Antitumor-drug-indnced apoptosis in leukemia cell lines HL-60 and U937 was quantitatively analyzed with TDT (terminal deoxynuleotidyl transferase-mediated nick-end labeling) approach, which allows to labal the ends of...Antitumor-drug-indnced apoptosis in leukemia cell lines HL-60 and U937 was quantitatively analyzed with TDT (terminal deoxynuleotidyl transferase-mediated nick-end labeling) approach, which allows to labal the ends of DNA broken strands in apoptotic cells by biotinlated dUTP which to avidin-FITC. In this way the apoptotic cells show nuorescent when the labeling cells were emitted by UV light microscope or laser-activated now cell sorting at r=480. In our study, HL-6o and U937 cell lines were cocultured respectively with cisdiaminodicaicroplatinum (CDDP), hydroxycamptothecinum (HCT) and vindesini sulfa (VCR) for 18 hours.By calculating percentages of apoptotic cells with TDT mothod, we were able to show that the two cell lines gave different sensitivity to the drugs. HL-60 showed high sensitivity to CDDP but U937 cells were more sensitive to other two drugs, HCT and VCR. Meanwhile we compared the results of obtained by DNA gel electrophoresis with that by TDT. We found that gel electrophoresis is not sensitive enough to reveal apoptosis since there was no ladder structure, a typical electrophoresis pattern for apoptosis, appeared until the apoptotic cells reached or over 13%. And we report in this paper as first time that three forms of apoptotic cells could be detected under fluorescent microscope, which we called as spot form and crescent form and assembling form in terms of distribution of light spots within cell nuclei. It seemed that the spot form was at an early stage of apoptosis and the crescent form rcprtscntcd a later stage of apoptosis.展开更多
OBJECTIVE: To probe insights into the reversal effect of bufalin on vincristine-acquired multidrug resistance(MDR) in human leukemia cell line K562/VCR.METHODS: Proliferative inhibition rate and the reversal index(RI)...OBJECTIVE: To probe insights into the reversal effect of bufalin on vincristine-acquired multidrug resistance(MDR) in human leukemia cell line K562/VCR.METHODS: Proliferative inhibition rate and the reversal index(RI) of bufalin were determined by Methyl thiazolyl tetrazolium assay. The uptake of Adriamycin(ADM) in K562/VCR cells, cell cycle and apoptosis rate were determined by flow cytometry(FCM). Cell morphologic changes were observed with Wright-Giemsa staining. The expression of P-glycoprotein(P-gp), multidrug-associated protein-1(MRP1), Bcl-x L and Bax protein were measured by immunocytochemistry.RESULTS: The human leukemia multidrug resistant K562/VCR cells showed no cross-resistance to bufalin. The RIs of bufalin at concentrations of 0.0002,0.001 and 0.005 μmol/L were 4.85, 6.94 and 14.77,respectively. Preincubation of 0.001 μmol/L bufalin for 2 h could increase intracellular ADM fluorescence intensity to 28.07%(P<0.05) and down-regulate MRP1 expression simultaneously, but no remarkable effect was found on P-gp protein. Cell cycle analysis indicated increased apoptosis rate and apparent decreased G2/M phase proportion after treatment with bufalin. When exposed to 0.01μmol/L bufalin, typical morphological changes of apoptosis could be observed. Down-regulation of Bcl-x L and up-regulation of Bax expression in K562/VCR cells could be detected by immunocytochemistry.CONCLUSION: Bufalin could partly reverse the MDR of K562/VCR cells, with a possible mechanism of down-regulating MRP1 expression and activating apoptosis pathway by altering Bcl-x L/Bax ratio.展开更多
Using Southern blot, Northern blot and Quick blot methods, we examined the rearrangement and expression of TCR βgene in four early differentiation stage cell lines from human hemopoietic system, namely HL-60, Jurkat,...Using Southern blot, Northern blot and Quick blot methods, we examined the rearrangement and expression of TCR βgene in four early differentiation stage cell lines from human hemopoietic system, namely HL-60, Jurkat, Daudi and Raji cells as well as lymphocytes from 17 acute lymphocytic leukemia （ALL） patients. The results showed. Ⅰ) Rearrangement of TCR βgene was seen in Jurkat cells. A germline pattern was observed in HL-60, Daudi and Raji cells. 2) Eight of 9 patients with T-ALL had cells with rearranged TCR βgene. But two of 3 patients with B-ALL and three of 5 patients with nonT, nonB-ALL also had cells with rearranged TCR βgene. 3) A 1.3 kb full-length transcript and a 1.0 kb truncated transcript were detected in Jurkat cells by probing with <sup>32</sup>P-TCR βcDNA. But some leukemic B cells also expressed an incompleted transcript. 4) TCR βmRNA was detected in six of 8 patients with T-ALL, four of 5 patients with nonT, nonB-ALL and one of 3 patients with B-ALL. But the level of expression was quite differ ent. The dual-rearrangement and the abnormal expression may give us a new clue for researching leukemogenesis.展开更多
