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Cell Growth & Differentiation Vol. 11, 501-506, September 2000
© 2000 American Association for Cancer Research

Articles

K562 Cells Resistant to Phorbol 12-Myristate 13-acetate-induced Growth Arrest: Dissociation of Mitogen-activated Protein Kinase Activation and Egr-1 Expression from Megakaryocyte Differentiation1

Candace Shelly, Lilli Petruzzelli and Roman Herrera2

Department of Cell Biology, Parke-Davis Pharmaceutical Research Division of Warner-Lambert Company, Ann Arbor, Michigan 48105 [C. S., R. H.], and Department of Internal Medicine, Division of Hematology/Oncology, University of Michigan and Veteran’s Affairs Medical Center, Ann Arbor, Michigan 48109 [L. P.]

Abstract

The K562 cell line undergoes megakaryocytic differentiation in response to phorbol 12-myristate 13-acetate (PMA) stimulation. This event correlates with mitogen-activated protein kinase activation, cell cycle arrest, and expression of the Egr-1 transcription factor. We have isolated K562 cells that are resistant to the growth-inhibitory action of PMA. Molecular characterization demonstrates that PMA resistance is downstream from PMA-induced activation of the mitogen-activated protein kinase pathway. Although the levels of Egr-1 expression and cyclic AMP-responsive element-binding protein phosphorylation are comparable in wild-type and PMA-resistant clones in response to PMA, the expression of megakaryocytic cell surface marker CD41 is detected only in the wild-type cells. The lack of differentiation of the PMA-resistant clones correlates with a failure of the PMA-treated cells to induce dephosphorylation and down-regulation of the retinoblastoma protein. These cells may provide a useful model system to distinguish those events that are connected to cell cycle arrest from those involved in the differentiation program initiated by PMA.

Introduction

Megakaryocytic differentiation of K562 cells induced by PMA3 mimics, in part, the physiological process that takes place in the bone marrow in response to a variety of stimuli (1) . The differentiation process is characterized by changes in cell morphology, adhesive properties, endomitosis, and expression of markers associated with megakaryocytes as well as cell growth arrest (1, 2, 3, 4, 5, 6) . The signaling cascade that leads to PMA-induced cell cycle arrest, polyploidy, and differentiation of K562 cells has been described in part, and a role for PKC in the megakaryocyte differentiation process has been established (7) . Studies carried out with K562 cells revealed that PMA induces the translocation of specific isoforms of PKC to particulate fractions (plasma and nuclear membranes), with a subsequent reduction in the levels of the ßll PKC isoform and an increase in the {alpha} PKC isoform (7) . PMA treatment also leads to the induction of transcription factors such as jun/fos (8) and of proto-oncogenes such as c-ski (9) and c-sis (2) . The induction of expression of Egr-1 by PMA has been implicated in the differentiation process (10) .

The mechanism of PMA-induced cell cycle arrest of leukemia cells is partially understood. Treatment of these cells with PMA leads to p53-independent expression of the cell cycle inhibitor p21waf1/cip1 (11 , 12) . In addition, PMA treatment leads to the complex regulation of cdk2 (13) and cyclin B/cdk1 (14) activities as well as regulation in the expression of cdc25 phosphatase (15) .

The Raf/MAPK pathway is involved in cell cycle arrest in response to PMA treatment of K562 cells (16, 17, 18) and is likely to be involved in the expression of p21waf1/cip1. Taxol-induced expression of p21waf/cip1 requires c-raf-1 (19) , and high-intensity Raf signaling in fibroblasts causes growth arrest that is mediated by p21waf/cip1 (20) . In addition, growth factor-mediated activation of MAPK leads to p21waf/cip1 expression (21, 22, 23) . These observations support the hypothesis that in K562 cells, PMA-induced activation of MAPK is required for cell cycle arrest and megakaryocyte differentiation by regulating the expression of the p21waf/cip1 and egr-1 genes.

We have begun to test this hypothesis by isolating K562 clones that are resistant to PMA-induced growth arrest and differentiation downstream from MAPK activation. Here we describe the properties of such clones and show that PMA-induced growth arrest correlates with dephosphorylation of the Rb protein. Furthermore, we demonstrate that induction of Egr-1 expression is not sufficient to mediate differentiation of K562 cells.

