DOCSLIB.ORG

Megakaryocytic Differentiation of K562 Cells Is Associated with Changes

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Megakaryocytic Differentiation of K562 Cells Is Associated with Changes [CANCER RESEARCH 50, 6323-6329. October 1. I990| Megakaryocytic Differentiation of K562 Cells Is Associated with Changes in the Cytoskeletal Organization and the Pattern of Chromatographically Distinct Forms of Phosphotyrosyl-specific Protein Phosphatases T. Martin Butler,1 Andrew Ziemiecki, and Robert R. Friis2 Institute for Clinical and Experimental Cancer Research, University of Bern, Tiefenaustrasse 120, CH-3004 Bern, Switzerland ABSTRACT entiation along an erythroid lineage. Herbimycin A, an inhibitor of protein phosphorylation, has been shown to induce differ We have analyzed morphological and biochemical changes occurring entiation of mouse embryonal carcinoma (F9), erythroleukemia during megakaryocytic differentiation of the human chronic myelogenous (MEL), and K562 cells (20, 21). Differentiation toward mega- leukemia cell line K562 induced by phorbol 12-myristate 13-acetate (I'M A). PMA-treated cells became growth arrested, were slightly larger karyocytes can be monitored by a reduction of growth potential and irregular in shape, adhered better to the culture flask surface, and (22), a reduction of expression of erythroid surface markers expressed the glycoprotein Ilia on their surfaces. The morphological (15), a reduction of c-myc expression (18, 23), an induction of changes induced by PMA treatment were associated with the disappear surface markers of the megakaryocytic lineage (15), an induc ance of actin from the cytosol and presumably reflect PMA-induced actin tion of c-sis and c-fos mRNA (23-25), and translocation of polymerization. Megakaryocytic differentiation was accompanied by protein kinase C from the cytoplasm to the membrane (22, 26). about a 3-fold decrease in the specific phosphotyrosine protein phospha- Erythroid differentiation is characterized by the induction of tase (PTPase) activity in the particulate membrane fraction, whereas the globin synthesis and the accumulation of hemoglobin together activity in the soluble cytosol fraction increased about 3-fold. The de with the expression of erythrocyte specific surface antigens (15) crease of PTPase activity in the particulate membrane fraction could be and a reduction of protein tyrosine phosphorylation (27). attributed to the disappearance of at least 1 distinct PTPase form displaying an apparent native M, of 200,000 and a reduction in activity Reversible tyrosine phosphorylation plays an important role of a M, 43,000 PTPase found associated with membranes of all cells in signal transduction in normal cells (28, 29) and has recently examined to date. The increase of PTPase activity in the cytosol fraction been implicated as crucial for differentiation and activation of manifested itself by the appearance of a new M, 40,000 PTPase and a hematopoietic cells (30-37). Frank and Sartorelli (38-40) have reduction of a A/, 60,000 PTPase. These results suggest the existence of observed a decrease in total tyrosine-phosphorylation during several growth- and/or differentiation-related PTPase activities in K562 the chemically induced differentiation of human HL-60 cells. cells. This decrease was shown to be associated with an overall activation of PTPase' activity. INTRODUCTION We have chosen the K562 cell line to study the changes in the PTPase activity during differentiation toward megakaryo- The K562 cell line was established in 1970 from a patient cytes induced by PMA. Differentiation was accompanied by an suffering from chronic myelogenous leukemia (1). The cell line arrest of growth, a change in morphology, and an increased exhibits the Philadelphia chromosome Ph1 (2-4), a chromo adherence of the cells. PMA-treated cells expressed glycopro somal aberration involving a 9:22 chromosomal translocation tein Ilia, a marker for differentiation along the megakaryocytic found in more than 90% of chronic myelogenous leukemia lineage. Interestingly, PMA treatment caused the