p53-mediated differentiation of the erythroleukemia cell · 2017-04-26 · p53-mediated...

p53-mediated Differentiation of the Erythroleukemia CellLine K5621

Kristina Chylicki,2 Mats Ehinger, Helena Svedberg,Gosta Bergh, Inge Olsson, and Urban GullbergDepartment of Hematology, Lund University, Sweden

AbstractThe tumor suppressor gene p53 can mediate bothapoptosis and cell cycle arrest. In addition, p53 alsoinfluences differentiation. To further characterize thedifferentiation inducing properties of p53, weoverexpressed a temperature-inducible p53 mutant(ptsp53Val135) in the erythroleukemia cell line K562.The results show that wild-type p53 and heminsynergistically induce erythroid differentiation of K562cells, indicating that p53 plays a role in the molecularregulation of differentiation. However, wild-type p53 didnot affect phorbol 12-myristate 13-acetate-dependentappearance of the megakaryocyte-related cell surfaceantigens CD9 and CD61, suggesting that p53 does notgenerally affect phenotypic modulation. The cyclin-dependent kinase inhibitor p21, a transcriptional targetof p53, halts the cell cycle in G1 and has also beenimplicated in the regulation of differentiation andapoptosis. However, transiently overexpressed p21 didneither induce differentiation nor affect the cell cycledistribution or viability of K562 cells, suggesting thattargets downstream of p53 other than p21 are criticalfor the p53-mediated differentiation response.

IntroductionThe tumor suppressor gene p53 is probably the most com-mon target for genetic alterations in human cancer, indicat-ing its importance for preserving a benign phenotype. p53 isknown to induce either cell cycle arrest or apoptosis ofpotentially malignant cells (1). Interestingly, p53 has alsobeen shown to participate in the differentiation process ofpancreatic carcinoma cells, muscle cells, keratinocytes, neu-rons, thyroid cells (2–4), and various leukemic cell lines, suchas leukemic L12 pre-B-cells, promyelocytic HL-60 cells, anderythroleukemic K562 cells (5–8). The molecular mecha-

nisms behind p53-mediated differentiation remain elusive. Inthe majority of experimental models, constitutive or perma-nent expression of p53 has been studied. There are, how-ever, certain disadvantages with the constitutive expressionof p53; apoptosis-inducing and cell cycle-arresting featuresof p53 may cause a subclonal selection for compensatorymechanisms, promoting cell survival and proliferation. Se-lected cells may have either mutations in p53 itself or otherchanges that might affect the differentiation response. More-over, because constitutive expression might select for cellswith an inherent capacity for differentiation that does notinvolve the expression of p53 as such, it cannot from thesestudies be concluded with certainty that p53 plays a directrole in the molecular regulation of differentiation. Therefore,to determine the role of p53 per se in leukemic cell differen-tiation, it is important to establish models with an inducibleexpression of p53. Along these lines, inducible expression ofp53 in Friend virus-transformed erythroleukemic cells andmonoblastic U-937 cells has been shown to induce signs ofdifferentiation (9, 10).

The cell cycle regulator p21 is a transcriptional target ofp53 (1, 11, 12). p21 arrests the cell cycle in the G1 phase, andexpression of p21 correlates to the induction of differentia-tion in a variety of tissues, including muscle cells, nerve cells,and leukemic cells (13–16). Moreover, induction of antisensep21 expression results in inhibition of induced differentiationof monoblastic U-937 cells (17, 18), suggesting a causalconnection between p21 and differentiation induction. How-ever, whether p21 is necessary for p53-mediated differenti-ation has not been demonstrated, although it has beenshown that p53-mediated differentiation correlates to ex-pression of p21 both in a constitutive K562 model and in aninducible U-937 model. (16, 19).

To further explore p53-mediated differentiation in leukemiccells, we decided to express p53 in an inducible mannerby transfecting the temperature-sensitive p53 mutantptsp53Val135 into erythroleukemic K562 cells. Normally,K562 cells can be induced to differentiate along both theerythroid and the megakaryocytic pathways. The resultsshow that although induced expression of wild-type p53 byitself causes very modest signs of differentiation, wild-typep53 indeed facilitates some aspects of the differentiationprogram. Hence, hemin-induced production of hemoglobin,but not phorbol ester-induced appearance of megakaryo-cytic markers of differentiation, was promoted by p53. Fur-thermore, a correlation between p53-mediated differentia-tion and high levels of p21 in the inducible K562 model wasobserved. However, transiently overexpressed p21 did nei-ther induce differentiation nor affect the cell cycle distributionor viability of K562 cells, suggesting that targets downstreamof p53 other than p21 are critical for the p53-mediated dif-ferentiation response.