Results

Isolation of K562 Cells Resistant to PMA-induced Growth Inhibition.
PMA treatment of K562 cells leads to prolonged activation of the MAPK pathway. This event is required for PMA-induced growth arrest and megakaryocytic differentiation of these cells (16, 17, 18) . We sought to generate a population of K562 cells that are resistant to PMA-induced growth arrest to further dissect the signaling events associated with PMA stimulation of these cells. K562 cells were randomly mutagenized with EMS and selected for their ability to grow in the presence of PMA as described in "Materials and Methods." We selected two clones (clones C1 and C4) from the resistant pool for further characterization. Fig. 1ACitation depicts the growth curve of these cells in the presence and absence of PMA. As seen in the Fig. 1Citation , the growth of wild-type K562 cells is markedly inhibited by PMA, whereas the growth of the isolated clones is minimally affected by PMA. Cell cycle analysis of these cells showed that the PMA-induced G1 arrest seen in wild-type K562 cells is not present in the resistant clones (Table 1)Citation . In contrast, morphological analysis of these cells after PMA treatment for 5 days did not reveal significant differences. The well-documented polylobulation of the cell nucleus in response to PMA is observed in both wild-type K562 cells and the resistant clones (Fig. 1B)Citation . At this time, it is not known whether the polylobulated cells in the resistant clones are capable of cell division.



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  		Fig. 1. Growth and morphological characteristics of PMA-resistant clones. A, growth curves of wild-type K562 cells and the EMS mutagenized clones, C1 and C4, in the presence (•, {blacktriangleup}, and {blacksquare}) or absence ({circ}, {triangleup}, and {square}) of PMA (40 nM). The growth rate of the cultured cells was determined as described in "Materials and Methods." B, wild-type K562 or C1 and C4 clones were treated with or without PMA (40 nM) for 4 days, affixed to the slide by cytospin, and stained using the May-Grunwald procedure as described in "Materials and Methods." The cells were analyzed and photographed at x100 under light microscopy.

 

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  		Table 1 Cell cycle analysis of K562 cells and clones C1 and C4

Cell cycle analysis of K562 cells and two clones derived by mutation of K562 was carried out as described in "Materials and Methods." Cells were fixed and labeled with propidium iodide, and staining was obtained by fluorescence-activated cell-sorting analysis as described in "Materials and Methods."

 
PMA-mediated Signaling in PMA-resistant K562 Cells.
To biochemically characterize the properties of the PMA-resistant K562 clones described above, we focused our studies on both early and late events associated with PMA stimulation of these cells. It is known that PMA stimulation of K562 cells leads to down-regulation of the expression level of PKC (7) and to increased expression of Pyk2 (24) . The levels of PKC and Pyk2 in the mutated clones were compared with those of wild-type K562 cells in response to PMA treatment. As seen in Fig. 2, A and BCitation , the response to long-term incubation of the resistant cells with PMA is comparable to the response obtained in wild-type cells. Down-regulation of PKC and enhanced expression of Pyk2 were observed in both the wild-type cell line and the resistant clones. Because prolonged activation of MAPK is required for PMA-induced growth inhibition of K562 cells, we compared the PMA-induced activation of MAPK of the resistant clones with that of wild-type cells. As shown in Fig. 2, C and DCitation , there is no significant difference between the mutant and wild-type cells. Because MAPK activation is intact in the PMA-resistant clones, it is likely that the resistant phenotype is downstream of PMA-induced MAPK activation.



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  		Fig. 2. PMA-mediated signaling in PMA-resistant K562 cells. The effect of PMA treatment on signal transduction pathways in both wild-type and PMA-resistant K562 cells was studied by measuring the levels of PKC (A) or Pyk2 (B) or the activation of the MAPK pathway (C and D). Data presented in D depict the C1 clone. Samples were processed as described in "Materials and Methods."

