ABSTRACT
Objective: To investigate the effect of nucleophosmin 1 (NPM1) mutant A on TGF-β1-induced K562 cell proliferation and AKT phosphorylation. Methods: K562 cells were infected with Ad5-NPM1 to create an NPM1 over-expression cell model. NPM1 levels were determined by ELISA and Western blot analysis. The levels of AKT and P-AKT were determined by Western blot. MTT was used to measure the proliferation of K562 cells. Results: NPM1 protein levels in K562 cells increased in an Ad5-NPM1-MOI-dependent manner. Cell proliferation and NPM1 levels in the supernatant were significantly increased in K562 cells infected with Ad5-NPM1-30 and Ad5-NPM1-100 compared to those infected with Ad5-vector-100 (P<0.01). Treatment with (10 ng/mL) TGF-β1 increased P-AKT levels, but not total AKT levels in K562 cells. TGF-β1-induced phosphorylation of AKT was significantly increased by infection of K562 cells with Ad5-NPM1-100. No significant differences were found in total AKT levels among all groups. TGF-β1 (10 ng/mL) treatment also in-creased the proliferation of K562 cells. TGF-β1-induced K562 cell proliferation was significantly increased by infection with Ad5-NPM1-100 (P<0.01). Conclusions: NPM1 improves TGF-β1-induced cell proliferation by up-regulating AKT phosphorylation levels.
ABSTRACT
<p><b>OBJECTIVE</b>To establish a real-time quantitative RT-PCR method for the detection of expression of bcl-2 mRNA.</p><p><b>METHODS</b>The vector containing bcl-2 gene as standard template was constructed with T-A cloning technique. The fluorogenic probe (i.e.,Taq man probe) was used to establish a real-time RT-PCR. bcl-2 mRNA expression level in Burkitt's lymphoma pre-and post-treated with bcl-2 antisense phosphothioate oligodeoxynucleotides (AS-PS-ODN) was determined with real-time quantitative RT-PCR, also with semi-quantitative RT-PCR.</p><p><b>RESULTS</b>The expression of bcl-2 mRNA in Burkitt's lymphoma treated with bcl-2 AS- PS-ODN decreased significantly and no changes of bcl-2-mRNA expression in group treated with nonsense ODN were noticed. The semi-quantitative RT-PCR results also demonstrated that bcl-2 level varied as detected with real-time fluorogenic quantitative RT-PCR, but less sensitive and accurate.</p><p><b>CONCLUSION</b>Detection of bcl-2 mRNA expression with the fluorogenic real-time quantitative RT-PCR is more accurate and sensitive than semi-quantitive RT-PCR.</p>
Subject(s)
Humans , Electrophoresis, Agar Gel , Methods , Gene Expression , Proto-Oncogene Proteins c-bcl-2 , Genetics , RNA, Messenger , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Objective:To investigate the feasibility of clinical application of HLA-DRB,DPB1 genotyping by PCR-SSP,RFLP for alloge-neic bone marrow transplantation(Allo-BMT) .Methods: Corresponding to each of the DRB alleles, nineteen different pairs of sequence specific primers were used to PCR amplify them from twenty samples in both recipients and donors and their amplified products were directly analyzed on agarose gel. At the same time, a pair of primer was used to PCR amplify the second exon of DPB1 and the restriction fragment length polymorphisms were analyzed on PAGE after their PCR products were separately digested by ten endonucleases. Results: Each DRB was typed in according to its clear and visualized electrophoresis band; the DPB1 genotype was also determined by analysis of all codes representing polymorphism fragment band with a computer. Four couples of recipient and donor were matched. Conclusion:PCR-SSP and RFLP are rapid, accurate and reliable genotyping approaches for Allo-BMT.