Résumé
Objective To investigate the effect of mouse double minute 2 (MDM2) mRNA ASON and mismatched oligonucleotide (ASONM) radiolabeled with 99Tcm on target gene expression in LNCaP cells.Methods The ASON and ASONM targeted to MDM2 mRNA were synthesized and radiolabeled by 99Tcm with the bifunctional chelator of HYNIC.The labeling efficiency,radiochemical purity,stability and molecular hybridization activity were investigated.The different concentrations of 99Tcm-HYNIC-ASON (0,100,500 nmol/L) and 99Tcm-HYNIC-ASONM (500 nmol/L) coated with lipofectamin 2000 were incubated with prostate cancer cells for 24 h,then RT-PCR and Western blot were carried out to assay the MDM2,p53 mRNA and the corresponding protein level.The variables of RT-PCR and Western blot were analyzed using one-way analysis of variance and q test.Results The labeling efficiency of ASON and ASONM were (65.15± 2.05)％ (n=5) and (64.93±2.18)％ (n=5),respectively.The radiochemical purity were both more than 90％.99Tcm-HYNIC-ASON had a good stability and could hybridize to the sense oligonucleotide (SON).The contents of MDM2 mRNA in 0,100,500 nmol/L 99Tcm-HYNIC-ASON and 500 nmol/L 99Tcm-HYNIC-ASONM groups were 0.458±0.035,0.250±0.026,0.174±0.032,0.463±0.033,respectively,and there were significant differences between each 2 groups except between 0 nmol/L 99Tcm-HYNIC-ASON and 500 nmol/L 99Tcm-HYNIC-ASONM groups (F=33.69,q =24.32-91.45,all P＜0.01).The average density of MDM2 protein in the 4 groups were 90.712±3.042,71.218±2.915,32.775±3.062,88.121±2.710,respectively (F=235.93,q=6.43-19.14,all P＜0.01; except 0 nmol/L99Tcm-HYNIC-ASON and 500 nmol/L 99Tcm-HYNIC-ASONM).The contents of p53 mRNA in the 4 groups were 0.185±0.046,0.203±0.040,0.213±0.027,0.163±0.049,respectively(F =2.18,P＞ 0.05).The average density of p53 protein was 33.865 ± 2.213,70.445±2.180,99.025±3.012,38.351±3.271,respectively (F=53.98,q =3.32-6.74,all P＜0.01 ; except 0 nmol/L 99Tcm-HYNIC-ASON and 500 nmol/L 99Tcm-HYNIC-ASONM).Conclusions The MDM2 antisense probe can accumulate in the prostate cancer cells,and specially hybridize to the MDM2 mRNA and inhibit target gene expression.This novel molecular probe has a promising potential for the diagnosis of prostate cancer at gene level.
Résumé
PURPOSE: This study was designed to investigate effect of the etoposide on expression of cell cycle regulatory proteins and apoptosis-related proteins in human skin fibroblast (HSF). MATERIALS AND METHODS: HSF cells were treated with etoposide. After treatment, expression patterns of cell cycle regulatory proteins and apoptosis-related proteins were analyzed by using Immunoprecipitation-Western blot method and RT-PCR. RESULTS: Immediately after etoposide treatment, E2F-1 was up- regulated following MDM2 down-regulation. After E2F-1 up-regulation, p53 and p21WAF1 level was markedly increased without or with mRNA up-regulation, respectively. Consistent with these results, cell cycles arrested in mainly G1 phase 24 hr after etoposide treatment. However, HSF cells progressed into apoptosis 72 hr after etoposide treatment. In this process, caspase-3 activation and Bax up-regulation were observed. CONCLUSION: In early response of etoposide treatment, E2F-1 plays an important role in p53 accumulation through MDM2 down- regulation. The increased p53 induce an increase of p21WAF1 level through p21WAF1 mRNA up-regulation. However, after long term treatment of etoposide, HSF cells resulted in apoptotic cell death through caspase-3 activation and Bax up-regulation.