 
Comparison of Rb Phosphorylation in Wild-Type and Mutant K562 Cells.
The mechanism of PMA-induced growth arrest of wild-type K562 cells is not fully understood. However, it has been proposed that PMA stimulation leads to p53-independent expression of the cyclin inhibitor p21waf/cip1 (25 , 26) . As such, one would expect a marked effect on the phosphorylation state of the cell cycle regulator, Rb, because its phosphorylation state is regulated by at least two distinct cyclin-cdk complexes (27) . Analysis of both the phosphorylated state and total Rb protein was carried out to correlate its expression with the growth properties of both wild-type and PMA-resistant K562 cells. As seen in Fig. 3ACitation , PMA stimulation of wild-type K562 cells leads to dephosphorylation and down-regulation of the Rb protein. Furthermore, this effect is partially blocked by inhibition of the MEK/MAPK pathway by the specific inhibitor PD098059 (28) . In contrast, the PMA-resistant cells fail to dephosphorylate or down-regulate Rb in response to PMA. This pattern was also seen when the analysis was carried out in synchronized cells (Fig. 3B)Citation . Because Rb phosphorylation is regulated in part by the activities of cdk2 and cdk4 gene products (27) , we analyzed the expression levels of both of these enzymes in wild-type and PMA-resistant synchronized cells. In wild-type K562 cells, expression of both cdk2 and cdk4 protein was modulated by PMA treatment (Fig. 3C)Citation . However, close analysis of the time course of Rb dephosphorylation indicates that Rb dephosphorylation occurs at earlier points than cdk4 or cdk2 disappearance. We have detected a modest inhibition of cdk4 activity at early points of PMA treatment (data not shown). In contrast, the resistant K562 cells failed to down-regulate either protein in response to PMA, thus providing a molecular explanation for the preserved phosphorylation of Rb observed in the PMA-resistant cells.



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  		Fig. 3. Regulation of Rb phosphorylation and cdk2 and cdk4 expression by PMA. Wild-type or PMA-resistant K562 clones were treated for 24 h with or without PMA in the presence or absence of the MEK inhibitor PD098059, and the level of phosphorylated and total Rb protein (A) was analyzed by immunoblot as described in "Materials and Methods." The analysis was also carried out in synchronized cell cultures of both wild-type K562 and C1 cells treated with or without PMA. Both phosphorylated Rb (B) and the expression level of cdk2 and cdk4 (C) were analyzed.

 
PMA-resistant Cells Fail to Differentiate.
The phenotypic changes associated with PMA treatment of K562 cells are consistent with differentiation toward the megakaryocytic cell lineage (8) . It has been proposed that expression of the egr-1 gene mediates some of the differentiation-associated changes induced by PMA (10) . In addition, it has been demonstrated that CREB phosphorylation is associated with megakaryocyte differentiation (29) . Therefore, we studied the association between Egr-1 expression, CREB phosphorylation, and differentiation of both wild-type and PMA-resistant cells in response to PMA. As shown in Fig. 4ACitation , CREB phosphorylation is observed in both wild-type and PMA-resistant clones in response to PMA. This event is not blocked by the MEK inhibitor, a treatment that otherwise induces blockade of the PMA-induced differentiation of wild-type K562 cells. Similarly, the PMA-induced expression of Egr-1 is observed in both wild-type and PMA-resistant K562 cells. However, this induction is partially dependent on the activity of the MEK/MAPK pathway. The level of expression correlates with the degree of MEK inhibition, as judged by the level of activated MAPK (Fig. 4B)Citation .



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  		Fig. 4. PMA induced CREB phosphorylation or Egr-1 expression in wild-type or PMA-resistant K562 cells. Wild-type or PMA-resistant K562 cells were treated with or without PMA in the presence or absence of PD098059, and the degree of CREB phosphorylation (A) or the induction of Egr-1 expression (B) was analyzed as described in "Materials and Methods."

 
To assess the differentiation profile of the PMA-resistant K562 clones, we measured the cell surface expression of one of the subunits of gpIIbIIa, CD41, which is a marker for megakaryocyte differentiation. As seen in the data presented in Fig. 5Citation , wild-type K562 cells express CD41 in response to PMA treatment. The PMA-resistant clones, however, failed to express this cell surface protein, suggesting that the PMA-induced differentiation of these cells is blocked.



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  		Fig. 5. Cell surface expression of differentiation marker CD41. Wild-type K562 cells and the mutant clones were treated with DMSO or PMA for 6 days before collection. Cells were stained with the control antibody ZPO-1 or with antibodies directed against CD41 and CD61. Bound antibody was detected with FITC-conjugated goat antimouse antibody. Histograms of the fluorescence intensity are reported for wild-type cells and for mutant clones C1 and C4.