disappearance cases (5, 6). The breakpoint on chromosome 22 is within the of soluble G-actin from the cytoplasm, a finding that is probably so-called breakpoint cluster region, which encodes an actively related to the morphological changes and that can be used as a transcribed gene of unknown function (7, 8). The translocated differentiation marker. Analysis of the PTPase activities present region of chromosome 9 encompasses the gene encoding the c- in the particulate membrane and the soluble cytosol fractions abl protooncogene tyrosine kinase (9). The newly formed tran from control and PMA-treated cells revealed quantitative and scription unit encodes a p210**â„¢*'fusion protein, consisting of qualitative differences. These results represent the first dem a NH2 terminus derived from the breakpoint cluster region onstration that cells may possess different PTPase activities fused to a jV-terminally truncated c-abl protein (10, 11). This depending on their growth and differentiation state. rearrangement results in activation of the c-abl cryptic tyrosine kinase activity (12). K562 cells can be regarded as pluripotent hematopoietic progenitor cells expressing markers for eryth- MATERIALS AND METHODS roid, granulocytic, monocytic, or megakaryocytic lineages as Materials. Hepes, <-amino-n-caproic acid, aprotinin, and bovine defined by surface-antigen expression (13). Treatment of K562 serum albumin were purchased from Sigma (St. Louis, MO); glycerol cells with phorbol esters (phorbol dibutyrate, phorbol 12-my was from Bethesda Research Laboratories (Gaithersburg, MD); acryl- ristate 13-acetate) can induce differentiation along a megakar- amide and AOY'-méthylènebis-acrylamidewere from Serva (Heidel yocytic-monocytic lineage (14, 15). In contrast, treatment with berg, Federal Republic of Germany); ammonium molybdate was from hemin (16), 5-azacytidin (17), l-/3-D-arabinofuranosylcytosine Fluka (Buchs, Switzerland); PMA and protein-grade Triton X-100 were (18), daunomycin (19), or herbimycin A (20) can induce differ- from Calbiochem (Luzern, Switzerland); and the remaining chemicals were from Merck (Darmstadt, Federal Republic of Germany). High- Received 8/7/89; accepted 6/27/90. performance liquid chromatography columns TSK-DEAE 3SW (7.5 x The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in 3The abbreviations used are: PTPase, phosphotyrosine protein phosphatase; accordance with 18 U.S.C. Section 1734 solely to indicate this fact. DEAE, diethylaminoethyl; 2-ME. 2-mercaptoethanol: Hepes. iV-2-hydroxylpiper- Received 8/7/89; revised 5/29/90. azine-A"-2-ethanesulfonic acid; HPLC. high-performance liquid chromatogra 1Present address: Department of Biochemistry, J405 Health Sciences Build phy; PMA, phorbol 12-myristate 13-acetate; SDS, sodium dodecyl sulfate; PTG, ing. SJ-70, University of Washington, Seattle, WA 98I95. 0.2% gelatin, 0.25% Triton X-100 in phosphate-buffered saline; DMSO, dimethyl 1To whom requests for reprints should be addressed. sulfoxide. 6323 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1990 American Association for Cancer Research. PHOSPHOTYROSYL PROTEIN PHOSPHATASES IN DIFFERENTIATION 150 mm), TSR G2000 SW (7.5 x 600 mm), and TSK-G2000 GSW Cell Fractionation of KS62 Cells. K562, a human chronic myeloid (21.5 x 600 mm) were obtained from LKB (Uppsala, Sweden). [y-"P] leukemia cell line, was grown in RPMI 1640 medium (GIBCO, Paisley, ATP was purchased from Amersham International (Amersham, United Scotland) supplemented with 10% fetal calf serum (Seromed, Berlin, Kingdom). Federal Republic of Germany) and penicillin (100,000 international Protease inhibitors used were <-amino-n-caproicacid (1 mivi),phenyl- units/liter)/streptomycin (100 mg/liter). Cells were collected by cen- methylsulfonyl fluoride (1 mM), and aprotinin (0.05 trypsin inhibitory trifugation, washed twice with phosphate-buffered saline, and resus- units/ml). pended in a small volume of hypotonie buffer (10 mM Hepes, pH 7.0; Substrate Phosphorylation. The isolation of membranes from the 10 mM NaCl; 10 mM NaF; 5 mM EDTA; 10 HIM2-ME; and protease human epidermoid