Apoptosis Induced by Bcl-2 Antisense Peptide Acid in HL60 Cells

Objective To study the differences and similarities of the antisense drugs with different structures on the biological functions of HL60 cells. Methods Cytotoxic effects were measured by cell viability assay. The expression levels of protein were assayed by immunofluorescence using fluoresce isothiocyanate label. The morphological changes in apoptotic cells were observed. Flow cytometric analysis of DNA fragmentation was also performed. Results Antisense peptide nucleic acid (PNA) targeting the coding region of the Bcl-2 mRNA could effectively inhibit the growth of HL60 cells, down-regulate the synthesis of Bcl-2 protein and induce apoptosis. After HL60 cells were treated with 10 μmol/L Bcl-2 antisense PNA or antisense oligonucleotide for 72 h respectively, apoptotic rates of HL60 cells were 17.80±1.53 and 13.17±1.12, respectively( P <0.05). Conclusion Antisense PNA targeting the coding region of Bcl-2 mRNA may have stronger antisense effects than the antisense oligonucleotides and could induce apoptosis of HL60 cells.

Pyrrolidine Dithiocarbamate (PDTC) Attenuates Luteolin-Induced Apoptosis in Human Leukemia HL-60 Cells

Studies have indicated that flavonoid luteolin is a potential inhibitor of tumor cell proliferation and may function as an anticarcinogenic agent. Pyrrolidine dithiocarbamate (PDTC), a synthetic compound, may exhibit biphasic effects on apoptosis depending on the experimental context. Previously, we found that luteolin induced the activation of the proapoptotic proteins, such as Bad, Bid, and Bax, in HL-60 human leukemia cells. We also explored the modulatory effects and molecular mechanisms of PDTC on the cytotoxicity of luteolin in HL-60 cells;PDTC could interfere with luteolin’s ability to cleave poly(ADP-ribose)-polymerase (PARP) and DNA fragmentation of factor-45 (DFF-45). In the current study, we further investigated the effect of PDTC on the luteolin-induced death-receptor pathway and the cleavage of the Bcl-2 family members. We found that the combination of luteolin and PDTC increased the survival of the HL-60 cells such that PDTC inhibited both extrinsic and intrinsic pathways in luteolin-induced apoptosis.

Regulation of Histone Acetylation and Apoptosis by Trichostatin in HL-60 Cells

Summary: In order to examine the strong anticancer action and low toxicity of Trichostatin A (TSA), the effect of TSA was examined on the growth inhibition, acetylation of histone H_3 and apoptosis in HL-60 cells by employing MTT, immunocytochemical techniques, and Annexin-V-FITC/PI assay. Our results showed that TSA could inhibit proliferation of HL-60 cells in a time-and dose-dependent manner, and the IC_~50 at the 36th h was 100 ng/ml. The apoptosis-inducing effect of TSA on HL-60 cells was also time-and dose-dependent. But it didn't demonstrate apparent apoptosis induction in NPBMNCs within specific dose and time range. Both of the acetylation of histone H_3 in HL-60 cells and NPBMNCs increased significantly (P<0.05) after treated with 100 ng/ml TSA for 4 h. However, there was no significant differences between the two groups (P>0.05). It is concluded that TSA can inhibit growth and induce apoptosis of HL-60 cells in a time-and dose-dependent manner, and is able to selectively induce apoptosis in HL-60 cells but does not respond in NPBMNCs under the same conditions. The difference of TSA between HL-60 cells and NPBMNCs can't be explained by the regulation of histone acetylation.

