ki67, p27 and p21(WAF1) in the 60 breast cancer lesions was detected by immunohistochemical method.</p><p><b>RESULTS</b>All 6 benign lesions, and 13 (72.2%) of the 18 breast cancers were NOEY2 positive by RT-PCR. By ISH, positive NOEY2 was obtained in all 10 benign lesions but only in 31 (51.7%) of the 60 breast cancer lesions. The difference was statistically significant (P = 0.025). NOEY2 positive rate tended to decrease with the increase of histological grade. However, NOEY2 expression was negatively correlated with the status of axillary lymph nodes. The positive NOEY2 rate was 75% in those without lymph node metastasis but only 26.7% in those with metastasis (P < 0.001). No correlation with other clinicopathological parameters including ER, Ki67, p27 or p21(WAF1) were found.</p><p><b>CONCLUSION</b>NOEY2 gene may be related with the pathogenesis of breast cancer. A link between NOEY2 loss expression and the spreading mechanism of breast cancer may possibly exist.</p>
Subject(s)
Female , Humans , Breast Neoplasms , Metabolism , Gene Expression , In Situ Hybridization , RNA, Messenger , Metabolism , Reverse Transcriptase Polymerase Chain Reaction , rho GTP-Binding Proteins , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To study the reversal effect of nomegestrol acetate (NOM) on mutidrug resistance (MDR) in MCF7/ADR and its mechanism.</p><p><b>METHODS</b>Using tetrazolium dye assay, effects of various concentrations of NOM on sensitivity to ADR in MCF7/ADR was studied. Expression of MDR related genes MDR1, glutathoine S-transferase Pi (GSTpi), Topoisomerase II alpha (Topo II alpha) and MDR related protein (MRP) were assayed by reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry assay. Using flow cytometry (FCM), intracellular ADR concentration effects on cell cycle were observed.</p><p><b>RESULTS</b>NOM significantly reversed MDR in MCF7/ADR. After NOM 20, 10 and 5 micromol/L treatment, the chemosensitivity to ADR increased to 21, 12 and 8 times. The reversal activity of NOM was stronger than that of the precursor compound megestrol acetate, and was comparable to that of verapamail. After treatment with NOM 5 micromol/L both MDR1 and GSTpi mRNA genes expression began to decline on D2 (P < 0.05, & P < 0.01) and reached the lowest level on D3 (both P < 0.01), but the expression levels began to rise on D6 again (both P < 0.05). The expression of MRP and Topo II alpha gave no significant change. Changes of P-gp and GSTpi protein expressions were similar to those of their mRNA expressions, showing early decline and late rise. Two hours after NOM 20, 10, and 5 micromol/L treatment, intracellular ADR concentration increased 2.7, 2.3 and 1.5 times, respectively. FCM data showed that after forty-eight hours, combined administration of NOM (20 micromol/L) and ADR (from low concentration to high concentration), MCF7/ADR cells showed gradual arrest in the G(2)M phase with the increase of ADR dose.</p><p><b>CONCLUSION</b>NOM has strong reversal effects on MDR in MCF7/ADR. The reversal takes place via different routes, i.e. down regulating mRNA and protein expression levels of MDR1 and GSTpi, increasing intracellular drug concentration, and enhancing the arrest of ADR in cells at G(2)M phase.</p>
Subject(s)
Humans , ATP Binding Cassette Transporter, Subfamily B, Member 1 , Genetics , Metabolism , Antigens, Neoplasm , Breast Neoplasms , Genetics , Pathology , Cell Survival , DNA Topoisomerases, Type II , Genetics , Metabolism , DNA-Binding Proteins , Drug Resistance, Neoplasm , Genetics , Gene Expression Regulation, Neoplastic , Glutathione S-Transferase pi , Glutathione Transferase , Genetics , Metabolism , Immunohistochemistry , Inhibitory Concentration 50 , Isoenzymes , Genetics , Metabolism , Megestrol , Multidrug Resistance-Associated Proteins , Genetics , Metabolism , Norpregnadienes , Pharmacology , Progesterone Congeners , Pharmacology , RNA, Messenger , Genetics , Metabolism , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured , Verapamil , PharmacologyABSTRACT
Purpose To explore the value of single cell isolation from tissue slides and single cell polymerase chain reaction(PCR) in the study of lymphy node germinal centers. Methods By single-cell isolation and single-cell PCR,rearranged immunoglobulin(Ig) genes were amplified from single Ki67(+) cells and single CD3(+) cells.The Ⅴ-region family specific primers were designed to Ig heavy chain leader region and light chain κ and λ framework region Ⅰ. Results PCR efficiency of Ki67(+) cells were 18.7% and 50.0% respectively in 2 lymph nodes.However,in 40 CD3(+) T cells,no Ig gene rearrangements were observed,which confirmed the efficiency and specificity of single cell isolation and single cell PCR. Conclusions Our study demonstrated the feasibility of single cell isolation from tissue slides and single cell PCR.This makes a sound basis for the use of technique in the further study of lymphoid diseases.
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Purpose:To clone and construct an eukaryotic expression vector of NOEY2 gene, and to observe the effects of NOEY2 transfection on the growth of human breast cancer cell line MDA MB 231. Methods:The coding region of NOEY2 was obtained with reverse transcription PCR, and then the PCR product was first cloned into pGEM T vector and further directionally subcloned into pcDNA3. Transfection reagent and selective antibiotic were lipofect AMINE and G418 respectively. The expression level of NOEY2 protein was detected by Western Blotting. The growth cures of the transfected and non transfected cells were recorded. The changes in cell cycle were analyzed by flow cytometry. Results:An eukaryotic expression vector of NOEY2 gene was constructed successfully. The cell transfected with NOEY2 showed definite expression of NOEY2, and the controls were negative. The growth of NOEY2 transfected cells was inhibited by 46.3%, compared with the parental cells on the seventh day after seeding. An obvious decrease in S phase and G2/M phase fraction and an increase in the percentage of G0/G1 phase cells were found in NOEY2 transfected cells. Conclusions:NOEY2 Transfection can inhibit the growth rate of MDA MB 231 cells in vitro, probably via the mechanism of G1 arrest. These data support the suggestion that NOEY2 is a tumor suppressive gene, which merits further investigation for its value as a therapy gene.
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Objetive: To observe the reversal effects of tumor necrosis factor (TNF) ? and interferon (IFN) ? on multidrug resistance (MDR) in K562 cell line resistant to adriamycin (ADR) (K562/A02). Methods: After treatment with TNF-? and IFN-? respectively, K562/A02 sensitivity to ADR was investigated using tetrazolium dye assay. MDR1 gene expression was assayed by semiquantitative reverse transcription-polymerase chain reaction and immunocytochemistry staining. Intracellular ADR concentration was also observed with flow cytometry. Results: The reversal activity after treatment with TNF-? or IFN-? was found to be increased up to 6 and 5-fold respectively at 24 h, and the peak with the increase of 10 and 8-fold respectively was seen at the 48 h (both P