Received 1/24/00; revised 4/13/00; accepted 4/24/00.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indi-cate this fact.1 This work was supported by the Swedish Cancer Foundation; the Swed-ish Childhood Cancer Foundation; The Tobias Foundation; The SwedishMedical Research Council (Project 11546); Funds of Lunds Sjukvårdsdis-trikt; The Gunnar, Arvid, and Elisabeth Nilsson Foundation; and The RoyalPhysiographic Society of Lund.2 To whom requests for reprints should be addressed, at Research De-partment 2, EB-block, University Hospital, S-221 85 Lund, Sweden.Phone: 46-46-173556; Fax: 46-46-184493; E-mail: [email protected].

315Vol. 11, 315–324, June 2000 Cell Growth & Differentiation

ResultsEstablishment and Characterization of K562 Clones In-ducibly Overexpressing p53. A temperature-sensitive formof p53 (ptsp53) was introduced into the erythroleukemiaK562 cell line by electroporation. K562 cells lack endoge-nous expression of p53 (20). At 32°C, the protein productfrom ptsp53 adopts a conformation permitting wild-type p53activity (i.e., the permissive temperature), whereas at 37°C,the protein adopts a conformation restricting wild-type p53activity (21, 22). Transfection of the K562 cells with ptsp53resulted in six clones growing under the selective pressure ofgeneticin. When screened for expression of ptsp53 by West-ern blot, four clones were shown to express high and com-parable amounts of ptsp53. These clones were designatedK562/ptsp53 A2, A4, A5, and A10, respectively (Fig. 1). Nop53 was detected in mock-transfected control clones(Fig. 1).

Wild-Type p53 Inhibits Proliferation and Induces CellDeath in K562 Cells. To analyze how expression of wild-type p53 affects the proliferation and viability of K562 cells,K562/ptsp53 clones and control clones were incubated atthe permissive temperature for 4 days. Each day, cells werecounted, and viability was assessed by trypan blue exclusion(Fig. 2). As expected, the wild-type p53-expressing clonesshowed a retarded proliferation rate as compared withmock-transfected clones measured as total number ofcells/ml (Fig. 2a). When incubated at 37°C (i.e., the temper-ature restrictive for wild-type p53 activity), no difference inproliferation rate was seen between mock-transfected andptsp53-expressing clones (data not shown). Although allptsp53-expressing clones showed extensive cell death at thepermissive temperature as compared with mock-transfectedcontrol clones, clones A2 and A5 differed from clones A4 andA10 in their higher resistance to p53-induced cell death,reflected by the higher fraction of viable cells in these clones(Fig. 2b). To determine whether the cell death was attribut-able to apoptosis, the cells were analyzed for expression of

Annexin V by FACS3 analysis concomitantly with propidiumiodide staining. This is a selective method for early detectionof apoptosis (Table 1; Ref. 23). As shown, the cells showedcharacteristics of apoptosis, as measured by expression ofAnnexin V. In accordance with the data on trypan blue ex-clusion, clones A4 and A10 showed higher rates of apoptosisthan clones A2 and A5. Thus, expression of wild-type p53 inK562 cells results in reduced proliferation and apoptosis tovarying degrees among the subclones.

Wild-Type p53 Promotes the Capacity for HemoglobinProduction Induced by Hemin. When incubated with he-min, K562 cells show signs of erythroid differentiation, asjudged by an increase of their hemoglobin content (24). Tostudy the role of p53 in erythroid differentiation without pre-vious selection against wild-type p53-dependent activities,

3 The abbreviations used are: FACS, fluorescence-activated cell sorter;PMA, phorbol 12-myristate 13-acetate; EGFP, enhanced green fluores-cent protein; IP, immunoprecipitation; CMV, cytomegalovirus; Rb, retino-blastoma.

Fig. 1. Expression of transfected p53 in K562 cells. p53-transfectedK562 clones A2, A4, A5, and A10 and mock-transfected clones M1 andM2 were subjected to Western blot, as described in “Materials and Meth-ods.” Arrow to the right, position of the p53 protein at Mr 53,000 in thetransfected clones. Left, positions of molecular weight standards.

