Intermedeol Isolated from the Leaves of Ligularia fischeri var. spiciformis Induces the Differentiation of Human Acute Promyeocytic Leukemia HL-60 Cells

• Abstract
• Introduction
• Materials and Methods
• Materials
• Determination of growth inhibition
• Differentiation assay
• Western blot analysis
• Results
• Cell growth inhibition
• Effect on differentiation of HL-60 cells
• The expression level of c-myc and p21^CIP1 during the
  intermedeol-induced differentiation of HL-60 cells
• Discussion
• Acknowledgements
• References

Abstract

The present work was performed to investigate the effects of intermedeol on proliferation and differentiation of human leukemia-derived HL-60 cells as well as the underlying mechanisms for these effects. Intermedeol exhibited a potent antiproliferative activity against HL-60 cells. In addition, this compound was found to be a potent inducer for HL-60 cell differentiation as assessed by nitroblue tetrazolium reduction test, esterase activity assay, phagocytic activity assay, morphology change, and expression of CD14 and CD66b surface antigens. These results suggest that intermedeol induces differentiation of human leukemia cells to granulocytes and monocytes/macrophage lineage. Moreover, the expression level of c-myc was down-regulated during intermedeol-dependent HL-60 cell differentiation, whereas p21^CIP1 was up-regulated. Taken together, our results suggest that intermedeol may have potential as a therapeutic agent in human leukemia.

Introduction

Imbalance between cell proliferation, apoptosis and differentiation leads to the development of clones of malignant cells. Based on the understanding of tumor biology in respect to the kinetics of cell populations, two new strategies, induction of differentiation and apoptosis, have recently emerged in the fields of cancer chemoprevention and chemotherapy. Differentiation from malignant or premalignant cells into more mature or normal-like cells as well as apoptosis in multistep carcinogenesis are theoretically amenable to preventive cancer intervention. Thus, compounds capable of inducing differentiation are considered as candidate agents for the prevention and/or treatment of cancer [1], [2].

The HL-60 cell line, derived from a patient with acute promyelocytic leukemia, provides a useful in vitro model system for studying the cellular and molecular events involved in the differentiation process [3]. An inducer of HL-60 differentiation is commonly considered to have a potentially therapeutic importance. Terminal differentiation of HL-60 cells can be monitored by changes of morphological, biochemical, and immunological properties. The differentiated HL-60 phenotype is characterized by growth inhibition, an increased adherence, a loss of cell-surface transferrin receptors, and increase in monocyte surface markers, induction of α-naphthyl acetate (non-specific) esterase and certain patterns of protein phosphorylation [4]. Certain compounds, known to be efficacious cancer preventative agents, such as interferon [5], retinoids [6], 1α,25-dihydroxyvitamin D₃ [7], [8] are potent inducers of HL-60 cell differentiation, and appear to be clinically effective against myeloproliferative disorders. Thus, the strategy that manipulates HL-60 cell differentiation has been used as a valid model to discover potential cancer chemopreventive agents in preclinical evaluation.

Thus, as a part of our screening program to evaluate the chemopreventive potential effect of natural compounds, we have investigated the effect of intermedeol, which was isolated from the leaves of Ligularia fischeri (Ledebour) Turcz. var. spiciformis, on HL-60 cell differentiation. The leaves of this plant have been used to treat jaundice, scarlet-fever, rheumatoidal arthritis and hepatic diseases [9]. We previously reported on the isolation of an eudesmane-type sesquiterpene, (+)-intermedeol (Fig. [1]) and 6-oxoeremophilenolide from the leaves of Ligularia fischeri var. spiciformis, and the cytotoxic effect of the former compound was shown [10]. Various biochemical and morphological examination performed in the present study indicated that intermedeol contains the inducing activity of HL-60 cell differentiation. Furthermore, we demonstrated that the expression of p21^CIP1 cyclin dependant kinase inhibitor (CDKI) was increased, whereas c-myc oncogene expression was down-regulated during intermedeol-induced differentiation of HL-60 cells.

