ABSTRACT
In order to study the effects of dominant negative human telomerase reverse transcriptase (DN-hTERT) on cell growth and telomerase activity in osteosarcoma cell line MG63, MG63 cells were transfected with DN-hTERT-IRES2-EGFP9 (DN) or IRES2-EGF (I, blank vector) with lipofectamine 2000. The stably transfected cells were selected with G-418. Cell growth properties were examined under a fluorescence microscope. The hTERT mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR). Telomerase activities were measured by TRAP-ELISE. The tumorigenicity was studied with tumor xenografts by subcutaneous injection of cancer cells into nude mice. The results showed that cell growth was suppressed in MG63 cells transfected with DN-hTERT. The hTERT mRNA was increased in N-hTERT transfected-MG63 cells (MG63/DN). The telomerase activity was 2.45±0.11 in MG63/DN cells, while 3.40±0.12 in the cells transfected with blank vector (MG63/I), (P＜0.05); DN-hTERT-expressing clones did not form tumors in 2 weeks, but the ratio of tumorigenesis was 30 % in nude mice bearing MG63/I (P＜0.01). It was concluded that DN-hTERT could specifically inhibit the cell growth and telomerase activity in MG63 cells.
ABSTRACT
In order to study the effects of dominant negative human telomerase reverse transcriptase (DN-hTERT) on cell growth and telomerase activity in osteosarcoma cell line MG63, MG63 cells were transfected with DN-hTERT-IRES2-EGFP9 (DN) or IRES2-EGF (I, blank vector) with lipofectamine 2000. The stably transfected cells were selected with G-418. Cell growth properties were examined under a fluorescence microscope. The hTERT mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR). Telomerase activities were measured by TRAP-ELISE. The tumorigenicity was studied with tumor xenografts by subcutaneous injection of cancer cells into nude mice. The results showed that cell growth was suppressed in MG63 cells transfected with DN-hTERT. The hTERT mRNA was increased in N-hTERT transfected-MG63 cells (MG63/DN). The telomerase activity was 2.45-0.11 in MG63/DN cells, while 3.40+/-0.12 in the cells transfected with blank vector (MG63/I), (P<0.05); DN-hTERT-expressing clones did not form tumors in 2 weeks, but the ratio of tumorigenesis was 30 % in nude mice bearing MG63/I (P<0.01). It was concluded that DN-hTERT could specifically inhibit the cell growth and telomerase activity in MG63 cells.
ABSTRACT
Objective: To explore the inhibitiory effect of antisense oligodeoxynucleotide(ASODN) of survivin gene on the cultured osteosarcoma cell line MG63 and its sensitization effect to chemotherapy.Methods: survivin phosphorothioate ASODN was synthesized and transfected into MG63 cells by lipofectamine 2000.MTT assay was used to detect cell inhibition ratio.Apoptosis was observed by flow cytometry.Survivin mRNA and protein expression were determined by RT-PCR and Western blot respectively.Results: The proliferation of the cells transfected by lipofectamine 2000 was inhibited by survivin ASODN in a dose and time dependent manner.A higher total apoptosis rate(81.12?3.2)% could be induced in MG 63 cells in group Lip-ASODN than in group Lip-SODN(27.09?2.1)%(P
ABSTRACT
Objective:To explore inhibition of telomerase activity and malignant phenotype on breast cancer cells MCF7 by Dominant Negative human telomerase reverase transcriptase gene(DN-hTERT).Methods:DN-hTERT eukaryotic expression vector DN-hTERT-IRES2-EGFP and empty vector(I) IRES2-EGFP were transfected into MCF7 by lipofectamine2000,after being selected by G418,positive clones were obtained;Tthe transfected cells growth was observed with inverted fluorescence microscope.The expression levels of hTERT mRNA of transfected cells were determined by reverse transcriptase polymerase chain reaction(RT-PCR).Telomerase activity of transfected cells was measured by TRAP-ELISE.Results:The proliferation of MCF7/DN cells were inhibited by DN-hTERT transtection;The expression levels of hTERT mRNA increased in MCF7/DN.Telomeric length was shorter in MCF7/DN than that in MCF7/I.The telomerase activity measured by TRAP-ELISE was 2.36?0.12 in MCF7/DN and was 3.32?0.14 in vacant vector MCF7/I.The telomerase activity in MCF7/DN was significantly lower than in vacant vector transfected MCF7/I(P