carcinoma cell line A431 containing the epidermal inhibitors). After 15 min on ice. the cells were homogenized with 20 growth factor-receptor has been described elsewhere (41). For substrate strokes in a glass Dounce homogenizer, and nuclei and cell debris were preparation, 10 ¿jgofA431 membranes were incubated with 2 ng of sedimented by centrifugation for 20 min at 2000 x g at 4°C.The epidermal growth factor (Collaborative Research Inc., Bedford, MA) clarified supernatant was centrifuged for 45 min at 100,000 x g at 4°C. for 15 min on ice in kinase buffer (20 mM Hepes, pH 7.2; 1 mM MnCh, The supernatant was used to assay for the soluble PTPase activity. The 5 mM MgCI2, 0.2% NP-40, 0.1 mg/ml bovine serum albumin. 10 m,M pellet was resuspended in Buffer A (without Triton X-100), extracted 2-ME, protease inhibitors, and 0.1 m\i vanadate). At this point, 20 ^Ci with 19c Triton X-100 (protein grade; Calbiochem, Luzern, Switzer [-y-32P]ATP(specific activity, 5000 Ci/mmol) were added and the in land) and centrifuged for 30 min at 30,000 x g. The resulting super cubation continued on ice for 15 min. Unincorporated labeled ATP natant was used to measure the paniculate PTPase activity. was separated from phosphorylated protein by passing over 2 small Phosphotyrosyl Protein Phosphatase Assay. A PTPase-containing Sephadex G-50 columns (4-ml bed volume) equilibrated with buffer A sample was incubated in a 100-^1 reaction volume at 30°Cin Buffer B (20 mM Hepes, pH 7.0; 1 mM NaF. 1 mM EDTA, 10 mM 2-ME, and (20 mM Hepes, pH 7.0; 5 mM EDTA; 20 mM NaF; 1 mM i-amino-n- protease inhibitors) containing 0.19o Triton X-100. caproic acid; 1 mM phenylmethylsulfonyl fluoride; and 10 mM 2-ME) containing 30,000 cpm of-"P-labeled substrate (4.5 fmol 12P).After 15 7.On min, the reaction was stopped by adding 0.1 mg of bovine serum albumin and 1:10 volumes of ice-cold 100% trichloroacetic acid and transferring the tube onto ice. The amount of released J2P¡wasdeter ,_ 6.5 control v mined by counting the molybdate extractable radioactivity following .
Load more

Recommended publications
  • ©Ferrata Storti Foundation
    original paper Haematologica 1997; 82:395-401 HUMAN LEUKEMIA K562 CELLS: INDUCTION TO ERYTHROID DIFFERENTIATION BY GUANINE, GUANOSINE AND GUANINE NUCLEOTIDES FABIO OSTI,* FEDERICA GINANNI CORRADINI,* STEFANIA HANAU,* MAURIZIO MATTEUZZI,* ROBERTO GAMBARI*° *Department of Biochemistry and Molecular Biology; °Biotechnology Center, University of Ferrara; Italy ABSTRACT Background and Objective. Human leukemic guanine, guanosine and guanine ribonucleotides K562 cells are able to undergo erythroid differenti- are effective inducers of K562 cell differentiation. ation in vitro when cultured with a variety of induc- Expression of both Hb Portland and Hb Gower 1 is ers, leading to increased expression of embryo-fetal increased in GTP-induced K562 cells. This increase globin genes such as the ␨, ⑀ and ␥-globin genes. is associated with greater ␥-globin mRNA accumu- Therefore the K562 cell line has been proposed as a lation. By contrast, ATP, CTP and UTP are not able very useful in vitro model system for determining the to induce erythroid differentiation. therapeutical potential of new differentiating com- Interpretaton and Conclusions. These findings sug- pounds as well as for studying the molecular mech- gest that guanine, guanosine and guanine ribonu- anism(s) that regulate changes in the expression of cleotides are inducers of erythroid differentiation of embryonic and fetal human globin genes. In this K562 cells. This is of some relevance since differen- study we explored whether nucleoside triphos- tiating compounds have been proposed as antitu- phates and related compounds are able to induce mor agents. In addition, inducers of erythroid dif- ferentiation that stimulate ␥-globin synthesis might differentiation of K562 cells. Methods. be considered in the experimental therapy of hema- K562 cell differentiation was studied tological diseases associated with a failure in the using the benzidine test; hemoglobins were charac- expression of adult ␤-globin genes.
