Effect of Nardostachyos Rhizoma on Apoptosis, Differentiation and Proliferation in HL-60 cells

Ju Sung-Min,Lee Jun,Choi Ho-Seung,Yoon Sang-Hak,Kim Sung-Hoon,Jeon Byung-Hun The Physiological Society of Korean Medicine and T 2006 동의생리병리학회지 Vol.20 No.1

Nardostachyos Rhizoma (N. Rhizoma) belonging to the family Valerianaceae has been anti-arrhythmic effect, and sedation to the central nerve and a smooth muscle. We reported that the water extract of N. Rhizoma induced apoptotic cell death and differentiation in human promyelocytic leukemia (HL-60) cells. Cytotoxicity of N. Rhizoma was detected only in HL-60 cells (IC50 is about 200 ${\mu}g/ml$). The cytotoxic activity of N. Rhizoma in HL-60 cells was increased in a dose-dependent manner. We used several measures of apoptosis to determine whether these processes were involved in N. Rhizoma-induced apoptotic cell death. The high-dose (200 ${\mu}g/ml$) treatment of N. Rhizoma to HL-60 cells showed cell shrinkage, cell membrane blobbing, apoptotic bodies, and the fragmentation of DNA, suggesting that these cells underwent apoptosis. Treatment of HL-60 cells with N. Rhizoma time-dependently induced activation of caspase-3, caspase-8, and caspase-9 and proteolytic cleavage of poly(ADP-ribose) polymerase. Also, we investigated the effect of N. Rhizoma on cellular differentiation and proliferation in HL-60 cells. Differentiation and proliferation of HL-60 cells was 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 assay, respectively. N. Rhizoma induced the differentiation of HL-60 at the low-dose (100 ${\mu}g/ml$) treatment, as shown by increased expression of differentiation surface antigen CD11b, but not CDl4 and increased reducing activity of NBT. When HL-60 cells were treated with N. Rhizoma at concentration of $50{\mu}g/ml\;and\;100{\mu}g/ml$, NBT-reducing activities induced approximately 1.5-fold and 20.0-fold as compared with the control. In contrast, HL-60 cells treated with the N. Rhizoma-ATRA combination showed markedly elevated levels of 26.3-fold at $50{\mu}g/ml$ N. Rhizoma-0.1 ${\mu}M$ ATRA combination and 27.5-fold at 50 ${\mu}g/ml$ N. Rhizoma-0.2 ${\mu}M$ ATRA combination than when treated with N. Rhizoma alone or ATRA alone. It may be that N. Rhizoma plays important roles in synergy with ATRA during differentiation of HL-60 cells. DNA flow-cytometry indicated that N. Rhizoma markedly induced a G1 phase arrest of HL-60 cells. N. Rhizoma-treated HL-60 cells increased the cell population in G1 phase from 32.71% to 42.26%, whereas cell population in G2/M and S phases decreased from 23.61% to 10.33% and from 37.78% to 33.98%, respectively. We examined the change in the $p21^{WAF1/Cip1}\;and\;p27^{Kip1}$ proteins, which are the CKIs related with the G1 phase arrest. The expression of the CDK inhibitor $p27^{Kip1},\;but\;not\;p21^{WAF1/Cip1}$ were markedly increased by N. Rhizoma. Taken together, these results demonstrated that N. Rhizoma induces apoptotic cell death through activation of caspase-3, and potently inhibits the proliferation of HL-60 cells via the G1 phase cell cycle arrest in association with $p27^{Kip1}$ and granulocytic differentiation induction .

