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'-mÃ©thylÃ¨nebis-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

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 Â¿jgofA431 membranes were incubated with 2 ng of sedimented by centrifugation for 20 min at 2000 x g at 4Â°C.The epidermal growth factor (Collaborative Research Inc., Bedford, MA) clarified supernatant was centrifuged for 45 min at 100,000 x g at 4Â°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 30Â°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 J2PÂ¡wasdeter ,_ 6.5 control v mined by counting the molybdate extractable radioactivity following .. the procedure of Antoniw and Cohen (42). One unit of PTPase activity was defined as the release of 1 fmol "Pi/min. Protein concentrations ! 6.0- were determined by the method of Bradford (43). Western Immunoblotting. A SDS-polyacrylamide gel (44) was blotted â€¢â€¢ PMA- - 5.5-1 trected onto nitrocellulose using the semidry electroblotting method described by Kyhse-Andersen (45). After blotting, the nitrocellulose was blocked in 0.5% gelatin (calfskin. Sigma) in phosphate-buffered saline for l h at 37Â°Cand rinsed twice in PTG for 10 min each at 37"C. The antibody 5.0 3 diluted in PTG was added in a minimal volume (5 ml) and incubated days at 37Â°Cfor l h in a rotating drum incubator (Bachofer, Reutlingen, Fig. I. Growth curve of K562 cells. K562 cells were grown in RPMI 1640 Federal Republic of Germany). Unbound antibody was washed away medium supplemented with \0rÂ¿fetal calf serum. On day I, 10 n\i PM.V in with PTG at 37Â°C(3-4 times, 10 min), '"I-labeled protein A (0.1 Â»Ci/ DMSO was added to induce differentiation cells. Control cells received DMSO ml) was added in a minimal volume of PTG (5 ml) and incubated at the same concentration. Medium was changed every day and fresh PMA or further at 37"C for 30 min. Unbound I25l-protein A was washed off DMSO was added.

Fig. 2. Morphology and glycoprotein Ilia expression of TPA-treated K562 cells. Cells were treated with TPA for 2 days as outlined in Fig. I. Details of indirect gp Ilia immunofluorescence are given in "Materials and Meth ods." a and c. Untreated cells; b and d. TPA- treated cells; c and Â¡I.gpIlia immunofluorescence. a and ft. x 200; c and