Materials and Methods

Materials

Intermedeol used for this study was isolated from the leaves of Ligularia fischeri var. spiciformis as previously described [10]. The physicochemical data of intermedeol were defined as follows: amorphous powder, [α]_D: + 12.1° (c 1.3, MeOH); EI-MS (70 eV) m/z (rel. int., %): 222.1 (24, M⁺), 204.1 (22, [M - H₂O]⁺). The intermedeol isolated was checked by HPLC and it was > 98 % pure. HL-60 human promyelocytic leukemia cell line was obtained from the Korean Cell Line Bank. RPMI 1640 medium, fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Life Technologies Inc. (Grand Island, NY, U.S.A.), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), nitrobluetetrazolium (NBT), genistein, 1α, 25(OH)₂D₃, trans-retinoic acid (ATRA), 12-O-tetradecanoylphorbol-13-acetate (TPA), α-naphthyl acetate esterase kit and 3-hydroxy-2-naphthoic acid o-toluidine (naphthol AS-D chloroacetate) esterase kit were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A). Fluorescence-isothiocyanate (FITC)-labeled anti-human CD14 mouse monoclonal antibody and anti-human CD66b mouse monoclonal antibody were obtained from Pharmingen (San Diego, CA, U.S.A.). Anti-human c-myc and p21^CIP1 mouse monoclonal antibody were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Determination of growth inhibition

HL-60 cells were grown at 37 °C in RPMI medium supplemented with 10 % FBS, penicillin (100 units/ml), and streptomycin sulfate (100 μg/ml) in a humidified atmosphere of 5 % CO₂. Cells were seeded at a concentration of 2 × 10⁵ cells/ml, maintained for logarithmic growth by passaging them every 2 - 3 d, and incubated for 1 - 4 days with intermedeol at various concentrations. Intermedeol dissolved in DMSO was added to medium in serial dilution (the final DMSO concentration in all assay did not exceed 0.1 %). Cell viability was checked by trypan blue exclusion method. We used genistein as a positive control [11].

Differentiation assay

NBT reduction test: The percentage of HL-60 cells capable of reducing NBT was determined by counting the number of cells which contained the precipitated formazan particles after cells had been incubated with NBT (1.0 mg/ml) at 37 °C for 30 min. TPA was used as a stimulator for the formation of formazan.

Phagocytosis test: HL-60 cells (1 × 10⁶ cells/ml) were suspended in serum-free RPMI 1640 medium containing 0.2 % latex particles (average diameter, 0.81 μM) and incubated at 37 °C for 4 h. After incubation, the cells were washed once with phosphate-buffered saline (PBS). Cells containing more than 10 latex particles were scored as phagocytic cells [11].

Esterase activity test: A smear preparation was chemically stained for α-naphthyl acetate esterase and naphthol AS-D chloroacetate esterase by the standard techniques [11].

Flow cytometry: HL-60 cells (2 × 10⁵ cells/ml) exposed to intermedeol were collected and washed twice with ice-cold PBS. Cells were then incubated with direct FITC-labeled anti-CD 14 or anti-CD-66b antibody on ice for 30 min, washed twice with PBS, and antibody binding to cells was quantified using a FACS flow cytometry (Becton Dickinson Co., Germany).

Western blot analysis

Cellular proteins were extracted from control and intermedeol-treated HL-60 cells. The washed cell pellets were resuspended in ELB buffer (50 mM HEPES, pH 7.0, 250 mM NaCl, 5 mM EDTA, 0.1 % Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, 5 mM NaF, 0.5 mM Na orthovanadate) containing 5 μg/ml each of leupeptin and aprotinin and incubated with 15 min at 4 °C. Cell debris was removed by microcentrifugation, followed by quick freezing of the supernatants. Protein concentration was determined by Bio-Rad (CA, U.S.A.) protein assay reagent (CA, U.S.A), as described by the manufacturer. Cellular proteins (50 μg) from treated and untreated cell extracts were electroblotted onto nitrocellulose membrane following separation on a 12 % SDS-polyacrylamide gel electrophoresis. The immunoblot was incubated overnight with blocking solution (5 % skim milk in PBS containing 0.02 % Tween 20) at 4 °C, and then incubated for 4 h with a 1 : 1000 dilution of monoclonal anti-c-myc or p21^CIP1 antibody. Blots were washed 2 times with PBS, and then incubated with a 1 : 1000 dilution of horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature, washed again three times with PBS, and then developed by enhanced chemiluminescence (Amersham Life Science, Arlington Heights, IL, U.S.A.).