    [Show full text]
  • Profiling of Metabolic Differences Between Hematopoietic Stem Cells
    H OH metabolites OH Article Profiling of Metabolic Differences between Hematopoietic Stem Cells and Acute/Chronic Myeloid Leukemia 1, 2, 1, 1 1 Byung Hoo Song y, Su Young Son y, Hyun Kyu Kim y, Tae Won Ha , Jeong Suk Im , Aeli Ryu 3, Hyeji Jeon 3, Hee Yong Chung 4, Jae Sang Oh 5,* , Choong Hwan Lee 2,6,* and Man Ryul Lee 1,* 1 Soonchunhyang Institute of Medi-Bio Science (SIMS), Soon Chun Hyang University, Cheonan 31151, Korea; [email protected] (B.H.S.); [email protected] (H.K.K.); [email protected] (T.W.H.); [email protected] (J.S.I.) 2 Department of Bioscience and Biotechnology, Konkuk University, Seoul 05029, Korea; [email protected] 3 Department of Obstetrics and Gynecology, Soonchunhyang University Cheonan Hospital, Chungcheongnam-do 31151, Korea; [email protected] (A.R.); [email protected] (H.J.) 4 Department of Microbiology, College of Medicine and Department of Biomedical Science, Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul 04763, Korea; [email protected] 5 Department of Neurosurgery, College of Medicine, Soonchunhyang University, Cheonan Hospital, Cheonan 31151, Korea 6 Research Institute for Bioactive-Metabolome Network, Konkuk University, Seoul 05029, Korea * Correspondence: [email protected] (J.S.O.); [email protected] (C.H.L.); [email protected] (M.R.L.); Tel.: +82-10-2918-3903 (J.S.O.); +82-2-2049-6177 (C.H.L.); +82-41-413-5013 (M.R.L.) These authors equally contributed to this work. y Received: 29 September 2020; Accepted: 22 October 2020; Published: 26 October 2020 Abstract: Although many studies have been conducted on leukemia, only a few have analyzed the metabolomic profiles of various leukemic cells.
    [Show full text]
  • Role of Ras/ERK-Dependent Pathway in the Erythroid Differentiation of K562 Cells
    EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 31, No 2, 76-82, June 1999 Role of Ras/ERK-dependent pathway in the erythroid differentiation of K562 cells Chi-Dug Kang1,2,5, In-Rok Do2, the elevated activity of ERK/MAPK to the level of Kwang-Woon Kim2, Byung-Kwon Ahn2, parental cells, the growth rate increased and the Sun-Hee Kim2, Byung-Seon Chung2, erythroid differentiation decreased slightly, and at Byung-Hak Jhun1,3 and Mi-Ae Yoo1,4 high concentration of PD98059, which inhibited the elevated activity of ERK/MAPK below that of the 1 Pusan Cancer Research Center, Pusan National University, Pusan 602-739, Korea parental cells, the growth rate turned down and the 2 Department of Biochemistry, College of Medicine, Pusan National University, Pusan erythroid differentiation was restored to the untreated 602-739, Korea control level. Taken together, these results suggest 3 Department of Pharmacy, College of Pharmacy, Pusan National University, Pusan that an appropriate activity of ERK/MAPK is required 609-735, Korea to maintain the rapid growth and transformed pheno- 4 Department of Molecular Biology, College of Natural Science, Pusan National type of K562 cells. University, Pusan 609-735, Korea 5 Corresponding author: Tel, +82-51-240-7739; Fax, +82-51-248-1118, E-mail, Keywords: K562, erythroid differentiation, Ras/ERK [email protected] pathway, PD98059 Accepted 17 May 1999 Introduction Abbreviations: CML, Chronic myelogenic leukemia; PTK, protein tyrosine kinase; The K562 cell line was established from pleural effusion MAPK/ERK-activating kinase, MEK; ERK, extracellular signal-regulated kinase; MAPK, of a patient with chronic myelogenous leukemia (CML) mitogen-activating kinase; SDS, sodium-dodecyl sulfate; PAGE, polyacrylamide gel- in blast crisis (Lozzio and Lozzio, 1975).