Fig. 2. Proliferation (A) and viability (B) of mock-transfected and p53-expressing K562 clones. Cells at an initial concentration of 0.2 3 106

cells/ml were grown in suspension culture at 32°C (i.e., the temperaturepermissive for the wild-type conformation of p53) for 4 days. Viability, asjudged by trypan blue exclusion, as well as the total number of cells, wasdetermined daily. Mean values are from four separate experiments. E,Mock 1; M, Mock 2; F, ptsp53/A2; f, ptsp53/A4; l, ptsp53/A5; Œ,ptsp53/A10.

316 p53 Promotes Differentiation

clones K562/ptsp53 A2 and K562/ptsp53 A5 were chosenfor additional experiments. The two remaining clones wereexcluded from differentiation studies because of extensivep53-mediated cell death, which made it difficult to examinethe differentiation response. To ascertain that the levels ofp53 remained high during differentiation induction with he-min, a Western blot was performed after 24 h, showingunaffected ptsp53 levels after incubation with 20 mM hemin(data not shown).

After 4 days at the permissive temperature with or without5 mM hemin, p53-expressing and mock-transfected cellswere subjected to a benzidine oxidation test (Fig. 3). This testis performed to determine the peroxidase activity of the cells,which reflects their content of hemoglobin. At this concen-tration of hemin, the maturation response of the mock-trans-fected control clones was weak, measured both as fraction(Fig. 3a) and as total number (Fig. 3b) of cells oxidizingbenzidine/ml. By contrast, the wild-type p53-expressingclones strongly responded to hemin, both measured as frac-tion (Fig. 3a) and total number (Fig. 3b) of benzidine-positivecells/ml. Regarded as fraction of cells, p53 increased thedifferentiation sensitivity of the K562 clones ;4-fold (Fig. 3a)and as absolute/total number of cells ;2-fold (Fig. 3b). How-ever, only a very modest differentiation inducing effect of p53per se was observed (Fig. 3).

The enhanced response to hemin was evident with con-centrations of hemin ranging from 5 to 20 mM, as shown forclone A5 in Fig. 4. The differentiation response of p53-expressing clones reached with 5 mM hemin (Fig. 4) wascomparable with the level of hemoglobin production in non-p53 producer clones attained with 20 mM hemin, regardedboth as fraction (Fig. 4a) and as total number (Fig. 4b) ofbenzidine-positive cells/ml. These data show that wild-typep53 promotes the capacity for hemoglobin production in-duced by hemin in K562 cells.

The Megakaryocyte-related Cell Surface Antigens CD9and CD61 Were Not Affected by Wild-Type p53. K562cells can be induced to express markers associated withmegakaryocytic differentiation when incubated with PMA(25). To further explore the role of p53 along different lin-eages of differentiation, K562/ptsp53 clones and controlclones were incubated with or without PMA at different con-centrations at the permissive temperature. Because PMAhas been shown to reduce the levels of p53 in some cells (26,

27), a Western blot was performed after 24 h in the presenceof the highest concentration of PMA (i.e., 10 nM) showingreduced, but clearly visible, levels of transfected p53 (Fig. 5).After 4 days, the cells were subjected to a FACS analysis ofthe megakaryocyte-related cell surface antigens CD9 (28)and CD61 (25), as described in “Materials and Methods.” Asshown in Fig. 6, both control K562 clones and wild-typep53-expressing K562 clones responded to PMA with up-regulation of CD9 and CD61 at comparable levels. Thus, ourresults do not support that p53 influences the regulation ofthe megakaryocyte-related cell surface antigens CD9 andCD61. In parallel with the FACS analysis, cells were counted,and viability was assessed by trypan blue exclusion. Thehighest concentration of PMA (i.e., 10 nM) completely abol-ished proliferation of both mock-transfected and wild-type

Fig. 3. Effects of p53 and hemin on differentiation of K562 cells, assayedby the capacity to oxidize benzidine. Cells at an initial concentration of0.2 3 106 cells/ml were incubated at 32°C (i.e., the temperature permis-sive for the wild-type conformation of p53) with or without hemin at 5 mM.After 4 days, cells were subjected to a benzidine oxidation test. Thefraction (A) or total number per ml (B) of benzidine-oxidizing cells areshown. Mean values from nine separate experiments are shown. M,culture medium; f, 5 mM hemin; bars, SE.