Since 1960, the tumor strains of L6565 viral leukemia, SRS lymphoma and L783 transplantable leukemia were established successively in our laboratory. Recently, derived from the strains of threse leukemia/lymphoma, SRS...Since 1960, the tumor strains of L6565 viral leukemia, SRS lymphoma and L783 transplantable leukemia were established successively in our laboratory. Recently, derived from the strains of threse leukemia/lymphoma, SRS-82 cell line, SACIIB2, SACIIC3 cell clones and a cell line infected with SRS leukemia virus (SRSV/3T3) were obtained at vitro. The main results of study on the biology, virology and Its Induction of differentiation are summarily reported.展开更多
Objective: To explore the effects of Taxol in inhibiting human leukemia k562 cell proliferation and inducing apoptosis in vitro. Methods: Human leukemia K562 cells were treated with Taxol of different concentrations f...Objective: To explore the effects of Taxol in inhibiting human leukemia k562 cell proliferation and inducing apoptosis in vitro. Methods: Human leukemia K562 cells were treated with Taxol of different concentrations for 12-72 hrs. Cell proliferation was evaluated by MTT assay and morphological changes of apoptosis were examined by microscopy. Cell apoptosis was determined by flow cytometry (FCM) and DNA gel electrophoresis. Results: Growth of K562 cells was inhibited by Taxol with an IC50 value of 0.84 μg/mi.Typical nuclear condensation and apoptosis bodies were observed as early as 24 hrs after a 0.5 μg/ml Taxol treatment; Apoptotic rate of the Taxol-treated K562 cells increased from 3.7% to 24.0% in 24 hrs. No DNA ladder was observed by DNA gel electrophoresis. Conclusion: Taxol could inhibit K562 cell growth and induce apoptosis in vitro.展开更多
Objective: To investigate the effects of anti-PML (promyelocytic leukemia) or anti-PML/RAR( (promyelocytic leukemia/retionic acid receptor() antisense oligonucleotides on cell growth, expression of PML-RAR( mRNA and P...Objective: To investigate the effects of anti-PML (promyelocytic leukemia) or anti-PML/RAR( (promyelocytic leukemia/retionic acid receptor() antisense oligonucleotides on cell growth, expression of PML-RAR( mRNA and PML-RAR(/PML protein location of NB4 cell lines. Methods: RT-PCR was used for detecting PML-RAR( mRNA expression, trypan blue exclusion for cell count, methylcellose assay for leukemic colony forming unit detection, immuno- fluorescence for PML-RAR(/PML protein location. Results: Both anti-PML start codon region antisence (STAS) and anti-PML-RAR( fusion region antisence (FUAS) could inhibit cell growth and the formation of acute myelocytic colony forming unit of cells(AML-CFU). Cells become partial differentiated at days 5, being more obvious in FUAS-treated cells than in STAS ones. Down regulation of PML-RAR( mRNA expression occurred at 24 hours in STAS and FUAS-treated cells and maintained for up to 72 hours. Immuno-fluorescence analysis with anti-PML monoclonal antibody showed a remarkable decrease even complete disappearance of microgranules. The residual granules became enlarged as discrete dots (<10 per cell), similar to normal POD structure in some STAS-treated cells at 24 hours. At 72 hours, nearly all the granules disappeared. Similar changes were observed in FUAS-treated cells. Conclusion: Both PML and PML-RAR( antisence oligonucleotides can specially block the expression of PML-RAR( at mRNA and protein levels. PML protein is implicated in the regulations of cell differentiation.展开更多
Objective: To investigate retroviral-mediated transfer and expression of human multidrug resistance (MDR) gene MDRI in leukemic cells. Methods: Human myeloid cells, K562 and NB4, were infected by MDR retrovirus from t...Objective: To investigate retroviral-mediated transfer and expression of human multidrug resistance (MDR) gene MDRI in leukemic cells. Methods: Human myeloid cells, K562 and NB4, were infected by MDR retrovirus from the producer PA317/HaMDR, and the resistant cells were selected with cytotoxic drug. The transfer and expression of MDRI gene was analyzed by using polymerasc chain reaction (PeR), now cytometry (FCM) and scmisolid colonies cultivation. Results: The resistant cells, K562/MDR and NB4/MDR, in which integration of the exogenous MDRI