    [Show full text]
  • Megakaryocytic Differentiation of K562 Cells Is Associated with Changes
    [CANCER RESEARCH 50, 6323-6329. October 1. I990| Megakaryocytic Differentiation of K562 Cells Is Associated with Changes in the Cytoskeletal Organization and the Pattern of Chromatographically Distinct Forms of Phosphotyrosyl-specific Protein Phosphatases T. Martin Butler,1 Andrew Ziemiecki, and Robert R. Friis2 Institute for Clinical and Experimental Cancer Research, University of Bern, Tiefenaustrasse 120, CH-3004 Bern, Switzerland ABSTRACT entiation along an erythroid lineage. Herbimycin A, an inhibitor of protein phosphorylation, has been shown to induce differ We have analyzed morphological and biochemical changes occurring entiation of mouse embryonal carcinoma (F9), erythroleukemia during megakaryocytic differentiation of the human chronic myelogenous (MEL), and K562 cells (20, 21). Differentiation toward mega- leukemia cell line K562 induced by phorbol 12-myristate 13-acetate (I'M A). PMA-treated cells became growth arrested, were slightly larger karyocytes can be monitored by a reduction of growth potential and irregular in shape, adhered better to the culture flask surface, and (22), a reduction of expression of erythroid surface markers expressed the glycoprotein Ilia on their surfaces. The morphological (15), a reduction of c-myc expression (18, 23), an induction of changes induced by PMA treatment were associated with the disappear surface markers of the megakaryocytic lineage (15), an induc ance of actin from the cytosol and presumably reflect PMA-induced actin tion of c-sis and c-fos mRNA (23-25), and translocation of polymerization. Megakaryocytic differentiation was accompanied by protein kinase C from the cytoplasm to the membrane (22, 26). about a 3-fold decrease in the specific phosphotyrosine protein phospha- Erythroid differentiation is characterized by the induction of tase (PTPase) activity in the particulate membrane fraction, whereas the globin synthesis and the accumulation of hemoglobin together activity in the soluble cytosol fraction increased about 3-fold.
    [Show full text]
  • Induction of Differentiation of the Acute Myeloid Leukemia Cell Line (HL-60) by a Securinine Dimer Wen Hou1, Zhen-Ya Wang1, Jing Lin1 and Wei-Min Chen 1
    Hou et al. Cell Death Discovery (2020) 6:123 https://doi.org/10.1038/s41420-020-00354-3 Cell Death Discovery ARTICLE Open Access Induction of differentiation of the acute myeloid leukemia cell line (HL-60) by a securinine dimer Wen Hou1, Zhen-Ya Wang1, Jing Lin1 and Wei-Min Chen 1 Abstract Differentiation therapy has been successfully applied clinically in cases of acute promyelocytic leukemia (APL), but few differentiation-induction agents other than all-trans retinoic acid (ATRA) have been discovered clinically. Based on our previously reported neuritogenic differentiation activity of synthetic dimeric derivatives of securinine, we explored the leukemia differentiation-induction activity of such as compound, SN3-L6. It was found that SN3-L6 induces transdifferentiation of both acute myeloid leukemia (AML) and chronic myelogenous leukemia (CML) cells but unexpectedly, a new transdifferentiation pathway from APL cells to morphologically and immunologically normal megakaryocytes and platelets were discovered. SN3-L6 fails to induce transdifferentiation of ATRA–produced mature granulocytes into megakaryocytes, indicating its selectivity between mature and immature cells. SN3-L6 induces CML K562 cells to transdifferentiate into apoptotic megakaryocytes but without platelet formation, indicating a desirable selectivity between different leukemia cells. Our data illuminate a differentiation gap between AML cells and platelets, and promises applications in leukemia differentiation therapy strategy. Introduction ATRA 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; differentiation-induction agents other than have Leukemia is characterized by a blockage of cell differ- since been discovered clinically. It is well known that entiation (Fig. 1). The most common method for treat- inducing APL cells to differentiate into mature granulo- ment of leukemia involves chemotherapeutic agents that cytes is the main pharmacologic action of ATRA.