Table 1 Expression of the apoptosis-related cell surface antigenAnnexin V in mock-transfected and wild-type p53-expressingK562 cells

K562 cells were incubated at an initial concentration of 200,000 cells/mlin culture medium at the permissive temperature. After 2 days, cells weresubjected to analysis of Annexin V by flow cytometry. Values shown arepercentage of cells expressing Annexin V.

Experiment 1 Experiment 2

ptsp53/A2 5.93 6.37ptsp53/A4 11.9 20.0ptsp53/A5 6.82 6.89ptsp53/A10 21.1 31.1Mock 1 2.67 2.75Mock 2 1.96 3.62

317Cell Growth & Differentiation

p53-expressing clones and also reduced viability to compa-rable levels in control clones and p53-expressing clones [theaverage viability among mock transfectants is 44% (SE is8%), and among wild-type p53-expressing clones, 49% (SEis 12%)].

p53-induced Cell Death Negatively Correlates to Ex-pression of p21. In addition to regulating the cell cycle, p21has been implicated in the regulation of differentiation andapoptosis (17, 29, 30). Because the sensitivity to p53-medi-ated apoptosis differed among the subclones (Fig. 2b), wewanted to determine whether this could be explained by p21.After incubation at the permissive temperature for 22 h, thedifferent clones were analyzed for expression of p21 (Fig. 7).No p21 was detected in mock-transfected control clones,whereas an induction of p21 at the permissive temperature

was confirmed in all transfected clones, indicating the pres-ence of transcriptionally active p53. The amount of p21 didnot correlate to the expression levels of p53 (compare Figs.1 and 7). Interestingly, however, the p53-mediated cell deathcorrelated inversely to the level of expression of p21, be-cause clones A2 and A5 not only were partially protectedagainst p53-mediated apoptosis (Fig. 2b) but also on re-peated Western blot analysis showed higher expression lev-els of p21 than did clones A4 and A10 (Fig. 7).

Transient Overexpression of p21 Does Not Protectagainst p53-mediated Apoptosis of K562 Cells. To deter-mine whether the observed correlation between high levelsof p53-induced p21 and protection against p53-mediatedapoptosis actually reflected a causal relationship, transienttransfection experiments with p21 were performed. K562 M1cells were transfected either with p21 cDNA or with a controlvector. The plasmid EGFP-N1 carrying the cDNA for EGFPwas cotransfected with each of the plasmids, thus allowingcell sorting based on the green fluorescent light emitted byEGFP. Twenty-four h after transfection, cells were sorted forexpression of EGFP by FACS analysis (for details, see “Ma-terials and Methods”). After another 36 h, cells were analyzedfor expression of p21 by Western blot (Fig. 8). As shown, nop21 was expressed in the mock-transfected cells, whereashigh levels of p21 was seen in cells transiently transfectedwith p21, demonstrating the production of p21 protein.

Hypophosphorylation of the retinoblastoma protein pRb isan important mechanism for p21-mediated cell cycle arrest(31, 32). Moreover, wild-type p53 activity causes a prompthypophosphorylation of pRb (16, 33). The phosphorylationstatus of pRb in control or p21-transfected K562 M3 cellswas therefore investigated by IP-Western blot to assure thatthe transfected p21 was functionally active (Fig. 9). Toachieve a positive control for induction of pRb hypophos-phorylation, K562/ptsp53/A5 cells were transiently trans-fected with control vector and incubated 24 h at the temper-ature that is permissive (i.e., 32°C) or nonpermissive (i.e.,37°C) for wild-type p53 activity. Expression of wild-type p53induced a shift from hyper- to hypophosphorylated Rb, asdemonstrated by the shift to faster migrating bands in thelane containing K562/ptsp53/A5 cells incubated at the per-missive temperature (Fig. 9). Moreover, expression of p21also caused a similar hypophosphorylation of pRb, an ob-servation shown on repeated Western blots. This suggests

Fig. 4. Dose dependency of the effect of hemin on the capability ofoxidizing benzidine for the p53-expressing K562 clone A5 and the mock-transfected clone M1. Cells at an initial concentration of 0.2 3 106 cells/mlwere incubated at 32°C (i.e., the temperature permissive for the wild-typeconformation of p53) with or without hemin at different concentrations.After 4 days, cells were subjected to a benzidine oxidation test. Thefraction (A) or total number per ml (B) of benzidine-oxidizing cells areshown. Mean values from three to nine separate experiments are shown.F, ptsp53/A5; M, Mock 1.