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100- O control 50-

o 2345 B) days 400-1 350- 300- o control Ã 250- o o 200-1 Fig. 3. Soluble G-actin disappears in PMA-treated K562 cells. A, SDS- polyacrylamide gel of the peak fractions (142) from gel permeation-HPLC of the g 150-I soluble fraction from control K562 cells (/) and from PMA-treated K562 cells o. PMA-treated (2). Fraction 142 of the gel permeation corresponds to an apparent M, of 43,000. " 100- B, autoradiograph of a Western blot of the soluble fractions from control (1-3) and PMA-treated (5-7) K562 cells. HPLC gel permeation fractions 130 (/ and 50- 5), 142 (2 and 6), and 154 (3 and 7) were loaded on a SDS-polyacrylamide gel, blotted onto nitrocellulose, and reacted with a chicken anti-rabbit smooth-muscle actin antiserum. Lane 4 was unloaded. days with 4 changes of PTG and the nitrocellulose blot was exposed to a Fig. 4. Specific PTPasc activity in the soluble and paniculate fraction of Kodak X-ray film. PMA-induced and control K562 cells. Cells were grown for 1 day in the absence of inducers. On day 1, 10 nM PMA in DMSO was added to half of the cells to Immunofluorescence. Indirect immunofluorescence was done using induce differentiation while control cells received DMSO alone. Fresh medium an anti-gp Ilia monoclonal antiserum from Dr. David Mason (Oxford, containing inducers was added every day. Each day, 1 flask of cells, induced and United Kingdom) kindly provided by Dr. William Vainchenker (Paris, control, was collected and cells were fractionated. Specific PTPase activity was France) (46). Unstimulated and 2-day PMA-stimulated cells were measured in the soluble and the paniculate fraction using the epidermal growth factor-receptor substrate incubated with 1 ng of PTPase sample for 15 min at stained, washed, and fluorescein isothiocyanate counterstained in sus 30Â°C.A, Specific PTPase activity in the soluble fraction; B. specific PTPase pension culture medium containing 5% fetal calf serum on ice. The activity in the paniculate fraction. cells were cytocentrifuged onto microscope slides, mounted, and micro scopically examined. treated K562 cells (Fig. 3A). This protein reacted in Western immunoblots with a rabbit antiserum raised against chicken RESULTS smooth-muscle actin (Fig. 3Ã„). Differentiation of K562 Cells in Culture. K562 cells were Specific PTPase Activities in K562 Cells after PMA Treat seeded in 6-well plates at a density of IO5 cells/well in RPMI ment. The changes in specific PTPase activity in the particulate 1640 medium containing 10% fetal calf serum. Following 1 day and the soluble fraction after cell fractionation of either control of culture, differentiation was induced by the addition of 10 nM or PMA-treated cells were analyzed (Fig. 4). In control cells, PMA in DMSO. Control cells received the same concentration the specific PTPase activity remained about constant in both of DMSO alone. Medium, containing PMA in DMSO or the particulate and the soluble fraction during the 4-day induc DMSO alone, was changed daily and cell number was deter tion period. In PMA-treated cells, however, a decrease in activ mined for 6 consecutive days. The addition of 10 nM PMA to ity of about 3-fold was observed in the particulate fraction, K562 cells caused an immediate growth arrest (Fig. 1). A whereas in the soluble fraction the specific PTPase activity distinct change in morphology could be observed upon micro increased about 3-fold (Fig. 4). scopic examination; the cell became slightly larger and adhered Partial Purification of the Particulate PTPase Activity from stronger to the plastic of the culture flask (Fig. 1). Indirect PMA-treated and Control K562 Cells. Membrane extracts of immunofluorescent staining using an anti-glycoprotein Ilia PMA-treated and DMSO control cells were injected on TSK monoclonal antibody revealed extensive cell surface expression DEAE 3SW HPLC columns equilibrated with Buffer C (25 on 12-O-tetradecanoylphorbol-13-acetate-treated cells (Fig. 2, mivi Hepes, pH 7.1; 5% glycerol; 0.1% Triton X-100; 1 mM compare c and d). NaF, 10 mM 2-ME; and protease inhibitors). A NaCl gradient Changes in the cytosolic protein pattern of PMA-treated and in Buffer C was developed up to 300 mM NaCl, 0.5-ml fractions control K562 cells were monitored by SDS-polyacrylamide gel were collected, and PTPase activity was assayed in alternate electrophoresis to detect possible markers for differentiation. A fractions (Fig. 5). In control K562 cell extracts, 2 PTPase major soluble protein with an apparent (gel permeation) and activities could be resolved: Peak P-l, eluting with 110 mM denatured (SDS-polyacrylamide gel electrophoresis) M, of NaCl, and Peak P-2, eluting with 140 mM NaCl (Fig. 5A). In 45,000 was lost completely from the soluble fraction of PMA- K562 cells induced to differentiate with PMA, the first peak P- 6325

HPLC-TSK G2000 SW colibrotion

14 16 18 22 ml elution volume B) 20 30 40 50 60 70 80 90 B) fraction number 40 -, 400 43 kD P-2 35- â€¢350 30- â€¢300 25- â€¢250 20- â€¢200 15- â€¢150

10- â€¢100 10 12 14 16 18 20 22 ml elution volume 5- 50 Fig. 6. Gel permeation-HPLC of the major paniculate DEAE PTPase peaks. 0 0 A, DEAE peak P-2 of membrane extracts from control cells was injected on a 0 10 20 30 40 50 60 70 80 90 HPLC TSK G200 SW gel permeation column equilibrated in Buffer D (Buffer C containing 300 mM NaCl). The column was run isocratically. 0.3-ml fractions fraction number were collected, and PTPase activity was assayed in every fraction. B, DEAE peak Fig. 5. Partial purification of the paniculate PTPase activity of PMA-induced P-2 of membrane extracts from PMA-treated cells was analyzed on a HPLC TSK and control K562 cells by high-performance liquid DEAE chromatography. G2000 SW gel permeation column as described above. Inset, column-calibration Membrane extracts of PMA-treated and DMSO-treated control cells were sepa run under the same conditions as described above with RNase A (M, 13,700), rated on a HPLC-TSK DEAE 3SW column equilibrated with Buffer C (see chymotrypsinogen A (M, 25,000). ovalbumin (M, 43,000), bovine serum albumin "Materials and Methods"). A NaCl gradient on Buffer C was developed up to (M, 67.000), and aldolase (M, 158,000) as marker proteins. 300 mM NaCl, 0.5-ml fractions were collected, and PTPase activity was assayed in alternate fractions. A, DEAE column of membrane extracts of control fraction cells; B. DEAE column of membrane extracts of PMA-differentiated K562 cells.

