Transfection Efficiency Comparison of Oligonucleotide and Plasmid to the HL-60 Cell Line with Liposomes

The transfection efficiency of oligonucleotide and plasmid to the HL-60 cell line with lipofectaminePLUS was compared through observing the transfection rate and the expression duration of exogenous gene in the target cells. The results showed that the transfection rate of oligonucleotide to the HL-60 was about 90 %—95 % and it had no obvious attenuation within 84 h. However, the plasmid transfection rate was only 5 %—25 % and it was decreased significantly within 60 h. It was suggested that the transfection of oligonucleotide with liposomes was better than that of plasmid.

Apoptotic Sensitivity to Irradiation Increased after Transfection of chk1 Antisense Chain to HL-60 Cell Line

Summary： The HL-60 cells were transfected with chkl antisense and sense chain, and 24 h later subjected to irradiation. Twenty-four h after irradiation, the changes in the chk1 protein expression was assayed by Western blot, and the cell cycles and apoptosis rate detected by FCM. The irradiated apoptosis sensitivity was increased by antisense blocking of chk1 gene in HL-60 cell line with the apoptosis rate being 26.31 %, significantly higher than that by the sense blocking （10.34 %, 0. 025〈P〈0.05）. In HL-60 cells transfected with chkl antisense chain, the G2/M phase arrest was attenua：ted and the cells in G2/M phase were accounted for 38.42 %, significantly lower than those of the cells transfected with chkl sense chain （54.64 %, 0. 005〈P〈0.01）. It was concluded that antisense blocking of chk1 gene could increase the apoptosis sensitivity to irradiation.

Effect of 8-Chloroadenosine on Undifferentiatied HL-60 Cell Line

Aim To study the effect of 8 chloroadenosine (8 CA)on undifferentiatied HL 60 cell line. Methods The IC 50 of cancer cell proliferation was determined using a microculture plate reader at 570 nm (MTT) and 540 nm (SRB). Morphology of HL 60 cells was observed under a scanning electron microscope and a transmission electron microscope. The differentiation of HL 60 cells was examined by nitro blue tetrazolium reduction (NBT) and acid phosphatase assay. The cycle of HL 60 cells was analyzed by flow cytometry. Results 8 CA inhibited proliferation of eight human cancer cell lines. The IC 50 ranked in the following order: KB (0 05 μmol·L -1 ) < HL 60 (0 25 μmol·L -1 ) < Bel 7402 (0 56 μmol·L -1 ) < MCF 7 (0 65 μmol·L -1 ) < HCT (0 79 μmol·L -1 ) < HeLa (0 89 μmol·L -1 ) < BGC 823 (1 149 μmol·L -1 ) < PG (2 50 μmol·L -1 ). The scanning and transmission electron microscopy showed that the microvilli of HL 60 cell surface shortened, and the shape of HL 60 cells nuclei changed to kidney shaped, horse shoe shaped and bilob ated after treatment with 8 CA. Meanwhile, 8 CA promoted NBT reduction and increased activity of acid phosphatase in HL 60 cells in a time and concentration dependent manner. Flow cytometry analysis indicated that 8 CA induced an appreciable increase of the cell population in G 1 phase with a marked reduction in S phase. Conclusion 8 CA can induce differentiation of HL 60 cells and block the cells at G 1 phase, thus inhibiting proliferation of HL 60 cells.

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.

Effect of Antisense Oligodeoxynucleotide Directed to NF-κB-RelA on Bcl-x_L mRNA in Extended Drug Resistance Leukemia Cell Line HL- 60/E6

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 x L 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 x L mRNA levels within 8 h. The inhibition rate of bcl x L 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.

1-Chloromethyl-6,7-dimethoxy-3,4-dihydro-1H-isoquinoline-2-sulfonic acid amide, a derivative of tetrahydroisoquinoline, induces granulocytic differentiation of the human leukemic HL-60 cells via G0/G1 phase arrest