HL-60 백혈령 세포주에서 분화유도물질로 전처치 후의 telomerase 활성

김인호,김숙자,정희정,박성규,이규택,원종호,서원석,백승호,홍대식 大韓血液學會 1999 大韓血液學會誌 Vol.34 No.1

청열소독음(淸熱消毒飮)이 HL-60 세포주의 Apoptosis에 미치는 영향

배진석,김종한,박수연,최정화,Bae, Jin-Sock,Kim, Jong-Han,Park, Su-Yeon,Choi, Jeong-Hwa 대한한방안이비인후피부과학회 2007 한방안이비인후피부과학회지 Vol.20 No.1

Objectives : This study was carried out to evaluate anti-tummor effect about apoptosis of Cheongyulsodok-Eum (CSE) Results : 1. Anti-tumor(HL-60 cells) effects of CSE water extracts(Exts) were more effective in high density.($IC_{50:}:572$ ${\mu}g/ml$) 2. The generation of $O_2\;^-$ in HL-60 cells were according to the concentration of CSE water Exts, specially more effective on 100 ${\mu}g/ml$ and 1000 ${\mu}g/ml$ concentration. 3. The SOD activities in HL-60 cells were in proportion as cytotoxicity against HL-60 cells of CSE water Exts. 4. The GPx activities in HL-60 cells were in proportion as cytotoxicity against HL-60 cells of CSE water Exts(more effective on 100 ${\mu}g/ml$ and 1000 ${\mu}g/ml$ concentration), but the catalase activities in HL-60 cells were not effective. 5. DPPH radical scavenging activity of CSE water Exts was effective.(3 ${\mu}g/ml:31.2{\pm}5.2$ %, 10 ${\mu}g/ml:49.6{\pm}7.3$ %, 30 ${\mu}g/ml:35.8{\pm}5.7$ % 100 ${\mu}g/ml:42.3{\pm}6.4$ %) 6. The results of cytotoxicity against HL-60 cells of CSE were as follows. 1) In hexane fraction, the cytotoxicity against HL-60 cells($IC_{50:}:592$ ${\mu}g/ml$) was more effective than against NIH3T3 cells. 2) In ethyl acetate fraction, the cytotoxicity against HL-60 cells was not effective. 3) In butanol fraction, the cytotoxicity against HL-60 cell($IC_{50:}:306$ ${\mu}g/ml$) was more effective than against NIH3T3 cells. 4) In $H_2O$ fraction, the cytotoxicity against HL-60 cells was not effective. Conclusion : These result suggest that CSE has antioxidative effects and anti-tumor effects by apoptosis of free radical($O_2\;^-$) activity, especially butanol and hexane fraction from water extract has more effective in anti-twnor effects.

HL60 세포주에서 방사선 조사에 의한 Apoptosis와 세포 주기 관련 유전자의 발현 변화

김진희(Jin Hee Kim),박인규(In Kyu Park) 대한방사선종양학회 1998 Radiation Oncology Journal Vol.16 No.4