HPLC-TSK G2000 GSWcalibration 1 was greatly reduced, whereas the second peak P-2 remained the same (Fig. 5B). The peaks of PTPase activity were further analyzed on a TSK G2000 SW HPLC gel permeation column equilibrated in Buffer D (Buffer C containing 300 mM NaCl); 0.3-ml fractions were collected and the PTPase activity was 13.7 kDa measured in every fraction. In 5 separate experiments, the PTPase activity in DEAE peak P-l could not be recovered from the gel permeation columns, probably because of its extreme lability. The DEAE peak P-2 from control and PMA-treated cells had an apparent M, of 43,000 as determined by gel 100 110 120 130 140 150 160 170 180 permeation (Fig. 6). This PTPase form has the same behavior ml elution volume on these 2 columns as the single PTPase form detected in membrane extracts of human A431 cells (41). 43 kD In further experiments, the DEAE chromatography was re placed by gel permeation on a TSK G2000 GSW column, equilibrated with Buffer D, as a first purification step. Mem brane extracts of control K562 cells revealed at least 3 different PTPase forms with molecular weights of 200,000, 60,000, and 43,000 (Fig. 1A). However, in membrane extracts of PMA- treated K562 cells, only the M, 43,000 PTPase form could be detected (Fig. IB). This PTPase eluted as a single peak at 140 mM NaCl when analyzed by DEAE-HPLC and represents the 80 90 100 110 120 130 140 150 160 170 180 DEAE peak P-2 in Fig. 5 (data not shown). The M, 200,000 ml elution volume PTPase from control cells could not be further analyzed by Fig. 7. Analysis of paniculate PTPase activity using gel permeation-HPLC as a first purification step. A, Freshly prepared membrane extracts of control K562 DEAE-HPLC since after rechromatography the activity was no cells were analyzed on a HPLC-TSK G2000 GSW column equilibrated with longer measurable. We discounted the possibility that the M, Buffer D. The apparent molecular weights were assessed from the calibration shown (inset) and are indicated above each activity-peak. EV. excluded volume. 200,000 PTPase is an aggregate form, since all buffers used for B. HPLC gel permeation analysis of membrane extracts from PMA-treated K562 gel permeation studies contained 0.1 % Triton X-100. We would cells. 6326

A) like to conclude that the M, 200,000 PTPase found in control 40-1 r 400 cells can no longer be detected after treatment of K562 cells S-1 350 with PMA. Partial Purification of the Soluble FITase Activity from PMA- Â§ 30- 300 treated and Control K562 Cells. A single PTPase activity was i - -250 5 detected in the soluble fraction of control K562 cells by DEAE- o HPLC, eluting at 100 mivi NaCI (peak S-1; Fig. 8A) and having I 20^ 200 z 2 an apparent A/r of 60,000 upon gel permeation-HPLC analysis I 15H 150 ^ (Fig. 9A). In contrast, the major soluble PTPase activity in Â» u 10 â€¢100 PMA-treated cells had an apparent M, of 40,000 (Fig. 9B) and eluted from a DEAE-HPLC column with 150 mM NaCI (peak l 5-I â€¢50 S-2; Fig. SB). Some residual PTPase activity could be observed at the position of the DEAE peak S-1 seen in extracts of control 10 20 30 40 50 60 70 80 90 cells, although it was greatly reduced. fraction number B) When the partial purification of the soluble PTPases from 35-, 400 S-2 PMA-treated and control cells was done in the reverse order, â€¢350 first gel permeation-HPLC and then DEAE-HPLC, the same â€¢300 result was obtained: a shift in molecular weight from a M, 25- 60,000 PTPase form to a M, 40,000 form with different Chro â€¢250 Ã¶ 51 20- S-1 matographie behavior on DEAE (data not shown). It should o â€¢200 z again be mentioned that all gel permeation buffers contained 15- 2 0.1% Triton X-100 in order to prevent aggregation. -150 E o ID o. - 100 DISCUSSION â€¢50 The KS62 myelogenous leukemia cell line was chosen to 10 20 30 40 50 60 70 80 90 investigate changes in PTPase activities accompanying differ fraction number entiation. Treatment of K562 cells with 10 nM PMA has been Fig. 8. Partial purification of PTPase activity present in the soluble fraction shown to induce megakaryocytic differentiation (15, 18, 22- by DEAE-HPLC. The soluble fraction containing 0.1rf Triton \-100 was ana 26). PMA treatment led to an almost immediate growth arrest lyzed using a TSK-DEAE 3SW HPLC column equilibrated with Buffer C. A, soluble fraction of control K562 cells. Peak S-1 eluted at a salt concentration of of the cells accompanied by a change in cell morphology (Figs. 100 mm NaCI. B, soluble fraction of PMA-treated K562 cells. Peak S-2 eluted at 1 and 2): the PMA-treated cells were slightly larger and flatter a salt concentration of 150 mM NaCI. than untreated cells and became adherent to the plastic of the cell culture flasks; control cells could be resuspended by shaking the culture flask whereas the PMA-treated cells had to be