gene was confirmed by PCR analysis, displayed a typical MDR phenotype. The expression of MDR1 transgenc was detected on truncated as well as full-length transcripts. Moreover, the resistant cells were P-glycoprotein positive at 78.0% to 98.7% analyzed with FCM. The transduction efficieny in K562 cells was studied on suspension cultures and single-cell colonies. The transduction was more efficient in coculture system (67.9%-72.5%) than in supernatant system (33.1%-46.8%), while growth factors may improve the efficiency. Conclusion: Retrovirus could allow a functional transfer and expression of MDR1 gene in human leukemia cells, and MDRI might act as a dominant selcctable gene for coexpression with the genes of interest in gene therapy.展开更多
Objective To explore the effect and mechanism of diallyl trisulfide on the activity of NADPH oxidase in Hl-60 cells.Methods HL-60 cells were treated with DATS at a indicated concentration for 0,1,3,6,12 hours,respecti...Objective To explore the effect and mechanism of diallyl trisulfide on the activity of NADPH oxidase in Hl-60 cells.Methods HL-60 cells were treated with DATS at a indicated concentration for 0,1,3,6,12 hours,respectively.The activity of NADPH oxidase was measured by the reduction of the yellow dye nitroblue tetrazolium(NBT).The mRNA expression of NADPH oxidase subunits,including gp91phox,p47 phox,p22 phox,Rac2 and Rac1,was detected by RT-PCR.The protein expression of p67 phox,gp91 phox and Rac2 was analyzed by Western blot.The cell membrane fractions were prepared according to the instruction of Mem-PER kit from Pierce Corp.Results The results showed that reduction ability of HL-60 cells for NBT markedly increased in a concentration-dependent manner following DATS incubation for 3 and 6 hours(P<0.05).HL-60 cells treated by DATS at a concentration of 150 μM for 3 hours have a maximal reduction effect for NBT.The results from RT-PCR indicated that mRNA expression of NADPH oxidase subunits,including p47phox,gp91 phox,p22 phox,Rac2 and Rac1,significantly increased in a concentration-dependent manner in HL-60 cells treated by DATS.The results from western blot showed that HL-60 cells following DATS incubation have a higher level expression of Rac2 and gp91phox,compared with untreated-HL-60 cells.Our results also indicated that a maximal expression level of p47phox,gp91 phox,p22 phox,Rac2 and Rac1in HL-60 cells is present at 3 hours following DATS incubation.We found that levels of both Rac2 and p67phox was reduced in the cytosolic fraction and meanwhile increased in the membrane fraction following HL-60 exposed to DATS,Which is dependent on the concentration and time of DATS treatment.Furthermore,the level of both Rac2 and p67phox located to the plasma membrane translocation was maximized following 150 μM of DATS incubation for 3 hours.Conclusions DATS induce the activation of NADPH oxidase by both up-regulating the expression of NADPH oxidase subunit and translocating the cytosolic Rac2 and p67 phox subunit to the plasma membrane in HL-60 cells.展开更多
文摘Antitumor-drug-indnced apoptosis in leukemia cell lines HL-60 and U937 was quantitatively analyzed with TDT (terminal deoxynuleotidyl transferase-mediated nick-end labeling) approach, which allows to labal the ends of DNA broken strands in apoptotic cells by biotinlated dUTP which to avidin-FITC. In this way the apoptotic cells show nuorescent when the labeling cells were emitted by UV light microscope or laser-activated now cell sorting at r=480. In our study, HL-6o and U937 cell lines were cocultured respectively with cisdiaminodicaicroplatinum (CDDP), hydroxycamptothecinum (HCT) and vindesini sulfa (VCR) for 18 hours.By calculating percentages of apoptotic cells with TDT mothod, we were able to show that the two cell lines gave different sensitivity to the drugs. HL-60 showed high sensitivity to CDDP but U937 cells were more sensitive to other two drugs, HCT and VCR. Meanwhile we compared the results of obtained by DNA gel electrophoresis with that by TDT. We found that gel electrophoresis is not sensitive enough to reveal apoptosis since there was no ladder structure, a typical electrophoresis pattern for apoptosis, appeared until the apoptotic cells reached or over 13%. And we report in this paper as first time that three forms of apoptotic cells could be detected under fluorescent microscope, which we called as spot form and crescent form and assembling form in terms of distribution of light spots within cell nuclei. It seemed that the spot form was at an early stage of apoptosis and the crescent form rcprtscntcd a later stage of apoptosis.