목 적 : 방사선조사에 의한 apoptosis에서 나타나는 각종 세포주기관련 유전자들의 발현 양상을 RNA와 단백 수준에서 분석하여 방사선조사에 의한 apoptosis에서의 세포주기 조절의 변화를 규명함으로서 방사선치료의 기전에 대한 분자생물학적 이해를 도모하고자 본 연구를 시행하였다. 대상 및 방법 : promyelocytic leukemia 세포주인 HL60 세포주를 배양하여 선형가속기(6MV X-선)로 세포에 8Gy의 방사선을 조사하였다. 조사후 다양한 시간 간격으로 Apoptotic DNAFragmentation Assay법을 이용하여 apoptosis를 확인하고 동시에 세포주기관련 유전자들(cyclin A, cyclin B, cyclin C, cyclin D1, cyclin E, cdc 2, CDK2, CDK4, p16INK4a , p21WAF1 , p27KIP1, E2F, PCNA와 Rb)을 단백질과 RNA 수준에서 분석하기위해 western blot analysis와 반정량적 RT-PCR을 시행하였다. 결 과 : 8 Gy의 방사선 조사에 의해 HL60세포에서 apoptosis가 관찰 되었다. 방사선 조사군에서 cyclin A단백은 조사후 48시간까지 시간이 갈수록 증가하였으며, cyclin E, E2F, CDK2 및 Rb 단백은 증가되었다가 다시 감소를 보였다. Rb단백의 증가는 대부분 비활성형인 ppRb(phosphorylated Rb protein)의 양적변화에 의한 것이었다. cyclin D1, PCNA, CDC2, CDK4, p16INK4a단백은 발현의 차이를 보이지 않았으며 p21WAF1과 p27KIP1 단백은 검출되지 않았다. cyclin A, B, C mRNA는 방사선 조사 직후 감소하였다가 12시간부터 발현이 증가되었으며 cyclin D1 mRNA는 조사후 바로 증가하여 48시간에 다시 감소하였다. cyclin E mRNA는 조사후 시간 이 경과함에 따라 감소하였다. CDK2 mRNA는 3시간째는 감소하다가 6시간부터 많은 증가를 보였으며 CDK4 mRNA는 조사후 6-12시간에 급격한 발현증가를 보였다. p16INK4a RNA는 발현의 변화가 없었으며, p21WAF1 및 p27KIP1 RNA의 발현은 관찰되지 않았다. 결 론 : 이상의 결과로 미루어볼 때, 방사선 조사에 의한 HL60세포의 apoptosis와 세포의 G1/S transition는 밀접한 관계가 있는 것으로 생각되며 Rb단백의 증가와 활성형 Rb단백의 감소 현상도 관련이 있는 것으로 사료된다. 이는 E2F의 비정상적인 과발현 및 cyclin E/CDK2의 발현 증가와 관련이 있는 것으로 추측된다. 또한 p21WAF1 및 p27KIP1는 방사선에 의한 apoptosis에는 관여되지 않는 것으로 사료된다. Purpose : To evaluate changes in expression of cell cycle related genes during apoptosis induced in HL60 cells by X-irradiation to understand molecular biologic aspects in mechanism of radiation therapy. Material and Methods : HL-60 cell line (promyelocytic leukemia cell line) was grown in culture media and irradiated with 8 Gy by linear accelerator (6 MV X-ray). At various times after irradiation, ranging from 3 to 48 hours were analyzed apoptotic DNA fragmentation assay for apoptosis and by western blot analysis and semi-quantitative RT- PCR for expression of cell cycle related genes (cyclin A, cyclin B, cyclin C, cyclin D1, cyclin E, cdc2, CDK2, CDK4, p16INK4a, p21WAF1, p27KIP1, E2F, PCNA and Rb). Results : X-irradiation (8 Gy) induced apoptosis in HL-60 cell line. Cycline A protein increased after reaching its peak 48 h after radiation delivery and cyclin E, E2F, CDK2 and RB protein increased then decreased after radiation. Radiation induced up-regulation of the expression of E2F is due to mostly increase of phosphorylated retinoblastoma proteins (ppRb). Cyclin D1, PCNA, CDC2, CDK4 and p16INK4a protein underwent no significant change at any times after irradiation. There was not detected p21WAF1 and p27KIP1 protein. Cyclin A, B, C mRNA decreased immediately after radiation and then increased at 12 h after radiation. Cyclin D1 mRNA increased immediately and then decreased at 48 h after radiation. After radiation, cyclin E mRNA decreased with the lapse of time. CDK2 mRNA decreased at 3 h and increased at 6h after radiation. CDK4 mRNA rapidly increased at 6 to 12 h after radiation. There was no change of expression of p16INK4a and not detected in expressin of p21WAF1 and p27KIP1 mRNA. Conclusion : We suggest that entry into S phase may contribute to apoptosis of HL60 cells induced by irradiation. Increase of ppRb and decrease of pRb protein are related with radiation induced apoptosis of HL60 cells and tosis of HL60 cells induced by irradiation. Increase of ppRb and decrease of pRb protein are related with radiation induced apoptosis of HL60 cells and this may be associated with induction of E2F and cyclinE/CDK2. These results support that p21WAF1 and p27KIP1 are not related with radiation induced- apoptosis.