Results

Cell growth inhibition

The effect of intermedeol on the proliferation of HL-60 cells was first examined. The cell growth of HL-60 cells was inhibited in a concentration- and time-dependent manner (Fig. [2]), suggesting that this chemical has an antiproliferative activity. The inhibitory effect became apparent at a concentration of 2.5 and 5 μM intermedeol, and no cytocidal effects were observed under these conditions. Thus, these concentrations were used throughout the present study.

Effect on differentiation of HL-60 cells

After 4 days treatment, the effect of intermedeol on HL-60 cell differentiation was compared with that of 1α,25(OH)₂D₃, and the results are summarized in Table [1]. When HL-60 cells were incubated with intermedeol at concentrations of 2.5 and 5 μM for 4 d, approximately 28.6 % and 39.4 % of HL-60 cells were stained with NBT, respectively, whereas only 6.6 % of the untreated cells were positive (Table [1]). 1α,25(OH)₂D₃ (20 nM) gave 41.5 % of NBT-reducible cells. In order to test whether intermedeol induces HL-60 cells to differentiate into monocyte/macrophage and granulocyte, the esterse activity was measured under identical conditions. Treatment of HL-60 cells with 2.5 and 5 μM of intermedeol for 4 d resulted in 18 % and 42.2 % increase of the α-naphthyl acetate esterase activity, respectively, but the effect of Intermedeol on the AS-D chloroacetate esterase activity was relatively mild. Moreover, cells treated with these compounds showed apparent phagocytic activity (Table [1]). In addition, as shown in Fig. [3], [2].5 to 5 μM intermedeol significantly increased the expression of both membrane antigen CD14 and CD 66b, whereas 1α,25(OH)₂D₃ (20 nM) mildly increased the expression of both CD14 and CD66b. Morphological evaluation of the cells after May-Grunwald Giemsa stain indicated that the intermedeol-treated cells exhibited granulation appearance, characteristic of differentiated cells, like as granulocytes and monocytes/macrophages (Fig. [4]).

The expression level of c-myc and p21^CIP1 during the
intermedeol-induced differentiation of HL-60 cells

To assess the altered expression of the characteristic cellular growth-related proteins of HL-60 cells by intermedeol, we examined the expression level of c-myc and p21^CIP1 using immnunoblot analysis. When HL-60 cells were incubated with intermedeol for 3 days, p21^CIP1 protein expression was clearly increased at 24 h and 48 h, and robust response was noted at 72 h. In contrast, c-myc expression was decreased about 50 % after 24 h incubation and sustained up to 72 h (Fig. [5]).[*]

Discussion

Currently, programs for the identification and testing of potential chemopreventive agents have been developed [12]. The preclinical laboratory work of these programs is designed to systemically test the efficacy and toxicity of chemopreventive agents in both in vivo and in vitro models [1], [2]. Among them, the in vitro model can be divided into three categories: inhibition of cell proliferation, blockage of transformation, and induction of differentiation. Many new agents that can selectively induce differentiation of premalignant and malignant cells into more mature cells have been identified in in vitro and in vivo models. The present study has demonstrated that intermedeol exerts a potent differentiation-inducing activity on promyelocytic leukemia HL-60 cells. This effect of intermedeol was confirmed with morphological analysis using Giemsa staining, NBT reduction test, esterase activity assay, phagocytosis and expression of cell surface antigens. Furthermore, p21^CIP1 expression was up-regulated by intermedeol whereas c-myc proteins were down-regulated. In addition, our previous study (unpublished data) demonstrated that intermedeol has an inducing effect on apoptosis in HL-60 cells. Taken together with the effects on apoptosis and differentiation in HL-60 cells, our results suggest that intermedeol could be used as a potent chemoprevention and/or chemotherapy agent in HL-60 human promyelocytic leukemia cells. However, it remains unclear whether intermedeol effectively induces the elimination of promalignant or malignant cells via differentiation and/or apoptosis in vivo (Fig. [5]).