Tetrahydroisoquinolines are known to have various biological effects, including antitumor activity. This study investigated the effect of 1-chloromethyl-6, 7-dimethoxy-3, 4-dihydro-1H-isoquinoline-2-sulfonic acid amide (CDST), a newly synthesized anticancer agent, on cellular differentiation and proliferation in HL-60 cells. Differentiation and proliferation of HL-60 cells were determined through expression of CD11b and CD14 surface antigens using flow cytometry and nitroblue tetrazolium (NBT) assay, and through analysis of cell cycle using propidium iodide staining, western blot analysis and immunoprecipitation, respectively. CDST induced the differentiation of HL-60, as shown by increased expression of differentiation surface antigen CD11b (but no significant change in CD14 expression) and increased NBT-reducing functional activity. DNA flow cytometry analysis indicated that CDST markedly induced a G0/G1 phase arrest of HL-60 cells. Subsequently, we examined the expre-ssion of G0/G1 phase cell cycle-related proteins, including cyclin-dependent kinases (CDKs), cyclins and cyclin dependent kinase inhibitors (CKIs), during the differentiation of HL-60. The levels of CDK2, CDK6, cyclin E and cyclin A were decreased, whereas steady-state levels of CDK4 and cyclin D1 were unaffected. The expression of the p27Kip1 was markedly increased by CDST, but not p21WAF1/Cip1. Moreover, CDST markedly enhanced the binding of p27Kip1 with CDK2 and CDK6, resulting in the reduced activity of both kinases. Taken together, these results demonstrate that CDST is capable of inducing cellular differentiation and growth inhibition through p27Kip1 protein-related G0/G1 phase arrest in HL-60 cells.

Antileukemic activity of jaspolide B,an isomalabaricane-type triterpene from marine sponge Jaspis sp. on human promyeloleukemic HL-60 cells

The antiproliferative activity and underlying mechanisms of jaspolide B, an isomalabaricane-type triterpene, isolated from the sponge Jaspis sp., were investigated using human promyeloleukemic HL-60 cells. Jaspolide B arrested HL-60 cells in the G2/M phase of the cell cycle and induced apoptosis of HL-60 cells in a dose- and time-dependent manner. Moreover, the poly （ADP-ribose） polymerase （PARP） protein was cleaved by jaspolide in a dose- and time-dependent way. Jaspolide B with an ICs0 value of 0.61 μmol/L was found to be comparable efficacy as that of paclitaxel （IC50：0.78 μmol/L）. These results implicate the potential ofjaspolide B as a promising anticancer agent in chemotherapy of leukemia by arresting cell cycle progression at G2/M phase and triggering apoptosis.

Polymethylenebis [acetamides] Analogues.Synthesis and Differentiation-Inducing Activity on HL-60 Cells

报导了一系列多亚甲基双［酰胺］类化合物的合成，由－摩尔多亚甲基二甲酰氯分别和二摩尔２－氨基噻唑啉及５－氨基－１－甲基吡啶酮反应制得。测定了其体外对ＨＬ－６０人早幼粒白血病细胞的分化诱导活性，初步结果表明：Ｎ，Ｎ｀－双吡啶酮基六二甲酰胺和Ｎ，Ｎ｀－双噻唑啉基八亚甲基二甲酰胺分别在０．１ｍｍｏｌ／Ｌ和０．５ｍｍｏｌ／Ｌ浓度时，诱导分化百分率可达６０％。此浓度下细胞存活率分别为２６％及２２％，其有效诱导浓度比ＨＭＢＡ低十倍。

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.

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.

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.

The Influence of Curcumin on the Cell Cycle of HL-60 Cells and Contrast Study

The mechanisms of curcumin inhibiting the proliferation of HL-60 cells were investigated. Acute myeloid leukemic cell line HL-60 was studied by using cell culture, NBT reduction, SABC method measuring BrdU incorporation rate, FCM measuring DNA contents and TUNEL method determining apoptotic cell percentage. Curcumin inhibited proliferation of HL-60 cells in a dose- and time-dependent manner. When the HL-60 cells were treated with 25 μmol/L curcumin for 48 h, the inhibitory rate was 60. 71 % ± 1. 20 %. The study on BrdU incorporation rate and the distribution of DNA content and NBT reduction indicated that curcumin arrested. the cells in G2/M phase of cell cy cle at first, then in G0/G1 phase, the whole cell cycle progression was slowed down and DNA synthesis activities was halted. The arrested cells went to apoptosis instead of differentiation. The comparative study indicated that the ability of curcumin regulating the cell cycle of HL-60 cells was stronger than As2O3 and the acting points of curcumin were not completely consistent with those of VP-16. It was suggested that curcumin was able to regulate, to some extent, the G1/S and G2/M transmit checkpoints and disturb the HL-60 cell cycle to induce apoptosis.