    [Show full text]
  • Selective Induction of Apoptosis in Philadelphia Chromosome-Positive Chronic Myelogenous Leukemia Cells by an Inhibitor of BCR ± ABL Tyrosine Kinase, CGP 57148
    Cell Death and Differentiation (1998) 5, 710 ± 715 1998 Stockton Press All rights reserved 13509047/98 $12.00 http://www.stockton-press.co.uk/cdd Selective induction of apoptosis in Philadelphia chromosome-positive chronic myelogenous leukemia cells by an inhibitor of BCR ± ABL tyrosine kinase, CGP 57148 Shingo Dan1, Mikihiko Naito1 and Takashi Tsuruo1,2,3 which is detected in virtually all cases. It is formed by a translocation between chromosome 9 and 22 (de Klein et al, 1 Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo 1982; Heisterkamp et al, 1983). This translocation results in a 2 Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, production of the BCR ± ABL fusion protein with enhanced Tokyo, Japan 3 tyrosine kinase activity, which is a major factor in the corresponding author: Institute of Molecular and Cellular Biosciences, The pathophysiology of CML (Heisterkamp et al, 1985; 1990). University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113, Japan. tel: 81-3-3818- 5341; fax: 81-3-3816-3592; e-mail: [email protected] The BCR ± ABL tyrosine kinase phosphorylates cellular proteins such as CRKL and CBL (Andoniou et al, 1994; Received 10.11.97; revised 6.3.98; accepted 2.4.98 Nichols et al, 1994; Oda et al, 1994). The phosphorylated Edited by: Y. Kuchino CRKL and CBL form a stable complex including BCR ± ABL and phosphatidylinositol-3' kinase (PI3K) (Sattler et al, 1996), which in turn activates PI3K to promote cell Abstract proliferation (Skorski et al, 1995). BCR ± ABL is also The BCR ± ABL tyrosine kinase has been implicated as the shown to activate Ras-dependent signaling pathways cause of Philadelphia chromosome (Ph1)-positive leukemias.
    [Show full text]
  • The Effect of BCR-ABL Specific Tyrosine Kinase Inhibitors
    Article The Effect of BCR-ABL Specific Tyrosine Kinase Inhibitors on the Thioredoxin System in Chronic Myeloid Leukemia Erin Clapper 1,2, Giovanna Di Trapani 1,* and Kathryn F. Tonissen 1,2,* 1 School of Environment and Science, Griffith University, Nathan, QLD 4111, Australia; E.Clapper@griffith.edu.au 2 Griffith Institute for Drug Discovery, Griffith University, Nathan, QLD 4111, Australia * Correspondence: G.DiTrapani@griffith.edu.au (G.D.T.); K.Tonissen@griffith.edu.au (K.F.T.) Abstract: Chronic myeloid leukemia (CML) is a myeloproliferative disorder that is caused by the formation of a fusion onco-protein, BCR-ABL. Since BCR-ABL plays a role in the progression of CML, the most common treatments of CML are tyrosine kinase inhibitors (TKIs) that specifically target BCR-ABL. However, resistance to TKIs is a major problem in CML treatment. A promising target in overcoming drug resistance in other cancers is the thioredoxin (TRX) system, an antioxidant system that maintains cellular redox homeostasis. The TRX system is upregulated in many cancers and this is associated with a poor prognosis. Analysis of a patient database showed that the expression of the TRX system was upregulated in CML patients compared to healthy donors. Our experiments revealed a significant link between the TRX and BCR-ABL systems since inhibition of BCR-ABL with chemical inhibitors and siRNA resulted in a decrease in the activity and expression of the TRX system in CML cells. This is notable as it shows that the TRX system may be a viable target in the treatment of CML. Citation: Clapper, E.; Di Trapani, G.; Keywords: chronic myeloid leukemia; BCR-ABL; thioredoxin; drug resistance; tyrosine kinase in- Tonissen, K.F.
    [Show full text]
  • Erythropoietin Promotes Resistance Against the Abl Tyrosine Kinase Inhibitor Imatinib (STI571) in K562 Human Leukemia Cells
    970 Vol. 1, 970–980, November 2003 Molecular Cancer Research Erythropoietin Promotes Resistance Against the Abl Tyrosine Kinase Inhibitor Imatinib (STI571) in K562 Human Leukemia Cells Karin M. Kirschner and Kurt Baltensperger Institute of Pharmacology, University of Bern, Bern, Switzerland Abstract malignant transformation of cells. Recent advances in the Chronic myeloid leukemia is characterized by the treatment of CML have largely relied on imatinib mesylate Philadelphia chromosome translocation that causes (STI571, Gleevec), a rationally designed inhibitor of the Abl expression of Bcr-Abl, a deregulated tyrosine kinase. tyrosine kinase (3). Imatinib potently suppresses the tyrosine Imatinib mesylate (STI571, Gleevec), a therapeutically kinase activity of the Bcr-Abl fusion protein and induces used inhibitor of Bcr-Abl, causes apoptosis of Bcr- apoptosis in Bcr-Abl-positive cells (4–9). The success of this Abl-positive cells. In the leukemia cell line K562, we novel drug is based on Bcr-Abl’s central place as the single observed spontaneous resistance to imatinib at very low most important component to maintain factor independence of frequencies when cells were exposed to the drug (1 MM) CML cells. The efficacy of imatinib in the treatment of chronic for more than 4 weeks. Surprisingly, in the presence of phase CML is well documented (8). Resistance resulting in erythropoietin (Epo), K562 cells were temporarily able to failure of treatment at initiation of therapy seems to be rare. sustain proliferation in the presence of imatinib, and However, several recent reports indicate that patients in the imatinib-resistant clones could be isolated with high accelerated phase and blast crisis that first responded well but frequencies.