Although the mechanism of differentiation induction by intermedeol is not clear, it appears that p21^CIP1 plays an important role in modulation of the cell differentiation activity of the compound. Induction of p21^CIP1, a downstream target of the tumor suppressor gene p53, by DNA damage is well-documented [13]. However, genotoxic insults also induce p21^CIP1 in a p53-independent manner [14]. Analogously, differentiation-inducing agents such as PMA stimulate p21 ^CIP1 expression in human leukemia cell line such as HL-60 and U937, which are p53-null [15]. Collectively, these findings indicate that p21^CIP1 induction occurs through both p53-dependent and -independent mechanisms and raise the possibility that DNA damage- and differentiation-associated stimuli may activate p21^CIP1 through distinct, although related, pathways. In this regard, it is noteworthy that intermedeol potentially induces p21^CIP1 after considerably longer exposure intervals (e. g., 72 h). In contrast, treatment of HL-60 cells with a high concentration of intermedeol (e. g., 20 μM) triggers apoptosis in HL-60 leukemia cells for relatively brief exposure intervals (data not shown). In this case, this observation indicates an ambiguous functional requirement for p21^CIP1 in intermedeol-mediated leukemic cell maturation.

The c-myc gene product is a nuclear protein and has been implicated in the control of normal cell growth as well as transformation, but its exact function is unknown. A decrease in c-myc mRNA has been demonstrated in vitro during chemically induced differentiation of various cell lines [16]. This decline was gradual or even biphasic as seen during the differentiation of the leukemia cell lines [17]. The inverse relationship between p21^CIP1 and c-myc expression after lower concentration of intermedeol treatment was anticipated, given the established role of c-myc in S-phase progression [18] as well as in promotion of apoptosis [19]. This finding indicates a cooperative interaction between c-myc down-regulation and p21^CIP1 induction in the maturation process. Such a notion is consistent with the results of a recent report demonstrating that c-myc can act as a negative regulator of p21^CIP1 expression [20].

In summary, our results suggest that the induction of HL-60 cell maturation by intermedeol may have a potential as a therapeutic approach for the treatment of leukemia although the doses are probably high for the chemoprevention. Furthermore, intermedeol-induced differentiation accompanying the increase of p21^CIP1 and decrease of c-myc expression suggest that the targeting of p21^CIP1 and c-myc regulation may have importance in the treatment of cancer.

Acknowledgements

Financial support from a grant (HMP-00-B-21 600 - 0124) of the 2000 Good Health R & D Project, Ministry of Health & Welfare, Korea is gratefully acknowledged.

