ABSTRACT
The effects of a novel immunosuppressive agent FTY720 on proliferation inhibition and apoptosis of acute leukemia cell lines HL-60 and U937, and the role of extracelluar regulated protein kinase (ERK) in the course of proliferation inhibition and apoptosis induced by FTY720 were studied. The proliferation inhibition rate of HL-60 and U937 cells by various concentrations of FTY720 was detected by MTT assay. Cell apoptosis was detected by DNA fragment analysis and flow cytometry. The phosphorylated ERK1/2 protein expression was observed by Western blotting. The change of intracellular distribution of ERK1/2 protein was identified by SP immunohistochemical staining. The results showed that FTY720 could inhibit the growth of HL-60 and U937 cells effectively in a dose-dependent manner. After incubation with FTY720 for 24 h, apoptosis was observed in HL-60 and U937 cells. The intracellular expression of phosphorylated ERK1/2 protein was also down-regulated and the distribution of ERK1/2 protein in cell nuclear was reduced during FTY720-induced apoptosis. So, that FTY720 inhibited ERK1/2 phosphorylation might mediate the role of FTY720-induced apoptosis and proliferation inhibition of leukemia cells.
Subject(s)
Humans , Apoptosis , Cell Division , Extracellular Signal-Regulated MAP Kinases , Metabolism , Fingolimod Hydrochloride , HL-60 Cells , Immunosuppressive Agents , Pharmacology , Mitogen-Activated Protein Kinase 1 , Metabolism , Mitogen-Activated Protein Kinase 3 , Metabolism , Phosphorylation , Propylene Glycols , Pharmacology , Signal Transduction , Sphingosine , U937 CellsABSTRACT
In order to investigate the regulative function of telomerase and phosphorylated (activated) extracellular regulated protein kinase (ERK) 1 and 2 in the leukemic cell lines HL-60 and K562 proliferation inhibition and apoptosis, three chemotherapeutic drugs Harringtonine (HRT), Vincristine (VCR) and Etoposide (Vp16) were selected as inducers. The proliferation inhibition rate was detected by MTT method, the cell cycle and cell apoptosis was analyzed by flow cytometry and the telomerase activity was detected by the telomeric repeat amplification protocol (TRAP) assay and bioluminescence analysis method. The phosphorylated ERK1/2 protein expression was detected by western blot method. The results showed that HRT, VCR and Vp16 could inhibit cell proliferation, induce apoptosis, inhibit telomerase activity and down-regulate the protein expression of phosphorylated ERK. It was suggested that ERK signal transduction pathway was involved in the down-regulation of telomerase activity and the onset of apoptosis in the leukemic cells treated by HRT, VCR and Vp16.
Subject(s)
Humans , Antineoplastic Agents , Pharmacology , Apoptosis , Cell Division , Down-Regulation , Etoposide , Pharmacology , HL-60 Cells , Harringtonines , Pharmacology , K562 Cells , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases , Metabolism , Phosphorylation , Telomerase , Metabolism , Vincristine , PharmacologyABSTRACT
To explore the effect of NF-κB on bcl-x gene transcription in extended drug resistance leukemia cell line HL-60/E6, drug-resistant subline HL-60/E6 was derived by intermittently exposing HL-60 cells to 6 ng/ml epirubicin. Indirect immunofluorescence was used to demonstrate the location of NF-κB-RelA in HL-60/E6 cells. FCM analysis and RT-PCR were used to detect the efficiency of liposome-mediated ODN transfection and the change of bcl-xL mRNA levels after 5 μmol/L phosphorothioate (PS)-derivatized antisense (AS) oligodeoxynucleotide (ODN) directed to RelA was transferred into HL-60/E6 cells. The results showed that RelA remained persistently active and located at the nuclei of HL-60/E6 cells，but in the cytoplasm of HL-60 cells, the efficiency of liposome-mediated ODN transfection was significantly higher than that of null ODN (P<0.01 in 4 h, 6 h, 12 h, 24 h). Exposure of HL-60/E6 cells to 5 μmol/L AS-PS-ODN directed to RelA led to a maximal 40 % decline of bcl-xL mRNA levels within 8 h. The inhibition rate of bcl-xL mRNA was (15±1.79) %, (28±2.34) %, (40±3.47) %, (20±1.54) % in 4 h, 6 h, 8 h, 15 h， respectively, but it was less than 15 % in control group. It was concluded that NF-κB was involved in regulating bcl-x transcription. It was suggested that NF-κB was an important factor for drug resistance in leukemia cells.