Fig. 5. Expression of transfected p53 with or without 10 nM PMA. Cellsat an initial concentration of 0.2 3 106 cells/ml were incubated with orwithout PMA at 10 nM at 32°C (i.e., the temperature permissive for thewild-type conformation of p53). After 24 h, the cells were subjected toWestern blot. Arrow to the right, position of p53 protein at Mr 53,000.


that the transiently transfected p21 was indeed functionallyactive under the experimental conditions.

Because cell cycle arrest has been reported to protectagainst apoptosis (34–36), we next asked whether expres-sion of p21 would affect the cell cycle distribution of K562cells. For this matter, mock-transfected K562 clones weretransiently transfected with p21 or with control vector asdescribed above. On days 1, 2, and 3 after cell sorting, thecell cycle distribution of transfected cells was investigated by

FACS analysis. No difference was observed between p21-expressing cells and control-transfected cells (Table 2 anddata not shown), indicating that expression of p21 does notaffect the cell cycle distribution of K562 cells. Moreover,transient overexpression of p21 did not affect the prolifera-tion rate of mock-transfected K562 cells (data not shown).

Fig. 6. Expression of megakaryocyte-related cell surface antigens incontrol cells and p53-expressing cells upon incubation with PMA. Cells atan initial concentration of 0.2 3 106 cells/ml were incubated with orwithout PMA at different concentrations at 32°C (i.e., the temperaturepermissive for the wild-type conformation of p53). After 4 days, the cellswere subjected to analysis of CD9 (A) and CD61 (B) by flow cytometry.Values shown are median fluorescence intensity for viable cells. Meanvalues from three to nine independent experiments are shown. E, Mock 1;M, Mock 2; L, Mock 3; ‚, Mock 4; F, ptsp53/A2; l, ptsp53/A5; bars, SE.

Fig. 7. Expression of the cell cycle regulator p21 in response to expres-sion of wild-type p53. Mock-transfected and p53-expressing K562 cloneswere incubated at 32°C (i.e., the temperature permissive for the wild-typeconformation of p53) for 22 h, after which the cells were subjected toWestern blot, as described in “Materials and Methods.” Arrow to the right,position of p21 protein at Mr 21,000. Left, positions of molecular weightstandards.

Fig. 8. Expression of p21 in p21 and control-transfected cells. Themock-transfected K562 clone M1 was transfected with control vector orthe cDNA for p21, respectively, as described in “Materials and Methods.”After 72 h, the cells were subjected to Western blot. Arrow to the right,position of p21 protein at Mr 21,000. Left, positions of molecular weightstandards.

Fig. 9. Degree of phosphorylation of pRb in p21 and control-transfectedcells. The mock-transfected K562 clone M3 was transfected with controlvector (C) or the cDNA for p21, respectively, as described in “Materialsand Methods.” K562/ptsp53/A5 cells transiently transfected with controlvector (C) and incubated 24 h at the temperature that is permissive (i.e.,32°C) or nonpermissive (i.e., 37°C) for wild-type p53 activity serve as apositive control for induction of pRb hypophosphorylation. Seventy-two hafter transfection, cells were subjected to analysis of pRb by IP-Westernblot. Arrow to the right, position of pRb. Left, positions of molecular weightstandards. One representative experiment of three performed is shown.


To examine whether transient expression of p21 in cellswith low expression of p53-induced p21 would protectagainst p53-mediated apoptosis, K562/ptsp53/A4 andK562/ptsp53/A10 and mock-transfected control clones weretransiently transfected with the cDNA for p21 or with a con-trol vector. Cells were sorted for expression of EGFP byFACS, and 16 h after cell sorting, cell clones were incubatedat the permissive (i.e., 32°C) temperature for 4 days. Eachday, cells were counted, and viability was assessed bytrypan blue exclusion. As expected, clones carrying ptsp53showed reduced viability. However, no difference in viabilitywas observed between clones transfected with p21 or withcontrol vector (Fig. 10), suggesting that forced expression ofp21 does not protect against p53-mediated apoptosis inK562 cells. Taken together, these data suggest that thep21-Rb axis is of minor importance for the regulation of cellcycle distribution and for the p53-induced cell death of K562cells.

