[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 .