白花蛇舌草 메탄올 抽出物의 抗腫瘍 效果 및 抗癌 棋戰에 關한 硏究

魯勳政,文九,文錫哉,元秦熹,文永昊,朴來佶 대한한방종양학회 2000 대한한방종양학회지 Vol.6 No.1

Objectives: This experimental study was carried out to evaluate the effects of aqueous and methanol extracts of Hedyotis diffusa which has long been used for cancer treatment in oriental medicines on the induction of apoptotic cell death in human lymphoid leukemia cell line, HL-60. Methods: Cells were treated with various concentrations (200 to 0.4㎍) and periods (6 to 30 hr) of H_2O and methanol extracts of Hedyotis diffusa. Then, cells were tested for viability by MTT assay. Cells wrere treated with 200㎍/ml of methanol extract fork various periods. Genomic DNA was isolated, separated, on 1.5% agarose gels, stained with ethidium bromide and visualized under UV light. Cells were treated with 200㎍/ml of each extract for 16hr.Then, cells were treated with various doses of each extract for 12 hr and 100㎍/ml of methanol extract for various periods. Lysate from the cells used to measure the activity of caspase-1 and-3 proteases by using fluorogenic peptide substrates including acetyl-YVAD-AMC and acetyl-DEVD-AMC, respectively, Cells were treated with 200㎍/ml of each extract for various periods. Cell lysates were immunoprecipated with anti-JNKl antibodies. The immune complex was reacted with 32^p-ATP and c-Jun as a substrate. The phosphotransferase activity of JNKI was measured by suing Phosphoimage analyzer (Fuji Co., Japan). Nuclear extracts were isolated and incubated with oligonucleotide probe of NF-kB was measured by using EMSA and visualized by PhosphoImage analyzer (Fuji Co, Japan). Cell lysates were prepared and analyzed by Western blotting with anti-Bc12 antibodies and anti-Bax antibodies. Cells were pretreated with various doses of methanol extract for 2 hr. Then, the extract was removed by centrifugation. Cells were resuspended with RPMI-1640 media containing 0.3% agarose, 10% FBS, overlayred onto bottom layer agarose and incubated at CO_2 incubator for 6 days. The number of colony was cunted under light microscopy (×100). Results: The death of HL_60 cells was markedly induced by the addition of methanol extract of Hedyotis diffusa in a dose and time-dependent manners. The apoptotic characteristic ladder pattern of DNA strand break was observed in death of HL_60 cells. In addition, it was shown nucleus chromatin condensation under Hoechst staining. Therefore, Hedyotis diffusa extract-induced death of HL-60 cells is mediated by apoptotic signaling processes. The activity of Caspaxe 3-like proteases remained in a basal level in HL-60 cells treated with aqueous extract of Hedyotis diffusa. However, it was markedly increased in HL-60 cells treated with methanol extract of Hedyotis diffusa. In addition, the phosphotransferase activity of JNKl was increased in HL-60 cells treated with methanol extract of Hedyotis diffusa. Furthermore, the activation of transcriptional activator, NF-kB was markedly induced by methanol extract of Hedyotis diffusa. Anti-apoptotic Bc12 was cleaved into 23Kda fragment by treatment of methanol extract of Hedyotis diffusa. However, expression of proapoptotic Bax protein was increased by treatment of methanol extract of Hedyotis diffusa in a time-dependent manner. Furthermore, methanol extract markedly inhibited the colony forming efficiency of HL-60 cells in semisolid agar culture. Conclusions: Above results suggest that methanol extract of Hedyotis diffusa induces the apoptotic death of human leukemic HL-60 cells via activations of Caspase-3 proteases, JNKI, transcriptional activator NF-κB, In addition, our results also suggest that methanol exthanol extract of Hedyotis diffusa reduces the malignant potential of HL-60 cells via down regulation of colony forming effciency through cleavage of Bc12 as well as induction of Bax.