Transient Overexpression of p21 Does Not Induce Dif-ferentiation of K562 Cells. p21 has been given a role in thedifferentiation process of a number of tissues, such as mus-cle cells, nerve cells, and a variety of hematopoietic cell lines(13–15, 17, 18, 37, 38). As mentioned above, expressionlevels of p21 correlates to p53-mediated facilitation of he-

min-induced differentiation of K562 cells, inasmuch as cellclones sensitive to p53-facilitated differentiation also showhigh levels of p21 (Figs. 3 and 7). To investigate whether p21per se induces differentiation of K562 cells, mock-trans-fected K562 control clones were transiently transfected withp21 or with control vector. Sixteen h after cell sorting byFACS, cells were seeded with or without hemin at differentconcentrations in culture medium. After 4 days, a benzidineoxidation test was performed (Fig. 11). As shown, control-transfected and p21-transfected cells respond with oxidationof benzidine at comparable levels when incubated both withand without hemin [5 mM hemin does not induce expressionof p21 in K562 cells (data not shown)]. Hence, these resultssuggest that expression of p21 per se does not induce orfacilitate the hemin-induced differentiation of K562 cells.

DiscussionOur results show that hemin and inducibly expressed wild-type p53 act in a cooperative manner to promote erythroiddifferentiation of K562 cells, in line with the previously ob-served cooperation of constitutively expressed wild-typep53 and tumor necrosis factor in K562 cells (8).

Fig. 10. Effect of p21 on the viability of wild-type p53-expressing andmock-transfected K562 clones. Cells transiently transfected with p21 orwith control vector were seeded in culture medium at a concentration of0.2 3 106 cells/ml and incubated at 32°C (i.e., the temperature permissivefor the wild-type conformation of p53) for 4 days. Viability, as judged bytrypan blue exclusion, as well as the total number of cells, was determineddaily. Viability was always .90% on day 0. Mean values from threeexperiments are shown. E, Mock 1/Control; F, Mock 1/p21; M, Mock2/Control; f, Mock 2/p21; M, A4/Control; f, A4/p21; ‚, A10/Control; Œ,A10/p21.

Fig. 11. Effects of p21 and hemin on the differentiation of K562 cells,assayed by the capacity to oxidize benzidine. The mock-transfected K562clones M1 and M2 were transfected with control vector or the cDNA forp21, respectively, as described in “Materials and Methods.” Forty-eight hafter transfection, cells at an initial concentration of 0.2 3 106 cells/mlwere incubated in culture medium with or without hemin at differentconcentrations. After 4 days, cells were subjected to a benzidine oxidationtest. The fraction of benzidine-oxidizing cells are shown. Mean valuesfrom three separate experiments are shown. M, culture medium; o, 1 mM

hemin; d, 5 mM hemin; f, 25 mM hemin; bars, SE.

Table 2 Cell cycle distribution of p21 or control-transfected K562 cells

Mock-transfected K562 clones were transiently transfected with p21 or control vector and sorted by a FACS analysis as described in “Materials andMethods.” Three days after FACS sorting, cells were explored for cell cycle distribution by a FACS analysis. Values show percentage of viable cells (i.e.,cells with sub-G1 DNA content are excluded). Values are from two independent experiments.

G0 1 G1 S G2

Control vector p21 Control vector p21 Control vector p21

Mock 1 35.5 39.5 56.4 52.1 8.1 8.5Mock 4 38.5 39.2 51.2 50.2 10.2 10.6


Wild-type p53 sensitizes K562 cells to hemin-induced dif-ferentiation ;4-fold, regarded as fraction of cells. Moreover,a better differentiation response of the p53-expressingclones was obvious also when it was determined as totalnumber of benzidine-oxidizing cells. This excludes the pos-sibility that p53-dependent growth arrest or apoptosis of asubpopulation of cells not prone for differentiation were thecause of the increased fraction of cells showing signs oferythroid differentiation and indicates that wild-type p53 in-deed has an inherent differentiation facilitating capacity. Fur-thermore, our results suggest that p53 and hemin have acooperative effect on erythroid differentiation of K562 cells,as judged by the finding that the differentiation responsebetween 1 and 10 mM hemin was higher than merely anadditive effect of the two separate differentiation inducers.This suggests that hemin and p53 operate in at least partiallyseparable pathways.