Effect of Concurrent Use of rh-IL-3 and rh-GM-CSFon Apoptosis of HL-60 Cells Induced by Ara-C

The myeloid leukemic cell line HL-60 was studied by using DNA gel electrophoresis, flow cytomery, McAb C-myc, McAb Bc1-2 and CFU-L. From zero to 36 h,the apoptosis rates of 8 different phases and other indexes were observed. The results showed that with the prolonged time of drug incubation,apoptosis of HL-60 cells increased progressively. This effect can be enhanced obviously by rh-IL-3 and rh-GM-CSF. At the same time,the killed rate of leukemic cells by Ara-C induction was increased. C-myc expression was decreased and Bc1-2 expression did not display apparent change. Interestingly, the normal hemopoietic cells were not affected by these two kinds of cytokine. The theoretical basis was provided for concurrent use of rh-IL-3, rh-GM-CSF and cytotoxic drugs whose purpose is to elevate remission rate during the phase of induced remission of leukemia.

Effect of N’-Acetylindirubin on Proliferation, Apoptosis and Cell Cycle in Acute Myeloid Leukemia HL-60 Cells

Acute promyelocytic leukemia (APL) is a severe type of acute leukemia and the prognosis of patients was poor. Indirubin is the active constituent of the traditional Chinese medicine qingdai and an indoline anti-tumor drug. N’-Acetylindirubin is a novel indirubin derivative with better curative effect and less side effect. In this study, the effects of N’-Acetylindirubin on proliferation, apoptosis and cell cycle of acute myeloid leukemia cell line HL-60 was examined. The results demonstrated that N’-Acetylindirubin significantly induced apoptotic cell death in a dose and time-dependent manner and arrested cell cycle in G2/M in HL-60 cells. N’-Acetylindirubin also suppressed cyclin D1. This study suggests that N’-Acetylindirubin may serves as a potential chemopreventive agent for acute promyelocytic leukemia.

CLONING AND EXPRESSION OF A GENE ASS0CIATE WITH HL_(60)CELL APOPTOSIS INDUCED BY INHIBITIONOF POLYAMINE BIOSYNTHESIS

Objective: To clone the gene associated with apoptosis induced by an inhibitor of polyamine biosynthesis, a-difluoromethylornithime(DFMO). Methods: The differential sufbtraction sereening was used for gene cloning from cDNA library of HL60 cells treated by DFMO. Northern blot,morphological observation, FCM assays and ladder map of DNA electrophoresis were performed. Results: The transfectiong gene expression and activity of inducing apoptosis in the cell transfected from recombinant plasmid containing the cloned fragment df4 wasproved. Conclusion: It is suggest that df4 gene cloned in the study coul be a gene regulating apoptosis of HL60 cells.

INHIBITION OF NF-κB ACTIVITY ENHANCED CYTOSINE ARABINOSIDE INDUCED APOPTOSIS IN LEUKEMIC CELL LINE HL60-N

Objective: To explore the effects of dexamethasone (DXM) and vincristine (VCR) on cytosine arabinoside (Ara-C) induced apoptosis and activation of nuclear factor-k-gene binding (NF-kB) in leukemic cell line HL60-n. Methods: Apoptosis of HL60-n cells was analysed by TdT-mediated X-dUTP nick and end labeling (TUNEL) and DNA electrophoresis. NF-kB activity of HL60-n cells was detected by electrophoretic mobility shift assay (EMSA). Results: There was slight activation of NF-kB in HL60-n cells without drug induction. Ara-C at 1 mmol/L significantly enhanced the activation of NF-kB in HL60-n cells. The level of NF-kB activation induced by DXM at 1 mmol/L or VCR at 0.1 mmol/L had no significant difference compared with that of the control group. However, in HL60-n cells pre-treated with 1 mmol/L of DXM or 0.1 mmol/L of VCR, the activation of NF-kB induced by 1 mmol/L of Ara-C was significantly suppressed with inhibition rates of 31.0% and 47.0%, respectively. The apoptosis rates of HL60-n cells induced by 1.0 mmol/L, 10 mmol/L and 100 mmot/L Ara-C were 45.003.16%, 61.883.40% and 77.624.75%, respectively. The apoptotic rates of HL60-n cells induced by DXM at 1 mmol/L or VCR at 0.1 mmol/L were similar to that of the control group. However, either DXM at 1 mmol/L or VCR at 0.l mmol/L could enhance the apoptosis of HL60-n cells induced by Ara-C at 1 mmol/L with rates of 39.1% and 59.2%, respectively. Conclusion: Ara-C can induce apoptosis and activation of NF-kB in HL60-n cells. The mechanism of increased apoptosis of HL60-n cells by DXM or VCR may be related to suppression of NF-kB activation.