基金Shanghai Municipal Health Bureau:Traditional Chinese Medicine in Treating with Advanced Hepatocellular Carcinoma(No.ZYSNXD-CC-ZDYJ032)
文摘OBJECTIVE: To probe insights into the reversal effect of bufalin on vincristine-acquired multidrug resistance(MDR) in human leukemia cell line K562/VCR.METHODS: Proliferative inhibition rate and the reversal index(RI) of bufalin were determined by Methyl thiazolyl tetrazolium assay. The uptake of Adriamycin(ADM) in K562/VCR cells, cell cycle and apoptosis rate were determined by flow cytometry(FCM). Cell morphologic changes were observed with Wright-Giemsa staining. The expression of P-glycoprotein(P-gp), multidrug-associated protein-1(MRP1), Bcl-x L and Bax protein were measured by immunocytochemistry.RESULTS: The human leukemia multidrug resistant K562/VCR cells showed no cross-resistance to bufalin. The RIs of bufalin at concentrations of 0.0002,0.001 and 0.005 μmol/L were 4.85, 6.94 and 14.77,respectively. Preincubation of 0.001 μmol/L bufalin for 2 h could increase intracellular ADM fluorescence intensity to 28.07%(P<0.05) and down-regulate MRP1 expression simultaneously, but no remarkable effect was found on P-gp protein. Cell cycle analysis indicated increased apoptosis rate and apparent decreased G2/M phase proportion after treatment with bufalin. When exposed to 0.01μmol/L bufalin, typical morphological changes of apoptosis could be observed. Down-regulation of Bcl-x L and up-regulation of Bax expression in K562/VCR cells could be detected by immunocytochemistry.CONCLUSION: Bufalin could partly reverse the MDR of K562/VCR cells, with a possible mechanism of down-regulating MRP1 expression and activating apoptosis pathway by altering Bcl-x L/Bax ratio.
文摘Using Southern blot, Northern blot and Quick blot methods, we examined the rearrangement and expression of TCR βgene in four early differentiation stage cell lines from human hemopoietic system, namely HL-60, Jurkat, Daudi and Raji cells as well as lymphocytes from 17 acute lymphocytic leukemia （ALL） patients. The results showed. Ⅰ) Rearrangement of TCR βgene was seen in Jurkat cells. A germline pattern was observed in HL-60, Daudi and Raji cells. 2) Eight of 9 patients with T-ALL had cells with rearranged TCR βgene. But two of 3 patients with B-ALL and three of 5 patients with nonT, nonB-ALL also had cells with rearranged TCR βgene. 3) A 1.3 kb full-length transcript and a 1.0 kb truncated transcript were detected in Jurkat cells by probing with <sup>32</sup>P-TCR βcDNA. But some leukemic B cells also expressed an incompleted transcript. 4) TCR βmRNA was detected in six of 8 patients with T-ALL, four of 5 patients with nonT, nonB-ALL and one of 3 patients with B-ALL. But the level of expression was quite differ ent. The dual-rearrangement and the abnormal expression may give us a new clue for researching leukemogenesis.
文摘Since 1960, the tumor strains of L6565 viral leukemia, SRS lymphoma and L783 transplantable leukemia were established successively in our laboratory. Recently, derived from the strains of threse leukemia/lymphoma, SRS-82 cell line, SACIIB2, SACIIC3 cell clones and a cell line infected with SRS leukemia virus (SRSV/3T3) were obtained at vitro. The main results of study on the biology, virology and Its Induction of differentiation are summarily reported.
文摘Objective: To explore the effects of Taxol in inhibiting human leukemia k562 cell proliferation and inducing apoptosis in vitro. Methods: Human leukemia K562 cells were treated with Taxol of different concentrations for 12-72 hrs. Cell proliferation was evaluated by MTT assay and morphological changes of apoptosis were examined by microscopy. Cell apoptosis was determined by flow cytometry (FCM) and DNA gel electrophoresis. Results: Growth of K562 cells was inhibited by Taxol with an IC50 value of 0.84 μg/mi.Typical nuclear condensation and apoptosis bodies were observed as early as 24 hrs after a 0.5 μg/ml Taxol treatment; Apoptotic rate of the Taxol-treated K562 cells increased from 3.7% to 24.0% in 24 hrs. No DNA ladder was observed by DNA gel electrophoresis. Conclusion: Taxol could inhibit K562 cell growth and induce apoptosis in vitro.
基金the National Natural Science Foundation of China(No. 39590291).