 
Discussion

We have isolated K562 cells that are resistant to PMA-induced growth inhibition. Although the exact molecular defect that results in PMA resistance of these cells is unknown, the initial signaling cascade initiated by PMA treatment is intact in the PMA-resistant cells. Like wild-type K562 cells, the mutated clones undergo prolonged and marked activation of the MAPK pathway, down-regulation of PKC expression, and up-regulation of Pyk2 expression. These signaling events have been described previously as being associated with PMA treatment of K562 cells (5 , 16, 17, 18 , 24) . The resistant phenotype of these cells has been maintained during successive passages (>40 passages); therefore, this phenotype is likely to be due to a permanent mutation(s) induced by EMS.

The PMA-resistant phenotype is due to a failure of the cells to arrest at the G1 stage of the cell cycle, a response observed in wild-type K562 cells (Table 1)Citation . Furthermore, this blockade appears to be downstream of MAPK activation because we observed sustained MAPK activation in both the wild-type and mutant cell lines (Fig. 2)Citation . It has been proposed that PMA-induced growth arrest of leukemia cells is mediated by a p53-independent, MAPK-dependent pathway that regulates the expression of the cell cycle regulator p21waf/cipi1 (11 , 12) . As expected, induction of p21waf/cip1 results in an accumulation of dephosphorylated Rb protein, a critical regulator of the G1-S-phase transition (30) . Once cells are stimulated to pass through G1 to S phase, Rb is inactivated by phosphorylation mediated by cyclin D/cdk4/cdk6 and cyclin E/cdk2 (27) .

Our analysis of Rb phosphorylation and expression in both the wild-type and PMA-resistant clones revealed that the most likely explanation for the failure of the resistance clones to undergo growth arrest in response to PMA stimulation is a lack of dephosphorylation of Rb (Fig. 3)Citation . The phosphorylation state of Rb is regulated by a cycle of kinases and phosphatase activities (30) . Therefore, a molecular explanation for the above-mentioned observation awaits the analysis of the expression and function of the known regulators of Rb phosphorylation. Although it has been described that PMA induces p21waf/cip1 in K562 cells, we have been unable to obtain reproducible results regarding p21 expression under our experimental conditions, thus precluding us from connecting the PMA-resistant phenotype to a lack of p21waf/cip1 expression. Alternatively, because we detected minor changes in cdk4 activity at the early points, we favor a model in which the PMA-resistant phenotype is due to a loss of PMA-modulated phosphatase activity directed against the Rb protein.

One of the goals that we hope to accomplish with the isolation of these PMA-resistant cells is to dissect the events correlated with the expression of the differentiated phenotype associated with PMA stimulation of K562 cells. Previous studies have shown that megakaryocytic differentiation of K562 cells is regulated by expression of the Egr-1 transcription factor (10) , whereas HEL cell differentiation correlates with selective phosphorylation at Ser133 of CREB (29) . Our analysis of both Egr-1 expression and CREB phosphorylation in the PMA-resistant clones revealed no difference from wild-type cells. However, when the expression of the cell surface marker for megakaryocyte differentiation was analyzed, those cells that are resistant to PMA failed to differentiate. These results suggest that Egr-1 expression and CREB phosphorylation are not sufficient to override the molecular block induced by EMS treatment of these cells. Although the block could be at the level of CD41 expression, the strict correlation between cell growth arrest and differentiation (1 , 5 , 6 , 31) makes this possibility less likely. These results suggest that the blockade of PMA-induced differentiation and cell cycle arrest is downstream from the phosphorylation of CREB or Egr-1 expression and reinforces the linkage between cell cycle arrest and differentiation of these cells.

Materials and Methods

Antibodies to phosphotyrosine (PY20) and {alpha} PKC (clone 3) used for immunoprecipitation and immunoblotting were from Transduction Laboratories (Lexington, KY). Antibody directed toward the phosphorylated forms of Erk1 and Erk2 was from Promega (Madison, WI), and phospho-specific Rb (Ser795) monoclonal antibody was obtained from New England Biolabs (Beverly, MA). Monoclonal antibodies directed against phosphotyrosine (4G10) and Pyk2 were from Upstate Biotechnology (Lake Placid, NY). Total Rb antibody (G3-245) was obtained from PharMingen (San Diego, CA), and antibodies directed against cdk2 (M2), cdk4 (H22), cyclin D1 (H295), Egr-1 (C19), and total Rb (C15) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