Generation of bcl-2 antisense RNA promotes apoptosis of human promyelocytic leukemia cell line HL-60

Objective: To analyze the effect of bcl-2 antisense RNA on the apoptosis of promyelocytic cell line HL-60. Methods: A plasmid pDOR-AB containing bcl-2 antisense cDNA was trans fected into HL-6O cells by lipofectin, and the effect of transfection was assured by DNA and RNA dot blottingI the change of bcl-2 expression and cell cycle was tested by flow cytometry; a morphologica1 change was observed by light microscope and electron microscope; and finally the sensitivity of trans fected cells to etoposide was compared with that of non-trans fected cells by gel electrophoresis. Results: pDOR-AB was successfully trans fected into HL-6o cells and its transcript was observed; Bcl-2 was down-regulated significantly ; apoptosis peak appeared before G1 phase in flow cy-tometry analysis: apoptotic cells could be seen by electron microscope, and during DNA gel electrophoresis the DNA ladder apppeared more frequently in the group trans fected with pDOR-AB than in transfected with pDOR and untransfected groups. Conclusion: Transient expression of bcl-2 antisense RNA can promote apoptosis of HL-60 cells and bco-2 plays a key role in the apoptosis of HL-60 cells.

Apoptosis Induced by N-Nitrosamines in Two Cancer Cell Lines

In the present study, we investigated the induction of apoptosis by N-nitrosopyrrolidine (NPYR) and N-nitrosodimethy-lamine (NDMA) in two human cell lines: HL-60 (leukemia) and HepG2 (hepatoma). Apoptotic cells were identified by: 1) chromatin condensation, 2) flow cytometry analysis and 3) poly (ADP-ribose) polymerase cleavage. Both cell lines exhibited morphological changes consistent with apoptotic events following treatment with N-nitrosamines. Flow cytometry analysis showed that both N-nitrosamines induced apoptotic cell death in a concentration and time dependent- manner. NPYR was stronger than NDMA, since it induced a significant apoptotic cell death after 72 h starting from a concentration of 10 mM, whereas NDMA was effective at 27 mM. Furthermore, NPYR and NDMA caused the cleavage of PARP in HL-60 cells whereas no PARP cleavage was detected in HepG2 cells. However, NPYR- and NDMA-induced cell death in HepG2 cells was prevented by specific caspase inhibitors. Caspase-8 mediated main pathway and was responsible for 76% (NPYR) and 64% (NDMA) inhibition of apoptosis. The data demonstrate that NPYR and NDMA induce apoptosis in HL-60 and HepG2 cell lines via caspase-dependent pathway.

Securinine induced apoptosis in human leukemia HL-60cells

目的：研究一叶秋碱能否诱导ＨＬ６０细胞凋亡．方法：用ＭＴＴ法检测一叶秋碱对细胞增殖影响；应用流式细胞仪检测凋亡细胞数；采用琼脂糖凝胶电泳法观测ＤＮＡ碎片；透射电镜观察凋亡的形态改变．结果：一叶秋碱５－８０ｍｇ·Ｌ－１能诱导ＨＬ６０细胞凋亡．电镜观察到典型的凋亡形态学改变，电泳呈现出阶梯状条带，流式细胞仪检测到凋亡率随剂量的增高而升高．ＭＴＴ法示一叶秋碱抑制ＨＬ６０细胞增殖，并且呈时间、剂量依赖性，药物作用１２ｈ的ＩＣ５０（９５％可信区间）分别为２７（１５－４７）ｍｇ·Ｌ－１．