References

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Dr. Kyung-Tae Lee

Department of Biochemistry, College of Pharmacy

Kyung-Hee University

Dongdaemun-Ku, Hoegi-Dong 130-701, Seoul

Korea

Email: ktlee@khu.ac.kr

Phone: +82-2-9610860

Fax: +82-2-9663885

References

• 1 Hong W K, Sporn M B. Recent advances in chemoprevention of cancer.  Science. 1997;  278 1073-7
  CrossrefPubMedSearch in Google Scholar
• 2 Suh N, Luyengi L, Fong H H, Kinghorn A D, Pezzuto J M. Discovery of natural product chemopreventive agents utilizing HL-60 cell differentiation as a model.  Anticancer Res.. 1995;  15 233-9
  PubMedSearch in Google Scholar
• 3 Collins S J, Ruscetti F W, Gallagher R E, Gallo R C. Terminal differentiation of human promyelocytic leukemia cells induced by dimethyl sulfoxide and other polar compounds. Proc Natl. Acad. Sci.  U S A.. 1978;  75 2458-62
  PubMedSearch in Google Scholar
• 4 Yam L T, Li C Y, Crosby W H. Cytochemical identification of monocytes and granulocytes. Am. J. Clin.  Pathol.. 1971;  55 283-90
  PubMedSearch in Google Scholar
• 5 Harris P E, Ralph P, Gabrilove J, Welte K, Karmali R, Moore M A. Distinct differentiation-inducing activities of gamma-interferon and cytokine factors acting on the human promyelocytic leukemia cell line HL-60.  Cancer Res.. 1985;  45 3090-5
  PubMedSearch in Google Scholar
• 6 Honma Y, Takenaga K, Kasukabe T, Hozumi M. Induction of differentiation of cultured human promyelocytic leukemia cells by retinoids. Biochem. Biophys. Res.  Commun.. 1980;  95 507-12
  PubMedSearch in Google Scholar
• 7 Koeffler H P, Amatruda T, Ikekawa N, Kobayashi Y, DeLuca H F. Induction of macrophage differentiation of human normal and leukemic myeloid stem cells by 1,25-dihydroxyvitamin D₃ and its fluorinated analogues.  Cancer Res.. 1984;  44 5624-8
  PubMedSearch in Google Scholar
• 8 Ostrem V K, Tanaka Y, Prahl J, DeLuca H F, Ikekawa N. 24- and 26-homo-1,25-dihydroxyvitamin D₃: preferential activity in inducing differentiation of human leukemia cells HL-60 in vitro. Proc. Natl. Acad. Sci.  U S A.. 1987;  84 2610-4
  PubMedSearch in Google Scholar
• 9 Choi O J. Usage and constituents of medicinal plants. Seoul; lweolseogak 1991: P 621
  Search in Google Scholar
• 10 Park H J, Kwon S H, Yoo K O, Sohn I C, Lee K T, Lee H K. Sesquiterpenes from the leaves of Ligularia fischeri var. spiciformis .  Planta Medica. 2000;  66 783-4
  Thieme ConnectPubMedSearch in Google Scholar
• 11 Lee K T, Sohn I C, Kim Y K, Choi J H, Choi J W, Park H J, Itoh Y, Miyamoto K. Tectorigenin, an isoflavone of Pueraria thunbergiana BENTH., induces differentiation and apoptosis in human promyelocytic leukemia HL-60 cells. Biol. Pharm.  Bull.. 2001;  24 1117-21
  PubMedSearch in Google Scholar
• 12 Fesus L, Szondy Z, Uray I. Probing the molecular program of apoptosis by cancer chemopreventive agents. J. Cell Biochem.  Suppl.. 1995;  22 151-61
  PubMedSearch in Google Scholar
• 13 Bunz F, Dutriaux A, Lengauer C, Waldman T, Zhou S, Brown J P, Sedivy J M, Kinzler K W, Vogelstein B. Requirement for p53 and p21 to sustain G2 arrest after DNA damage.  Science.. 1998;  282 1497-501
  CrossrefPubMedSearch in Google Scholar
• 14 Loignon M, Fetni R, Gordon A J, Drobetsky E A. A p53-independent pathway for induction of p21^waf1cip1 and concomitant G1 arrest in UV-irradiated human skin fibroblasts.  Cancer Res.. 1997;  57 3390-4
  PubMedSearch in Google Scholar
• 15 Danova M, Giordano M, Mazzini G, Riccardi A. Expression of p53 protein during the cell cycle measured by flow cytometry in human leukemia. Leuk.  Res.. 1990;  14 417-22
  PubMedSearch in Google Scholar
• 16 Filmus J, Buick R N. Relationship of c-myc expression to differentiation and proliferation of HL-60 cells.  Cancer Res.. 1985;  45 822-5
  PubMedSearch in Google Scholar
• 17 Todokoro K, Ikawa Y. Sequential expression of proto-oncogenes during a mouse erythroleukemia cell differentiation.  Biochem Biophys Res Commun.. 1986;  135 1112-8
  CrossrefPubMedSearch in Google Scholar
• 18 Baker S J, Pawlita M, Leutz A, Hoelzer D. Essential role of c-myc in ara-C-induced differentiation of human erythroleukemia cells.  Leukemia.. 1994;  8 1309-17
  PubMedSearch in Google Scholar
• 19 Askew D S, Ashmun R A, Simmons B C, Cleveland J L. Constitutive c-myc expression in an IL-3-dependent myeloid cell line suppresses cell cycle arrest and accelerates apoptosis.  Oncogene.. 1991;  6 1915-22
  PubMedSearch in Google Scholar
• 20 Shi Y, Glynn J M, Guilbert L J, Cotter T G, Bissonnette R P, Green D R. Role for c-myc in activation-induced apoptotic cell death in T cell hybridomas.  Science.. 1992;  257 212-4
  PubMedSearch in Google Scholar

Dr. Kyung-Tae Lee

Department of Biochemistry, College of Pharmacy

Kyung-Hee University

Dongdaemun-Ku, Hoegi-Dong 130-701, Seoul

Korea

Email: ktlee@khu.ac.kr

Phone: +82-2-9610860

Fax: +82-2-9663885