인간의 급성 비임파성 백혈암세포(HL60)의 표면항원에 결합하는 재조합 single-chain Fv (ScFv)의 개발

김철홍,한승희,김형민,한재용,임명운,김진규,Kim, Cheol Hong,Han, Seung Hee,Kim, Hyeong Min,Han, Jae Yong,Lim, Myeong Woon,Kim, Jin-Kyoo 한국미생물학회 2015 미생물학회지 Vol.51 No.2

단일클론항체 AP64 IgM은 인간의 급성 비임파성 골수암(ANLL) 세포주 HL60에 결합하며 쥐의 ANLL 세포에도 교차결합(cross-react)한다. 또한 complement에 의해 매개되어지면 골수암 억제효과를 나타낸다. 본 연구에서는 RT-PCR에 의해 AP64 IgM을 분비하는 하이브리도마의 $V_H$ 및 $V_L$ cDNA로부터 유래된 재조합 single-chain variable domain fragment (ScFv)를 제조하였다. $V_H$ 및 $V_L$은 15개 아미노산으로 구성된 linker $(G_4S)_3$으로 연결되었다. 재조합 ScFv는 Escherichia coli BMH 71-18에서 single polypeptide chain으로 발현되었다. Periplasmic extract를 $Ni^+$-NTA-agarose affinity column에 가하여 발현된 재조합 ScFv를 정제하였으며 westernblot으로 정제된 단백질을 탐지하였다. 정제된 재조합 ScFv는 AP64 IgM 모항체가 탐지하는 항원과 같은 HL60 세포의 표면항원(약 30 kDa)을 인지하였다. 그러나 HL60의 표면항원에 대한 ScFv의 결합력은 AP64 IgM 모항체보다 낮아서 추후 이에 대한 개선이 필요하다. 종합하여 볼 때 HL60 세포주에 특이적인 재조합 ScFv는 진단 또는 치료목적으로 유용한 생물학적 제재가 될 수 있을 것이다. A monoclonal antibody AP64 IgM binds to human acute nonlymphocytic leukemia (ANLL) cell line HL60 and also cross-reacts with the homologous antigen in a rat ANLL cell. This antibody mediated by complement, has leukemia a suppression effect. In this study, we generated a recombinant single-chain variable domain fragment (ScFv) which were derived from $V_H$ and $V_L$ cDNA of AP64 IgM-secreting hybridoma by RT-PCR. The two variable regions were joined with a single 15 amino acid linker $(G_4S)_3$. This recombinant ScFv was expressed as a single polypeptide chain from Escherichia coli BMH 71-18. The recombinant ScFv was purified by applying the periplasmic extract to $Ni^+$-NTA-agarose affinity column and detected with westernblot. The purified recombinant ScFv recognized a surface antigen (about 30 kDa) of HL60 cell line which is the same antigen detected by parental AP64 IgM. But the affinity of ScFv for a surface antigen of HL60 was lower than that of the parental AP64 IgM, which needs to be further improved. Overall, the recombinant ScFv specific to HL60 might be a useful bioreagent for either diagnostic or therapeutic purposes.

백화사설초(白花蛇舌草) 메탄올 추출물(抽出物)의 항종양(抗腫瘍) 효과(效果) 및 항암(抗癌) 기전(機轉)에 관(關)한 연구(硏究)

노훈정,문구,문석재,원진희,문영호,박래길,No, Hoon-Jeong,Moon, Gu,Moon, Seok-Jae,Won, Jin-Hee,Moon, Young-Ho,Park, Rae-Gil 대한암한의학회 2000 大韓癌韓醫學會誌 Vol.6 No.1