Induction of apoptosis in HL-60 cells by harringtonine

Recently, many reports showed that most of cytotoxic anticancer drugs in current usesuch as DNA topoisomerase Ⅰinhibitor camptothecin (CAM), or DNA topoisomerase Ⅱinhibitor teniposide (TEN) could induee apoptosis in susceptible cells. Apoptosis (Apo),which is different from necrosis (Nec), is a specific mode of cell death recognized by thecharacteristic pattern of condensation of chromatin, integrity of plasma membrane and

Inhibition of NF-κB by mutant IκBα enhances TNF-α-induced apoptosis in HL-60 cells by controlling bcl-x_L expression

Backgound The aim of this study was to explore whether the inhibition of nuclear factor-κB (NF-κB) activation by mutant IκBα (S32,36→A) can enhance TNF-α-induced apoptosis of leukemia cells and to investigate the possible mechanism.Methods The mutant IκBα gene was transfected into HL-60 cells by liposome-mediated techniques. G418 resistant clones stably expressing mutant IκBα were obtained by the limiting dilution method. TNF-α-induced NF-κB activation was measured by electrophoretic mobility shift assay (EMSA). The expression of bcl-x L was detected by RT-PCR and Western blot after 4 hours exposure of parental HL-60 and transfected HL-60 cells to a variety of concentrations of TNF-α. The percentage of apoptotic leukemia cells was evaluated by flow cytometry (FCM). Results Mutant IκBα protein was confirmed to exist by Western blot. The results of EMSA showed that NF-κB activation by TNF-α in HL-60 cells was induced in a dose-dependent manner, but was almost completely inhibited by mutant IκBα repressor in transfected cells. The levels of bcl-x L mRNA and protein in HL-60 cells increased after exposure to TNF-α, but changed very little in transfected HL-60 cells. The inhibition of NF-κB activation by mutant IκBα enhanced TNF-α-induced apoptosis. The cytotoxic effects of TNF-α were amplified in a time- and dose-dependent manner.Conclusions NF-κB activation plays an important role in the resistance to TNF-α-induced apoptosis. The inhibition of NF-κB by mutant IκBα could provide a new approach that may enhance the anti-leukemia effects of TNF-α or even of other cytotoxic agents.

Effect of adenovirus-mediated p27 gene expression on the proliferation and apoptosis of HL-60 and Raji cell lines

Background p27 is an essential mediator of cell cycle control,which plays a key negative role in the proliferation and tumorigenesis of certain cell types. Here, we designed this study to explore the possible effects of p27 on the proliferation and apoptosis of HL-60 and Raji cell lines.Methods HL-60 and Raji cells were transfected with p27 via an adenovirus-mediated approach. The efficiency of Adp27 infection and the expression of p27 mRNA and protein were evaluated by X-gal staining, RT-PCR, and flow cytometry. The proliferation and apoptosis of HL-60 and Raji cells were estimated by means of trypan blue staining, MTT assay, Annexin V/PI, and DNA ladder electrophoresis. Results The infection efficiencies in HL-60 and Raji cells were 40.3% and 32.0%, respectively. RT-PCR and flow cytometry showed that there was significant expression of p27 mRNA and protein in HL-60 and Raji cells infected with Adp27; on the other hand, uninfected HL-60 cells showed faint traces of p27 mRNA and protein and Raji cells showed nearly no signs of p27 mRNA and protein. As demonstrated by a cell growth curve and by an MTT assay, strong time-dependent proliferation inhibition was apparent in HL-60 and Raji cells infected by Adp27. After 72 hours of infection, the Annexin V+/PI- apoptotic cell rates in HL-60 and Raji cell lines were 46.9% and 35.7%, respectively, significantly higher than in the control groups (4.7% and 5.6%, respectively). Typical DNA ladder bands were detectable in HL-60 and Raji cells after 48 hours of Adp27 infection. Conclusions Adenoviral vector-mediated p27 gene transfection of HL-60 and Raji cells leads to the inhibition of cellular proliferation and the promotion of cell apoptosis. This technique may provide an approach to gene therapy for leukemia or lymphoma.