kDo HPLC-TSK G20OO SW calibration scraped off using a rubber policeman. Cell surface expression of glycoprotein Ilia, an indicator of differentiation along the megakaryocytic lineage, was clearly evident in PMA-treated cells (Fig. 2). One aspect of the morphological changes observed was a change in the extent of actin polymerization. Actin is the major component of microfilaments (or thin filaments), which shape cells. Changes in microfilament organization (actin depolymer- ization) have been reported to accompany transformation of cell culture fibroblasts and epithelial cells by various oncogenes

12 14 16 18 20 (47) and by phorbol ester tumor promoters (48). In growing ml elution volume K562 cells, cytoplasmatic actin existed in part as monomeric

B) G-actin, which disappeared upon PMA-induced differentiation (Fig. 3). Thus, with respect to actin polymerization, PMA- 4-0 kD treated cells resemble more the situation seen in untransformed fibroblasts or epithelial cells, whereas logarithmically growing K562 cells resemble transformed cells. Megakaryocytic differentiation of K562 cells was accom panied by changes in the pattern of PTPase activity. Treatment of K562 cells with 10 nM PMA for 2 days resulted in a decrease of specific PTPase activity in the paniculate fraction and an increase in the soluble fraction (Fig. 4). This change does not 10 12 14 16 18 20 22 seem to be due to a redistribution of PTPase molecules between ml elution volume the particulate and the soluble fraction, but rather reflects the Fig. 9. Gel permeation-HPLC of the major soluble DEAE PTPase peaks. A, disappearance of a M, 200,000 and a 60,000 PTPase form from soluble DEAE peak S-1 analyzed on a TSK-G2000 SW HPI.C column equili the particulate fraction (Fig. 7) and a shift of a M, 60,000 brated with Buffer D. The apparent molecular weights were assessed from the calibration curve shown and are indicated. B, soluble DEAE peak S-2 analyzed PTPase form to a M, 40,000 form in the soluble fraction (Figs. by gel pcrmeation-HPLC. 8 and 9). It is unclear whether the M, 60,000 forms in the 6327