Objectives: This experimental study was carried out to evaluate the effects of aqueous and methanol extracts of Hedyotis diffusa which has long been used for cancer treatment in oriental medicines on the induction of apoptotic cell death in human lymphoid leukemia cell line, HL-60. Methods: Cells were treated with various concentrations (200 to $0.4{\mu}g$) and periods (6 to 30 hr) of $H_2O$ and methanol extracts of Hedyotis diffusa. Then, cells were tested for viability by MTT assay. Cells wrere treated with $200{\mu}g/ml$ of methanol extract fork various periods. Genomic DNA was isolated, separated, on 1.5% agarose gels, stained with ethidium bromide and visualized under UV light. Cells were treated with $200{\mu}g/ml$ of each extract for 16 hr. Then, cells were treated with Hoechst dye 33342 and observed by fluorescence microscopy. Cells were treated with various doses of each for 12 hr and $100{\mu}g/ml$ of methanol extract for various periods. Lysate from the cells used to measure the activity of Caspase-1 and-3 proteases by using fluorogenic peptide substrates including acetyl-YVAD-AMC and acetyl-DEVD-AMC, respectively. Cells were treated with $200{\mu}g/ml$ of each extract for various periods. Cell lysates were immunoprecipated with anti-JNKl antibodies. The immune complex was reacted with $32^p-ATP$ and c-Jun as a substrate. The phosphotransferase activity of JNKI was measured by using PhosphoImage analyzer (Fuji Co., Japan). Nuclear extracts were isolated and incubated with oligonucleotide probe of $NF-{\kappa}B$. Transcriptional activation of ${\kappa}B$ was measured by using EMSA and visualized by PhosphoImage analyzer (Fuji Co, Japan). Cell lysates were prepared and analyzed by Western blotting with anti-Bc12 antibodies and anti-Bax antibodies. Cells were pretreated with various doses of methanol extract for 2 hr. Then, the extract was removed by centrifugation. Cells were resuspended with RPMI-1640 media containing 0.3% agarose, 10% FBS, overlayred onto bottom layer agarose and incubated at $CO_2$ incubator for 6 days. The number of colony was counted under light microscopy ($\time100$). Results: The death of HL-60 cells was markedly induced by the addition of methanol extract of Hedyotis diffusa in a dose and time-dependent manners. The apoptotic characteristic ladder pattern of DNA strand break was observed in death of HL-60 cells. In addition, it was shown nucleus chromatin condensation and fragmentation under Hoechst staining. Therefore, Hedyotis diffusa extract-induced death of HL-60 cells is mediated by apoptotic signaling processes. The activity of Caspase 3-like proteases remained in a basal level in HL-60 cells treated with aqueous extract of Hedyotis diffusa. However, it was markedly increased in HL-60 cells treated with methanol extract of Hedyotis diffusa. In addition, the phosphotransferase activity of JNKl was increased in HL-60 cells treated with methanol extract of Hedyotis diffusa. Furthermore, the activation of transcriptional activator, $NF-{\kappa}B$ was markedly induced by methanol extract of Hedyotis diffusa. Anti-apoptotic Bc12 was cleaved into 23Kda fragment by treatment of methanol extract of Hedyotis diffusa. However, expression of proapoptotic Bax protein was increased by treatment of methanol extract of Hedyotis diffusa in a time-dependent manner. Furthermore, methanol extract markedly inhibited the colony forming efficiency of HL-60 cells in semisolid agar culture. Conclusions: Above results suggest that methanol extract of Hedyotis diffusa induces the apoptotic death of human leukemic HL-60 cells via activations of Caspase-3 proteases, JNKI, transcriptional activator $NF-{\kappa}B$, In addition, our results also suggest that methanol extract of Hedyotis diffusa reduces the malignant potential of HL-60 cells via down regulation of colony forming

Dimethylsulfoxide (DMSO) induces downregulation of heme oxygenase-1 (HO-1) in HL-60 cells: involvement of HO-1 in HL-60 cell differentiation

( Eun Mi Noh ),( Dong Hyu Cho ),( Young Rae Lee ),( Young Ju Jeong ),( Jong Hyeon Kim ),( Hee Suk Chae ),( Jin Ny Park ),( Won Seok Jung ),( Sung Joo Park ),( Jong Suk Kim ) 생화학분자생물학회(구 한국생화학분자생물학회) 2011 BMB Reports Vol.44 No.11

Heme oxygenase-1 (HO-1), an inducible enzyme with broad tissue expression, is wel1-regulated in response to hematopoietic stress and preserves vascular homeostasis. We investigated the involvement of HO-1 in HL-60 cell differentiation. Dimethyl sulfoxide (DMSO) completely decreased HO-1 expression in a time-dependent manner, but clearly induced HL-60 cell differentiation, as evidenced by a marked increase in CD11b expression. Interestingly, zinc protoporphyrin (ZnPP), a strong inhibitor of HO-1, induced HL-60 cell differentiation. In contrast, treatment with cobalt protoporphyrin (CoPP), an activator of HO-1, decreased CD11b expression. Additionally, ZnPP downregulated HO-1 protein expression in HL-60 cells, whereas CoPP induced upregulation. These results suggest that HO-1 might have a negative function in DMSO-induced HL-60 cell differentiation. This study provides the first evidence that HO-1 plays an important role in DMSO-induced HL-60 cell differentiation. [BMB reports 2011; 44(11): 753-757]