Inhibition of haringtonine-induced apoptosis by tetradecanoylphorbol acetate in human leukemia HL-60

目的：研究十四酰佛波乙酸酯（ＴＡ）预处理后，三尖杉酯碱（Ｈａｒ）和喜树碱（Ｃａｍ）诱导人白血病ＨＬ６０细胞凋亡的变化．方法：染色质凝集观察，流式细胞术，ＤＮＡ琼脂糖凝胶电泳和点杂交．结果：ＴＡ２００ｎｍｏｌ·Ｌ－１预处理ＨＬ６０细胞６ｈ，明显抑制Ｈａｒ０１ｍｇ·Ｌ－１作用３ｈ诱导的细胞凋亡，但只部分抑制Ｃａｍ０２ｍｇ·Ｌ－１作用３ｈ诱导的细胞凋亡．ＴＡ预处理ＨＬ６０细胞明显降低ｃｍｙｃ基因的表达．结论：ＴＡ明显抑制由Ｈａｒ和部分抑制由Ｃａｍ诱导的ＨＬ６０细胞凋亡，而且ＴＡ预处理的细胞中ｃｍｙｃ基因表达明显下降，这可能导致了对诱导细胞凋亡的抑制．

ARSENIC TRIOXIDE DOWNREGULATES TELOMERASE ACTIVITY IN HL-60 CELLS

Objective: To evaluate whether arsenic trioxide (AS2O3) could downregulate human telomerase reverse transcriptase (hTERT) gene expression and telomerase activity during induction of apoptosis of HL-60 cells. Methods: Apoptosis was detected by morphological observation and flow cytomertric cell cycle analysis. The expression of hTERT at mRNA and protein levels was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) and immunofluorescence using fluoresce isothiocyanate (FITC) label, respectively. Telomerase activity was determined by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). Results: Treatment of 2 μmol/L at As2O3 could induce apoptosis of HL-60 cells. hTERT was decreased at both mRNA and protein levels during apoptosis of HL-60 cells. Telomerase activity of HL-60 cells was significantly inhibited. Conclusion: It is suggested that telomerase activity of HL-60 cells might be specifically inhibited by AS2O3 through the downregulation of hTERT gene expression.

EFFECTS OF ANTISENSE OLIGODEOXYNUCLEOTIDES ON EXPRESSION OF CASPASE-3 IN Γ-RADIATION INDUCED APOPTOTIC HL-60 CELLS

Objective To study the inhibitory effects of caspase-3 mRNA antisense oligodeoxynucleotides (ASODN) on expressions of caspase-3 and it's mRNA in γ-radiation induced apoptotic HL-60 cells, and screen the effective ASODN. Methods ASODN-1 and ASODN-2 targeting 5′-noncoding region and initial translation region of caspase-3 mRNA were respectively designed, synthesized and introduced into HL-60 cells by means of liposome-mediated transfection followed by 10Gy γ-radiation exposures. TUNEL assay was conducted to investigate the morphologic change and apoptotic percentage of HL-60 cells 18 h later. Immunocytochemical staining and one step RT-PCR were respectively performed to detect the expressions of caspase-3 and it's mRNA. Mismatched oligodeoxynucleotide (MODN) transfected and un-transfected HL-60 cells were taken as control. Results TUNEL assay found that the apoptotic percentages in ASODN-1 and ASODN-2 groups were significantly reduced compared with the control groups (P<0.01) when the final concentration of both ASODNs was ≥3μmol/L. Immunocytochemistry showed that caspase-3 positive cell percentages were reduced but the average gray values increased significantly compared with the control groups (P<0.01). RT-PCR showed expressions of caspase-3 mRNA was decreased after ASODN transfection. Furthermore, ASODN-1 proved more effective in inhibiting HL-60 cell apoptosis than ASODN-2 (P<0.01). Conclusion Caspase-3 mRNA ASODNs can prevent HL-60 cells from apoptosis induced by γ-radiation and reduce expression of caspase-3 and its mRNA. These effects are dose dependent in a certain range.