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1990 American Association for Cancer Research. PHOSPHOTYROSYL PROTEIN PHOSPHATASES IN DIFFERENTIATION paniculate and the soluble fraction of untreated cells are iden cell line K562 contains a breakpoint in ber and produces a chimeric bcr/c- abl transcript. Mol. Cell. Biol., 6: 607-616, 1986. tical. The A/r 43,000 membrane-associated PTPase is believed 11. Stivelman, E., Lifshitz, B.. Gale, R. P., and Canaani. E. Fused transcript of to represent the PTPase form also detected in membrane ex abl and ber genes in chronic myelogenous leukemia. Nature (Lond.), 315: tracts of A431 cells (41). The paniculate M, 43,000 PTPase is 550-554, 1985. 12. Konopka. J. B.. and Witte, O. N. Detection of c-abl tyrosine kinase activity probably similar or identical to PTPase IB observed in the in vitro permits direct comparison of normal and altered abl gene products. paniculate fraction of human placenta, and the M, 40,000 Mol. Cell. Biol., 5: 3116-3123, 1985. 13. Leary, J. F., Ohlsson-Wilhelm, B. M., Giuliano. R., LaBella. S., Farley, B., soluble PTPase in K562 cells is probably similar or identical to and Rowley, P. T. Multipotent human hematopoietic cell line K562: lineage- the placental soluble PTPase IB as defined by Tonks et al. (49). specific constitutive and inducible antigens. Leuk. Res., //: 807-815, 1987. The most interesting observation is the disappearance of a M, 14. Tetteroo, P. A. T., Massaro, F., Mulder, A., Schreuer-van Gelder, R., and von dem Borne, A. E. G. K. Megakaryoblastic differentiation of proerythro- 200,000 PTPase form from the paniculate fraction of PMA- blastic K562 cell line cells. Leuk. Res., 8: 197-206, 1984. treated K562 cells (Fig. 7). It is attractive to suggest that the 15. Sutherland, J. A., Turner. A. R., Mannoni, P., McGann, L. E.. and Turc, J.- M. Differentiation of K562 leukemia cells along erythroid, macrophage, and M, 200,000 PTPase present in the paniculate fraction of un megakaryocyte lineages. J. Biol. Response Modif., 5: 250-262, 1986. treated K562 cells is identical to CD45, since: (a) CD45 has 16. Andersson, L. C., Jokinen, M., and Gahmberg. C. G. Induction of erythroid been shown to be expressed on the surface of K562 cells (50); differentiation in the human leukaemia cell line K562. Nature (Lond.), 278: 364-365. 1979. (b) CD45 has been shown to have PTPase activity (51); and (c) 17. Gambari. R., delSenno. L.. Barbieri. R., Viola. L., Tripodi. M., Raschella, CD45 has been shown to be phosphorylated in vitro by protein G., and Fantoni. A. Human leukemia K-562 cells: induction of erythroid kinase C, and in peripheral I -cells following stimulation with differentiation by 5-azacytidine. Cell Differ., 14: 87-97, 1984. 18. Bianchi ScarrÃ , G. L., Romani, M., Coviello, D. A., CarrÃ©,C., Ravazzolo, phorbol esters, known activators of protein kinase C (52, 53). R., Vidali, R., and Ajmar, F. Terminal erythroid differentiation in the K-562 The suggestion that the M, 200,000 PTPase is identical to cell line by 1-fi-D-arabinofuranosylcytosine: accompaniment by c-myc mes CD45 is further supported by the fact that megakaryocytes are senger RNA decrease. Cancer Res., 46: 6327-6332, 1986. 19. Tonini, G. P., Radzioch, D., Gronberg, A., Clayton, M., Blasi, E., Benetton, the precurser cells of platelets, and it is known that CD45 is G., and Varesio, L. Erythroid differentiation and modulation of c-myc not present on platelets (54). expression induced by antineoplastic drugs in the human leukemic cell line In this article, we show the existence of several growth- and/ K562. Cancer Res., 47:4544-4547. 1987. 20. Honma, Y., Okabe-Kado, J., Hozumi, M., Uehara, Y., and Mizumo, S. or differentiation-related PTPase activities in K562 cells. Pres Induction of erythroid differentiation of K562 human leukemic cells by ently, it is not known whether the different PTPase forms are herbimycin A. an inhibitor of tyrosine kinase activity. Cancer Res., 49: 331- encoded by different genes, are derived by differential splicing, 334, 1989. 21. Kondo, K.. Watanabe, T., Sasaki, H., Uehara, Y., and Oishi, M. Induction or arise by association of a single PTPase form with different of in vitro differentiation of mouse embryonal carcinoma (F9) and erythro- cellular components. The isolation of cDNA clones for PTPases leukemia (MEL) cells by Herbimycin A, an inhibitor of protein phosphorylation. J. Cell Biol., 109: 285-293, 1989. will help to shed light on these open questions. 22. Forsbeck, K., Nilsson, K.. Hansson, A., Skoglund, G., and Ingelman-Sund- berg, M. Phorbol ester-induced alteration of differentiation and proliferation in human hematopoietic tumor cell lines: relationship to the presence and ACKNOWLEDGMENTS subcellular distribution of protein kinase C. Cancer Res., 45: 6194-6199, 1985. We would like to thank Dr. Thomas Kreis of the European Molecular 23. Colamonici, O., Trepel, J. B., and Neckers, L. A. Megakaryocyte differentia Biology Laboratory', Heidelberg, Federal Republic of Germany, for the tion: studies at the molecular level using the K562 cell line. In: R. Levine, N. generous gift of the rabbit anti-chicken smooth-muscle actin antiserum. Williams, J. Levin, and B. Evatt. eds. Megakaryocyte Development and Function, pp. 187-191. New York: Alan R. Liss, Inc.. 1986. The authors are grateful to the Ludwig Institute for Cancer Research, 24. MÃ¤kelÃ¤,T.P.. Alitalo. R., Paulsson. Y., Weslermark. B.. Heldin. C.-H., and ZÃ¼rich,for financial support of this work and for a doctoral stipend to Alitalo, K. 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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1990 American Association for Cancer Research. 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 Bütler, Andrew Ziemiecki and Robert R. Friis

Cancer Res 1990;50:6323-6329.

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