HL-60 세포주에서 myeloblastin mRNA 발현의 하향조절

이대희 고신대학교(의대) 고신대학교 의과대학 학술지 2005 고신대학교 의과대학 학술지 Vol.20 No.1

Background : Recent clinical studies have shown that a high proportion of patients with acute promyelocytic leukemia (APL) achieves complete remission after treatment with all-trans retinoic acid (ATRA). However, most patients who receive continuous treatment with ATRA relapse and develop ATRA-resistant leukemia. In this study, the author investigated the strategies to overcome ATRA resistance of acute promyelocytic leukemia (APL) cells by inducing the differentiation of dendritic cells (DCs) from human leukemic cell lines for the development of adoptive immunotherapy. Myeloblastin (mbn) was used as one of the indicators of differentiation in this study. Methods : To study the biochemical and enzymatic charicteristics of the human myeloblastin the enzyme was extracted from human leukocytes and purified by a combination of Ultrogel AcA 54 and Bio-Rex 70 chromatographies. To evaluate the mbn protein expression in cells, anti-mbn antibody was prepared by two-step procedure including ammonium sulfate precipitation and DEAE-cellulose ion exchange chromatography. HL-60 cell differentiation was induced by the addition of all-trans retinoic acid (ATRA), dimethyl sulfoxide (DMSO), phorbol 12-myristate 13-acetate (PMA), cholecalcitriol (VD) to the media for 6 days and the expression of mbn mRNA and mbn protein were determined by RT-PCR method and ELISA, respectively. HL-60 cells, K-562 cells, NC-37 and RPMI 7666 cells were cultured in RPMI 1640 supplemented with 10% fetal calf serum for 7 days, with various agents or ligands such as calcium ionophore (CI), Flt3-ligand (FL) and PMA to generate dendritic cells from the cell lines. A portion of each cell lines was harvested and the rest of them was cultured in the new RPMI 1640 supplemented with 10% fetal calf serum, with Flt3-ligand for 7 days more. RNA was extracted and gene expression from each cell lines was determined by RT-PCR method. The morphology of the cells was evaluated from cytospin slide preparations with Wright's stain. Results : 3.8 mg of proteinase-3 was isolated from 67 mg of leukocyte extract. 6g of anti-mbn polyclonal antibody was raised. PMA induced a significant inhibition of mbn mRNA expression in HL-60 cells. The cells exposed to ATRA or DMSO or VD show a little change in the mbn mRNA expression. Thus the terminal differention of HL-60 cells and K-562 cells by ATRA, DMSO, VD, and hemin was imcomplete and a large fraction of the cells was in undifferentiated or premature states. The promyelocytic leukemic cell line HL-60, B lymphoblast cell lines RPMI 7666 and NC-37 could be induced to dendritic cells in vitro. Treatment of HL-60 ith PMA resulted in the expression of myeloid-related DC phenotypes, while treatment of RPMI 7666 with FL and treatment of NC-37 with PMA and FL lead to the expression of lymphoid-related DC phenotypes. Conclusion : In conclusion, myeloid-related DC phenotypes and lymphoid-related DC phenotypes can be generated from HL-60, NC-37, and RPMI 7666 cell lines, respectively. These DC phenotypes can potentially be used as a cellular leukemia vaccine in vivo or to generate antileukemic T cells in vitro for adoptive immunotherapy.