ABSTRACT
To explore the effect of NF-κB on bcl-x gene transcription in extended drug resistance leukemia cell line HL-60/E6, drug-resistant subline HL-60/E6 was derived by intermittently exposing HL-60 cells to 6 ng/ml epirubicin. Indirect immunofluorescence was used to demonstrate the location of NF-κB-RelA in HL-60/E6 cells. FCM analysis and RT-PCR were used to detect the efficiency of liposome-mediated ODN transfection and the change of bcl-xL mRNA levels after 5 μmol/L phosphorothioate (PS)-derivatized antisense (AS) oligodeoxynucleotide (ODN) directed to RelA was transferred into HL-60/E6 cells. The results showed that RelA remained persistently active and located at the nuclei of HL-60/E6 cells，but in the cytoplasm of HL-60 cells, the efficiency of liposome-mediated ODN transfection was significantly higher than that of null ODN (P<0.01 in 4 h, 6 h, 12 h, 24 h). Exposure of HL-60/E6 cells to 5 μmol/L AS-PS-ODN directed to RelA led to a maximal 40 % decline of bcl-xL mRNA levels within 8 h. The inhibition rate of bcl-xL mRNA was (15±1.79) %, (28±2.34) %, (40±3.47) %, (20±1.54) % in 4 h, 6 h, 8 h, 15 h， respectively, but it was less than 15 % in control group. It was concluded that NF-κB was involved in regulating bcl-x transcription. It was suggested that NF-κB was an important factor for drug resistance in leukemia cells.
ABSTRACT
AIM: To explore whether inhibition of NF-?B by antioxidant pvrrolidine dithiocarbamate (PDTC) sensitizes leukemia cells to cytotoxic drugs and its mechanism. METHODS: The indirect immunofluorescence method and electrophoretic mobility shift assay (EMSA) were used to measure the activation of NF-?B. The apoptotic cells were evaluated by flow cytometry (FCM) and the in vitro growth inhibitory effect was performed using a MTT assay. RESULTS: EMSA showed that NF-?B was activated by daunorubicin (DNR), VP-16 and then was inhibited by PDTC in a dose-dependent manner. NF-?B activation was further verified because of subunit RelA of NF-?B locating in the nuclei. FCM analysis showed that apoptotic index of HL-60 cells was up to (8.97?0.81)%, (16.01?1.06)%, (22.96?1.33)% from (5.34?0.62)%, (10.16?0.42)%, (17.32?1.15)% after exposure of HL-60 cells to 2.5-10 mg/L VP-16 combined with PDTC. VP-16 added with PDTC produced greater growth inhibitory effect to HL-60 cells than did VP-16 or DNR only (P
ABSTRACT
ObjectiveTo investigate the visual condition and causes of blindness and low vision of ov er 60 years old people found by routine physical examination. Methods A total of 892 cases over 60 years old people took a yearly routine examinatio n, such as far and near vision in daily life, anterior segment and fundus of the eye and intraocular pressure. If the vision were under 0 3, the refractive vis ion needs to be corrected. The blindness and low vision patients were selected a nd taken further related examinations to analyse statistically the causes of bli ndness. Results Among 892 cases investigated, blindness together with low vision were 42 cases (4 71%). The morbidity rate of blindness and low vision were 1 01% (9 cases) a nd 3 70% (33 cases) respectively. Diseases caused blindness were macular lesio ns (57 14%), glaucoma(11 91%), cataract (9 52%), retinal diseases, optic nerv e atrophy, keratopathy etc. Refractory eye diseases were obviously more than t he eye diseases which can be treated. The morbidity rate of over 80 years old pe ople were obviously more than that of under 80 years group. Conclusions With ageing, the old people easily suffer from eye diseases and results in bli ndness. For the prevention and treatment of blindness for the elderly, much atte ntion to treatment of eye diseases should not only paid, active training is also important.
ABSTRACT
AIM: To study the role of plk1 in the ant i-cancer effect of colcemid and vincristine against K562 cells. METHODS: K562 cells were treated with colcemid and vincristine a nd antisense oligonucleotide of plk1, then expression of plk1 and ?-tubulin wer e investigated by Western blotting and confocal microscopy. RESULTS: Treatment of K562 cells with colcemid and vincristine i nfluenced the condensation of plk1 and assembly of ?-tubulin, though without ch ange of protein quantity. Treatment with antisense oligonucleotide of plk1 not o nly reduced the expression of plk1 without influence on protein quantity of ?-t ubulin detected by Western blotting, but also disturbed the formation of centros ome observed by confocal microscopy. CONCLUSION: The function of colcemid and vincristine destructing the spindle might be realized through the mechanism of restraining condensation of plk1 and assembly of ?-tubulin, which might be dependent on plk1. plk1 may be a potential target in anti-cancer therapy.