The present modest differentiation-inducing activity of p53alone in K562 cells is consistent with previous results oninducible expression of p53 in these cells (39). However,K562 cells with constitutively expressed p53 can show ashigh as 40% benzidine positivity (7). The divergence in dif-ferentiation response between cells exhibiting inducible orconstitutive expression of p53 may be explained by a sub-clonal selection during establishment of cell clones consti-tutively expressing p53, which affects the differentiation re-sponse. Constitutive expression of p53 might select for cellswith an inherent capacity for differentiation that is not directlyconnected to p53. For example, it has been demonstratedthat cells engaged in a differentiation program can be pro-tected against cell death (40). Thus, although activation ofp53 probably provokes apoptosis in the majority of the cells,a small differentiation-prone subpopulation could surviveand proliferate. Another possible explanation is that differ-entiation depends on a certain pace of proliferation, and thatthe absence of cell cycle-arresting features of p53 duringconstitutive expression allows the differentiation-promotingeffects of p53 to be more pronounced. This view is sup-ported by the observation that differentiation synergisticallyinduced by p53 and IFN-g in U-937 cells correlates to adecreased fraction of cells in the G1 phase of the cell cycle,as compared with cells treated with p53 alone (19).

Furthermore, wild-type p53 activity did not affect the PMA-induced up-regulation of megakaryocyte-related cell surfacemarkers on K562 cells. This suggests that p53 does notunspecifically influence phenotypic modulation. Rather,these data might suggest distinctive interactions with thedifferentiation response, perhaps reflecting specific molecu-lar interactions pertaining to specific differentiation pro-grams, because p53 facilitated differentiation induced byhemin but not by PMA of K562 cells. Although the levels ofp53 declined in response to PMA in the K562 cells, the cellsstill expressed considerable amounts of p53 protein. Hence,it is very likely that the p53 levels present suffice for facilita-tion of differentiation but do not participate in the response toPMA, because low levels of p53 can induce differentiation(41) and that the transcriptional activity of p53 can increaseconcomitantly with decreasing levels of p53 protein duringdifferentiation of mouse keratinocytes (42).

The cell cycle regulator p21 is a transcriptional target ofp53 and arrests the cell cycle in the G1 phase. Moreover, anumber of studies demonstrate that p21 can protect againstapoptosis of monoblastic U-937 cells as well as muscle cells(17, 30) and also against p53-mediated apoptosis of humanmelanoma cells (29). Furthermore, a correlative connectionbetween high levels of p21 and differentiation has beenshown in a number of tissues, such as muscle cells, nervecells, and hematopoietic cells (13–15).

In the present study, high levels of p53-induced p21 cor-related to protection against p53-mediated apoptosis, allow-ing p53-mediated differentiation to proceed. Thus, levels ofp21 seemed discriminative between p53-mediated differen-tiation and death. To examine whether this connection re-flected a causal relationship or was merely correlative, p21was transiently overexpressed in K562 cells expressing wild-type p53 and low levels of p53-induced p21 (i.e., clones A4and A10). Overexpression of p21 did not affect the responseto p53-mediated apoptosis, indicating that p21 does notprotect against p53-mediated apoptosis of K562 cells. Toanalyze the role of p21 in the induced differentiation of K562cells, p21 was transiently overexpressed in p53-null K562cells with and without hemin. No p21-related effects on thedifferentiation response were demonstrated, suggesting thatp21 alone does not induce or facilitate hemin-induced dif-ferentiation of K562 cells. However, a potential cooperationbetween p21 and p53 in the differentiation of p53-expressingcells proved impossible to analyze because of the low via-bility in the studied cell clones. Hence, because p21 wasalways expressed during the p53-mediated differentiation, itcannot be excluded that p21 and p53 cooperate for induc-tion of differentiation.

Furthermore, despite the ability of the transfected p21 toactivate the retinoblastoma protein by dephosphorylation, nop21-related effects on the cell cycle were observed. Takentogether, these data suggest that the p21-Rb axis is of minorimportance in the regulation of differentiation, cell cycle dis-tribution and apoptosis of K562 cells. Interestingly, it wasshown recently that inducible expression of pRb does notaffect the growth of mouse lymphoid cells or human myeloid32D cells. Instead, growth is inhibited by ectopic expressionof p130 (43), indicating distinct cell cycle regulatory functionsfor the members of the Rb family, possibly depending on thecellular context. Moreover, because p21 alone does not in-duce differentiation of K562 cells, other molecules than p21are probably critical for mediating p53-induced differentia-tion.