급성전골수성백혈병 HL- 60 세포주에서 방사선조사에 의한 세포고사기전

김혜정(Hye Jung Kim),문성근(Sung Keun Moon),이재훈(Jae Hoon Lee),문성록(Sun Rock Moon) 대한방사선종양학회 2001 Radiation Oncology Journal Vol.19 No.2

목 적 :방사선 조사에 의하여 유발되는 세포고사의 신호전달기전, 특히 caspase계 cysteine protease의 활성화, Bcl2 및 Bax 단백질, cytochrome c의 세포질내로의 방출, Fas 와 Fas-L 단백질의 발현양상 등의 조사를 통하여 방사선조사에 의하여 유발되는 세포고사기전을 규명하고자 하였다. 대상 및 방법 :HL-60 세포주에 6 MV의 X-선을 조사하고 세포생존율, caspase의 활성도, Bcl2 및 Bax 단백질, cytochrome c의 세포질내로의 방출여부, 및 Fas 와 Fas- L 단백질의 발현양상을 조사하였다. 결 과 :방사선조사 후 세포의 생존율은 조사선량과 조사 후 시간경과에 따라 감소되었다. 세포고사의 특징인 사다리형 DNA 분절은 방사선조사 4시간 후부터 시간경과에 따라 증가하였으며, 조사선량이 증가할수록 더욱 현저하였다. 방사선조사 후 caspase계 cysteine proteases 중 caspase-2, 3, 6, 8 및 9의 활성화가 시간경과에 따라 증가하였으며, 16 Gy의 방사선량조사 4시간 후에 poly (ADP- ribosyl) polymerase (PARP)의 분절과 Western blot을 이용한 procaspase-3의 분절을 확인함으로서 caspase-3의 활성을 간접적으로 증명할 수 있었다. Bcl2 단백질은 방사선조사 후 시간경과에 따라 감소하였으며, Bax 단백질은 시간경과에 따라 발현이 증가하는 양상을 관찰할 수 있었다. 방사선조사 후 cytochrome c의 세포질내로의 방출을 확인하였다. 또한 Fas 및 Fas-L 단백질 모두 방사선조사 후 발현이 증가하는 양상을 관찰할 수 있었다. 결 론 :HL-60 세포주에서 방사선 조사에 의해 유발되는 세포사멸이 세포고사기전에 의해서 매개됨을 확인하였으며, 이는 세포내 caspase계 cysteine proteases, Bcl2, Bax, 세포질내로의 cytochrome c 방출 그리고 Fas, Fas-L가 관여하는 신호전달경로의 활성화에 의한 것임을 의미하였다. Purpose : The mechanical ins ights of death of cancer cells by ionizing radiation are not yet clearly defined. Recent evidences have demonstrated that radiation therapy may induce cell death via activation of s ignaling pathway for apoptos is in target cells . This study is des igned whether ionizing radiation may activate the s ignaling cascades of apoptos is including caspase family cysteine proteases , Bcl2/Bax, cytochrome c and Fas/Fas- L in target cells . Materials and Methods : HL-60 cells were irradiated in vitro with 6 MV X- ray at dose ranges from 2 Gy to 32 Gy. The cell viability was tested by MTT assay and the extent of apoptos is was determined using agarose gel electrophores is . The activities of caspase proteases were measured by proteolytic cleavages of substrates . Western blot analys is was used to monitor PARP, Caspase- 3, Cytochrome- c, Bcl- 2, Bax, Fas and Fas- L. Results : Ionizing radiation decreases the viability of HL- 60 cells in a time a nd dose dependent manner. Ionizing radiation- induced death in HL- 60 cells is an apoptotic death which is revealed as characteristic ladder- pattern fragmentation of genomic DNA over 16 Gy at 4 hours . Ionizing radiation induces the activation of caspase-2, 3, 6, 8 and 9 of HL-60 cells in a time- dependent manner. The activation of caspase- 3 protease is also evidenced by the digestion of poly (ADP- ribose) polymerase a nd procaspase-3 with 16Gy ionizing irradiation. Anti- apoptotic Bcl2 express ion is decreased but apoptotic Bax express ion is increased with mitochondrial cytochrome c release in a time- dependent manner. In additon, express ion of Fas and Fas- L is also increased in a time dependent manner. Conclusion : These data suggest that ionizing radiation- induced a poptos is is mediated by the activation of various s igna ling pathways including caspase family cysteine proteases , Bcl2/Bax, Fas a nd Fas- L in a time and dose dependent manner.