K562 Cell Mutagenesis and Clone Isolation.
K562 cells (American Type Culture Collection; 5 x 105 cells/ml; 2 x 107 cells) were treated with 150 µg/ml EMS (Sigma, St. Louis, MO) for 48 h as described previously (32 , 33) . After a recovery period (3–4 days), the EMS-treated cells were cultured in RPMI 1640 supplemented with 10% FCS in the presence of 50 nM PMA (Sigma). PMA was replaced every 2 days. After 7–9 days, PMA was withdrawn, and the cells were left to recover in RPMI 1640 supplemented with 10% FCS for approximately 1 month, by which time their growth rate had recovered to near that of the wild-type cells, with a 90% viability as determined by blue dextran exclusion. These cells were then treated again with PMA (40 nM) for 2 days and seeded for single cell clone selection in 96-well plates in the presence of 50 nM PMA. Positive clones were selected by two additional rounds of this method to ensure that they represented a single clone.

Growth Curve.
Wild-type K562 cells or the EMS-derived clones were grown in RPMI 1640 supplemented with 10% FCS in the presence or absence PMA (40 nM). At the indicated times, the number of cells was assessed. Duplicate samples were counted in triplicate on a Coulter Z1 Particle Counter (Coulter Corp., Hialeah, FL).

Cell Synchronization.
Cells (1 x 106) were incubated in the presence of nocodazole (1 µg/ml) for 18 h. After washing cells in RPMI 1640 supplemented with 10% FCS, cells were seeded at 4 x 105 cells/ml and, after 4 h, treated in the presence or absence of PMA (40 nM). Cells were collected at the time of initial seeding after nocodazole treatment (t = -4), at the time of addition of PMA (t = 0) and then at 3, 6, 9, 12, and 24 h of incubation. Cells were lysed into radioimmunoprecipitation assay buffer as described previously (34) , the protein concentration in each sample was determined using the protein assay kit from Bio-Rad (Hercules, CA), and an equal amount of protein was applied in each sample subjected to SDS-PAGE.

Cytospin and May-Grunwald Staining.
Approximately 3 x 104 cells were spun onto a microscope slide for 4 min at 800 rpm under medium acceleration in a Cytospin 3 (Shandon, Pittsburgh, PA). After air drying, slides were stained with May-Grunwald stain (Sigma) according to the manufacturer’s instructions.

Immunoprecipitation and Immunoblotting.
Preparation of cell lysates, immunoprecipitation, and immunoblotting were performed as described previously (18 , 24 , 34 , 35) .

Cell Cycle Analysis and Differentiation Markers.
Cell cycle progression of wild-type and mutated K562 cells was analyzed as described previously, and the data were processed using Multicycle Software (Phoenix Flow Systems, San Diego, CA) as described previously (24 , 36) . Expression of differentiation marker CD41 was assessed by flow cytometry according the methods described previously (18 , 37) .

Acknowledgments

We thank Lisa Cummins for help in preparing the manuscript.

Footnotes

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 accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by NIH Grants CA46592 (to the University of Michigan-Comprehensive Cancer Center) and AR20557 (to the University of Michigan-Multipurpose Arthritic Center) and by the University of Michigan-BRCF Core Flow Cytometry facility. L. P. was supported by the American Society of Hematology. Back

2 To whom requests for reprints should be addressed, at Department of Cell Biology, Parke-Davis Pharmaceutical Research Division of Warner-Lambert Company, Ann Arbor, MI 48105. Phone: (734) 622-5963; Fax: (734) 622-5668; E-mail: Roman.Herrera{at}wl.com Back

3 The abbreviations used are: PMA, phorbol 12-myristate 13-acetate; MAPK, mitogen-activated protein kinase; CREB, cyclic AMP-responsive element-binding protein; Rb, retinoblastoma; PKC, protein kinase C; cdk, cyclin-dependent kinase; EMS, methanesulfonic acid ethyl ester; MEK, mitogen-activated protein/extracellular signal-regulated kinase kinase. Back

Received for publication 3/24/00. Revision received 8/ 1/00. Accepted for publication 8/ 3/00.

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Cancer Research Clinical Cancer Research
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