Altered expression of nuclear matrix proteins in etoposide induced apoptosis in HL-60 cells

The events of cell death and the expression of nuclear matrix protein (NMP) have been investigated in a promyelocytic leukemic cell line HL-60 induced with etoposide. By means of TUNEL assay, the nuclei displayed a characteristic morphology change, and the amount of apoptotic cells increased early and reached maximun about 39% after treatment with etoposide for 2 h. Nucleosomal DNA fragmentation was observed after treatment for 4 h. The morphological change of HL-60 cells, thus, occurred earlier than the appearance of DNA ladder. Total nuclear matrix proteins were analyzed by 2-dimensional gel electrophoresis. Differential expression of 59 nuclear matrix proteins was found in 4 h etoposide treated cells. Western blotting was then performed on three nuclear matrix acssociated proteins, PML, HSC70 and NuMA. The expression of the suppressor PML protein and heat shock protein HSC70 were significantly upregulated after etoposide treatment, while NuMA, a nuclear mitotic apparatus protein, was down regulated. These results demonstrate that significant biochemical alterations in nuclear matrix proteins take place during the apoptotic process.

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.

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.

Apoptosis of human leukemic HL-60 cells induced to differentiate by treatment with RA or DMSO

In present study we studied the effect of all-trans retinoic acid(ATRA) and dimethylsulfoxide (DMSO) on the induction of apoptosis in HL-60 cell line. Based on morphological changes by Hochest 33342 staining and identification of internucleosomal DNA cleavage by gel electrophoresis, we observed aberrant nuclear chromatin eondensation and ladder-like pattern of DNA degradation. Using Flow Cytometric method, we found sub-G1 peak in RA-treated HL-60 cells starting 5 to 6 d after the initiation of the treatment. However, such an obvious apoptotic peak was not identified in DMSO-differentiated cells. Combining the research accomplished before, our study approves further that apoptosis could be a common mode of death of terminally differentiated HL-60 cells.

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.

Effects of Concurrent Use of rh-IFN-γ and Curcumin on the Anti-proliferative Capacity of HL-60 Cells

To understand the effect of rh-IFN-γ on the ability of curcumin to kill HL-60 cells in vitro, the myeloid leukemic cell line HL-60 was studied by using cell culture. BrdU incorporation rate was examined by SABC, DNA content was determined by flow cytometry and apoptotic cell percentage was determined by TUNEL method. The results showed that curcumin inhibited proliferation of leukemic cells in a dose-dependent manner. When HL-60 cells were treated with 25 μmol curcumin for 24 h, the proliferative inhibitory rate was 43. 75±2. 00 %.This effect could be enhanced obviously by IFN-γ, the combined proliferative inhibitory rate increased to over 80 %. The 5-BrdU incorportion rate and the distribution of DNA content indicated that curcumin could arrest cells in the G1/G0 and G2/M phase of cell cycle. At the same time, the sub-G1 peak (apoptotic peak) appeared. After IFN-γ combined with curcumin, DNA synthesis rate decreased further. It showed a significant difference when compared with single drug group (Pr< 0. 05). Meanwhile, sub-G1 peak also increased. The percentage of TUNEL positive cells was aslo increased. It is concluded that IFN-γ can enhance the antiproliferative ability of curcumin against HL-60 cells.

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.

EFFECTS OF THE HYPERTHERMIA AND HARRINGTONINE ON TELOMERASE ACTIVITY OF HL-60 CELLS

Objective To understand the mechanism of the hyperthermia and harringtonine in purging of leukemia cells In Vitro. telomerase activity of HL-60 cells treated by hyperthermia ( 42℃ for one hour) and different concentrations of Harringtonine were investigated.Methods Using Telomeric Repeats Amplification Protocol (TRAP) and ELISA techniques to analyze the telomerase activity of HL-60 cells. Results Our results showed that harringtonine inhibited the telomerase activity of HL-60 cells in a dosage related manner. Moreover, the telomerase activity of HL-60 cells was significantly decreased after the hyperthermia treatment as compared with untreated cells.Conclusion The effect of the hyperthermia and Harringtonine on purging leukemia cells In Vitro may be mediated by down regulation of telomerase activity of tumor cells.