    [Show full text]
  • And Imatinib-Resistant Chronic Myelogenous Leukemia Cells Seiichi Okabe, Tetsuzo Tauchi, and Kazuma Ohyashiki
    Cancer Therapy: Preclinical Characteristics of Dasatinib- and Imatinib-Resistant Chronic Myelogenous Leukemia Cells Seiichi Okabe, Tetsuzo Tauchi, and Kazuma Ohyashiki Abstract Purpose: Althoughdualsrc-familykinase/BCR/ABLinhibitor,dasatinib (BMS-354825),provides therapeutic advantages to imatinib-resistant cells, the mechanism of dasatinib resistance was not fully known. Experimental Design: We used TF-1 BCR/ABLcells, by introducing the BCR/ABL gene into a leukemia cell line,TF-1and K562, and established dasatinib- (BMS-R) and imatinib-resistant (IM-R) cells.We characterized chronic myelogenous leukemia drug-resistant cells and examined intracellular signaling. Results: The IC50 of dasatinib was 0.75 nmol/L(TF-1BCR/ABL), 1nmol/L(K562), 7.5 nmol/L (TF-1 BCR/ABLIM-R), 10 nmol/L(K562 IM-R), 15 Amol/L(TF-1 BCR/ABLBMS-R), and 25 Amol/L(K562 BMS-R).The number of BCR/ABLcopies in resistant cell lines was the same as the parental cell line by fluorescence in situ hybridization analysis. There was no mutation in Abl kinase.We found that protein levels of BCR/ABLwere reduced in dasatinib-resistant cell lines. BCR/ABLprotein was increased by treatment of an ubiquitin inhibitor.The Src kinase, Lck, as well as mitogen-activated protein kinase and Akt were activated, but p21WAF, phosphatase and tensin homologue was reduced in K562 BMS-R cells. Removal of dasatinib from the culture medium of K562 BMS-R cells led to apoptosis, and activated caspase 3 and poly (ADP-ribose) polymerase. Conclusion:These results suggest that the expression and protein activation signatures identified in this study provide insight into the mechanism of resistance to dasatinib and imatinib and may be of therapeutic chronic myelogenous leukemia value clinically.
    [Show full text]
  • Induction of Erythroid Differentiation in K562 Cells by Inhibitors of Inosine Monophosphate Dehydrogenase1
    [CANCER RESEARCH 49. 5555-5560. October 15. 1989] Induction of Erythroid Differentiation in K562 Cells by Inhibitors of Inosine Monophosphate Dehydrogenase1 John Yu,2 Victor Lemas, Theodore Page, James D. Connor, and Alice L. Yu Department of Molecular and Experimental Medicine, Research Institute of Scripps Clinic, La Jolla, California 92037 fj. Y., V. L.], and Department of Pediatrics, University of California at San Diego, La Jolla, California 92093 [T. P., J. D. C., A. L. Y. ] ABSTRACT by incubation with ribavirin were also analyzed. Preliminary reports of this investigation have been presented (15, 16). The effects of three inhibitors of inosine monophosphate (IMP) de- hydrogenase on a human erythroleukemic cell line, K562, were studied. Following incubation with these inhibitors, K562 cells underwent differ MATERIALS AND METHODS entiation and accumulated hemoglobins. The induction of hemoglobin accumulation was dose dependent; maximum induction was observed at Cell Culture. Stock cultures of K562 (American Type Culture Col 100, 25, and 3 MM,respectively, for ribavirin, tiazofurin, and mycophen- lection, Rockville, MD) were grown in RPMI 1640 medium supple olic acid. The induction was associated with reduction of intracellular mented with 50 lU/ml of penicillin, 50 Mg/ml of streptomycin, and GTP content and was blocked by adding guanosine within 24 h after 15% fetal calf serum (Hyclone Laboratories, Inc., Logan, UT), as adding inducer. The effective dose for half-maximum induction by riba previously described (17). Cells were caused to differentiate by addition virin was 3 times less than that for 50% inhibition of K562 proliferation; of inducers to final concentrations as specified in the text.