雷公藤红素下调HL-60细胞P-Akt与Cyclin D1蛋白表达及其诱导细胞凋亡的效应

本研究探讨雷公藤红素诱导HL-60细胞凋亡及其可能的作用机制。以不同浓度雷公藤红素(0.25-8.0μmol/L)分别作用于HL-60细胞24-72小时,采用MTT法检测细胞增殖活性;TUNEL荧光染色、流式细胞术观察雷公藤红素对HL-60细胞凋亡及周期的影响;Western blot、RT-PCR法分别检测雷公藤红素对HL-60细胞内Akt(P-Akt)及其下游分子Cyclin D1的蛋白、基因的表达水平。结果表明,雷公藤红素能明显抑制HL-60细胞增殖,具有浓度依赖和时间依赖性。此外,雷公藤红素以浓度依赖性方式诱导HL-60细胞凋亡,并伴随明显的凋亡细胞形态学改变。雷公藤红素的凋亡诱导可能与其诱导HL-60细胞周期阻滞于G0/G1期有关。雷公藤红素对P-Akt及CyclinD1蛋白及基因表达水平均有不同程度的抑制作用,该抑制作用呈明显的量效和时效关系。结论:雷公藤红素明显抑制HL-60细胞的增殖,并诱导其凋亡,其抗白血病效应可能与其下调P-Akt和Cyclin D1蛋白表达有关。

芦笋有效成分熊果酸诱导HL-60细胞凋亡的实验研究

目的：探讨芦笋有效成分熊果酸对ＨＬ６０细胞增殖、凋亡的影响。方法：用ＭＴＴ法、ＤＮＡ凝胶电泳、形态学方法等测定熊果酸对ＨＬ６０细胞增殖、凋亡的影响。结果：熊果酸抑制ＨＬ６０细胞增殖，对ＨＬ６０细胞的半数生长的抑制剂量（ＩＣ５０）为８２６μｍｏｌ／Ｌ，并能诱导其发生凋亡。结论：熊果酸通过抑制ＨＬ６０细胞增殖和诱导其凋亡发挥抗肿瘤作用。

鸦胆子油乳诱导HL-60细胞凋亡的研究

目的 :研究鸦胆子油乳诱导HL 60细胞凋亡的作用。方法 :鸦胆子油乳 1∶1 0 0 ,1∶40 ,1∶2 0 (V/V)分别处理HL 60细胞 6h ,琼脂糖凝胶电泳法 ,流式细胞仪法 ,荧光显微镜法 ,电镜分别观察药物诱导HL 60细胞凋亡的作用。结果 :鸦胆子油乳 1∶40组产生明显DNA梯状条带 ;流式细胞仪检测药物浓度为 1∶40 ,1∶2 0组的凋亡细胞百分率分别为 86 .8%和 97% ;鸦胆子油乳 1∶40组在荧光显微镜呈现明显凋亡细胞特征 ;药物浓度为 1∶40 ,1∶2 0组的细胞经电镜观察呈现明显凋亡细胞特征。结论 :鸦胆子油乳 1∶2 0和 1∶40组有明显的诱导HL

冬凌草甲素诱导HL-60细胞凋亡

目的 研究冬凌草甲素诱导人白血病HL 6 0细胞凋亡的作用。方法 形态学观察 ,DNA凝胶电泳及流式细胞术。结果 冬凌草甲素能显著地诱导HL 6 0细胞发生凋亡 ,其作用呈明显的浓度效应关系和时间依赖性。形态学观察可见凋亡小体的形成 ,琼脂糖凝胶电泳可见明显的DNA梯带 ;流式细胞仪检测到G1亚峰。结论 冬凌草甲素能诱导HL 6 0细胞凋亡 ,并与其细胞杀伤活性相互平行 。