文摘Objective: To investigate the effects of anti-PML (promyelocytic leukemia) or anti-PML/RAR( (promyelocytic leukemia/retionic acid receptor() antisense oligonucleotides on cell growth, expression of PML-RAR( mRNA and PML-RAR(/PML protein location of NB4 cell lines. Methods: RT-PCR was used for detecting PML-RAR( mRNA expression, trypan blue exclusion for cell count, methylcellose assay for leukemic colony forming unit detection, immuno- fluorescence for PML-RAR(/PML protein location. Results: Both anti-PML start codon region antisence (STAS) and anti-PML-RAR( fusion region antisence (FUAS) could inhibit cell growth and the formation of acute myelocytic colony forming unit of cells(AML-CFU). Cells become partial differentiated at days 5, being more obvious in FUAS-treated cells than in STAS ones. Down regulation of PML-RAR( mRNA expression occurred at 24 hours in STAS and FUAS-treated cells and maintained for up to 72 hours. Immuno-fluorescence analysis with anti-PML monoclonal antibody showed a remarkable decrease even complete disappearance of microgranules. The residual granules became enlarged as discrete dots (<10 per cell), similar to normal POD structure in some STAS-treated cells at 24 hours. At 72 hours, nearly all the granules disappeared. Similar changes were observed in FUAS-treated cells. Conclusion: Both PML and PML-RAR( antisence oligonucleotides can specially block the expression of PML-RAR( at mRNA and protein levels. PML protein is implicated in the regulations of cell differentiation.
基金a grant from the Public Health Bureau of Jiangsu Province (H9549).
文摘Objective: To investigate retroviral-mediated transfer and expression of human multidrug resistance (MDR) gene MDRI in leukemic cells. Methods: Human myeloid cells, K562 and NB4, were infected by MDR retrovirus from the producer PA317/HaMDR, and the resistant cells were selected with cytotoxic drug. The transfer and expression of MDRI gene was analyzed by using polymerasc chain reaction (PeR), now cytometry (FCM) and scmisolid colonies cultivation. Results: The resistant cells, K562/MDR and NB4/MDR, in which integration of the exogenous MDRI gene was confirmed by PCR analysis, displayed a typical MDR phenotype. The expression of MDR1 transgenc was detected on truncated as well as full-length transcripts. Moreover, the resistant cells were P-glycoprotein positive at 78.0% to 98.7% analyzed with FCM. The transduction efficieny in K562 cells was studied on suspension cultures and single-cell colonies. The transduction was more efficient in coculture system (67.9%-72.5%) than in supernatant system (33.1%-46.8%), while growth factors may improve the efficiency. Conclusion: Retrovirus could allow a functional transfer and expression of MDR1 gene in human leukemia cells, and MDRI might act as a dominant selcctable gene for coexpression with the genes of interest in gene therapy.
文摘Objective To explore the effect and mechanism of diallyl trisulfide on the activity of NADPH oxidase in Hl-60 cells.Methods HL-60 cells were treated with DATS at a indicated concentration for 0,1,3,6,12 hours,respectively.The activity of NADPH oxidase was measured by the reduction of the yellow dye nitroblue tetrazolium(NBT).The mRNA expression of NADPH oxidase subunits,including gp91phox,p47 phox,p22 phox,Rac2 and Rac1,was detected by RT-PCR.The protein expression of p67 phox,gp91 phox and Rac2 was analyzed by Western blot.The cell membrane fractions were prepared according to the instruction of Mem-PER kit from Pierce Corp.Results The results showed that reduction ability of HL-60 cells for NBT markedly increased in a concentration-dependent manner following DATS incubation for 3 and 6 hours(P<0.05).HL-60 cells treated by DATS at a concentration of 150 μM for 3 hours have a maximal reduction effect for NBT.The results from RT-PCR indicated that mRNA expression of NADPH oxidase subunits,including p47phox,gp91 phox,p22 phox,Rac2 and Rac1,significantly increased in a concentration-dependent manner in HL-60 cells treated by DATS.The results from western blot showed that HL-60 cells following DATS incubation have a higher level expression of Rac2 and gp91phox,compared with untreated-HL-60 cells.Our results also indicated that a maximal expression level of p47phox,gp91 phox,p22 phox,Rac2 and Rac1in HL-60 cells is present at 3 hours following DATS incubation.We found that levels of both Rac2 and p67phox was reduced in the cytosolic fraction and meanwhile increased in the membrane fraction following HL-60 exposed to DATS,Which is dependent on the concentration and time of DATS treatment.Furthermore,the level of both Rac2 and p67phox located to the plasma membrane translocation was maximized following 150 μM of DATS incubation for 3 hours.Conclusions DATS induce the activation of NADPH oxidase by both up-regulating the expression of NADPH oxidase subunit and translocating the cytosolic Rac2 and p67 phox subunit to the plasma membrane in HL-60 cells.