    [Show full text]
  • Induction of Terminal Differentiation in Human K562 Erythroleukemia Cells by Arabinofuranosylcytosine
    Induction of terminal differentiation in human K562 erythroleukemia cells by arabinofuranosylcytosine. C Luisi-DeLuca, … , D Spriggs, D W Kufe J Clin Invest. 1984;74(3):821-827. https://doi.org/10.1172/JCI111498. Research Article We have previously demonstrated that continuous exposure of human HL-60 human promyelocytes to 1-beta-D- arabinofuranosylcytosine (ara-C) results in the induction of terminal differentiation to monocyte-like cells. The present study extends these findings by demonstrating that ara-C induces hemoglobin synthesis in human K562 erythroleukemia cells. This effect occurs maximally at an ara-C concentration (5 X 10(-7) M) that results in K562 cytostasis. In contrast to the reversible effects of hemin and hydroxyurea on globin synthesis in this cell line, we have found that the induction of K562 hemoglobin synthesis by ara-C is irreversible. An induction of K562 hemoglobin synthesis also occurs with aphidicolin, another inhibitor of S-phase DNA synthesis, but not with vinblastine, an inhibitor of mitosis. Finally, ara-C induction of a differentiated K562 phenotype is accompanied by the loss of self-renewal capacity, a finding consistent with terminal differentiation. Find the latest version: https://jci.me/111498/pdf Induction of Terminal Differentiation in Human K562 Erythroleukemia Cells by Arabinofuranosylcytosine Cynthia Luisi-DeLuca, Thomas Mitchell, David Spriggs, and Donald W. Kufe Division of Medicine, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115 Abstract. We have previously demonstrated inhibition of replicative DNA synthesis by ara-C causes DNA that continuous exposure of human HL-60 human fragmentation (4). Furthermore, there is a highly significant promyelocytes to 1-fl-D-arabinofuranosylcytosine (ara- relationship between the incorporation of ara-C into DNA and C) results in the induction of terminal differentiation to the loss of leukemic cell clonogenic survival (2, 5, 6).
    [Show full text]
  • Detection of BCR-ABL Proteins in Blood Cells of Benign Phase Chronic Myelogenous Leukemia Patients1
    ICANCER RESEARCH 51. 3048-3051. June 1. 19911 Advances in Brief Detection of BCR-ABL Proteins in Blood Cells of Benign Phase Chronic Myelogenous Leukemia Patients1 Jie Qiang Guo, Jean Y. J. Wang, and Ralph B. Arlinghaus2 Department of Molecular Pathology, The University of Texas, M. D. Anderson Cancer Center, Houston, Texas 77030 fJ. Q. C., R. B. A.], and Department of Biology, The University of California at San Diego. La lolla, CA 92093 [J. Y. J. W.¡ Abstract protein in cell lines derived from blast crisis CML patients. The BCR-ABL protein kinase assay has also been used to detect More than 95% of patients with chronic myelogenous leukemia (CML) P210 BCR-ABL in uncultured cells from patients in blast crisis contain an abnormal chromosome termed the Philadelphia chromosome (Ph1). I'll1 and the resulting BCR-ABL fused genes are markers for this (13). Attempts to detect the BCR-ABL protein in chronic phase type of leukemia. The product of the fused BCR-ABL genes is a protein patients by this assay have been hindered by large numbers of of about 2000 amino acids termed P210 BCR-ABL. Although the BCR- mature cells in blood and bone marrow samples in these pa ABL protein can be routinely detected in blood cells from blast crisis tients, which contain high concentrations of degradative en CML patients by assaying for its activated tyrosine kinase activity, zymes (13). Extracts of mature granulocytes, from either nor detection of P210 BCR-ABL in early stage CML patients (chronic phase) mal or CML patients, rapidly destroy P210 kinase activity from has not yet been possible (S.
    [Show full text]