In conclusion, our results show that even if p53 does nothave a pronounced differentiation inducing capacity on itsown, it facilitates hemin-induced erythroid differentiation ofK562 cells. This indicates that p53 plays a role in the molec-ular regulation of specific differentiation programs. More-over, other downstream targets than p21 are probably criticalfor p53-mediated differentiation of K562 cells.

Materials and MethodsCells and Culture Conditions. The human erythroblastic leukemia cellline K562 (44) was cultured in RPMI 1640 (Gibco Ltd., Paisley, UnitedKingdom), supplemented with 10% heat-inactivated FCS (Gibco Ltd.) in a


humidified CO2 atmosphere at 37°C. Exponentially growing cells wereused for all experiments. The number of cells and viability, as judged bytrypan blue exclusion, were determined by counting the cells in a Burkerchamber.

Vector Constructs. The eukaryotic expression vector ptsp53(pLTRp53cGval135) carrying the cDNA for a murine temperature-sensitivemutant of p53, driven by the long terminal repeat from Harvey murinesarcoma virus, was generously provided by Dr. Moshe Oren (Rehovot,Israel). At 32°C, the protein product from p53cGval135 cDNA adopts aconformation permitting wild-type p53 activity, whereas at 37°C, theprotein adopts a conformation restricting wild-type p53 activity. The eu-karyotic expression vector pRC-CMV was from InVitrogen (AMS Biotech-nology, Oxon, United Kingdom). It provides a CMV promoter-driven ex-pression of introduced cDNA and confers resistance to geneticin, allowingfor selection of recombinant cells. Because ptsp53 lacks a selectablemarker for eukaryotic cells, it was cotransfected with pRC-CMV to selectfor ptsp53-containing cells. Control clones were obtained by transfectionwith pRC-CMV alone. The p21 cDNA was cloned by reverse transcription-PCR on total RNA from vitamin D3-induced myelo-monoblastic U-937cells as described (45). After control sequencing, the p21 cDNA wascloned into the eukaryotic expression vector pcDNA3, providing a CMVpromoter-driven expression of p21. The pEGFP-N1 plasmid expressingEGFP under the control of a CMV promoter was from Clontech Labora-tories, Inc. (Palo Alto, CA).

Transfection Procedure. The transfection was performed as de-scribed previously (46). Briefly, for constitutive expression cells wereresuspended in 37°C culture medium (RPMI 1640 1 10% FCS) to aconcentration of 10 3 106 cells/ml. The plasmid was introduced into thecells by electroporation using the Bio-Rad gene-pulser (Bio-Rad, Melville,NY) with electrical settings of 260 V and 960 mF. After 2 days, cells wereseeded together with Geneticin (Boehringer Mannheim, Mannheim, Ger-many) at a concentration of 1.5 mg/ml in 96-well plates to allow forselection of transfected cells. After 2–3 weeks, individual cell clones wereexpanded to mass cultures and assayed for expression of p53. Fortransient transfections, electroporation was performed as above but withelectrical settings of 300 V and 960 mF. p21/pcDNA3 and pcDNA3 (controlvector), respectively, were cotransfected with pEGFP-N1. After 24 h,transfected cells were separated from nontransfected cells by a FACSsorting based on the fluorescent light emitted by EGFP. The transfectionefficiency (i.e., the percentage fluorescent cells) varied between 32 and48% among the individual cell sortings. For viability experiments, FACS-sorted cells were incubated 16 h in culture medium at 37°C prior toincubation at 32°C. For cell cycle experiments, FACS-sorted cells wereincubated 16 h in culture medium at 37°C before FACS analysis. Fordifferentiation and proliferation experiments, FACS-sorted cells were in-cubated 16 h in culture medium at 37°C prior to seeding at 0.2 millioncells/ml or addition of hemin. For analysis of p21 or pRb protein, FACS-sorted cells were incubated for 36 h in culture medium at 37°C prior toWestern and IP-Western blot.

IP-Western Blot. Expression and phosphorylation status of the reti-noblastoma protein pRb was detected by immunoprecipitation followedby Western blot. One million cells were lysed at 4°C in lysis buffer [50 mM

Tris-HCl (pH 8.0), 0.15 M NaCl, 5 mM EDTA (pH 8.0), and 0.5% NP40;KEBO, Stockholm, Sweden] including a protease inhibitor cocktail (Com-plete; Boehringer Mannheim). Lysates were vortexed for 10 s, after whichthey were incubated on ice for 1 h. DNA was then removed by centrifu-gation at 14,000 3 g for 1 h. Lysates were subjected to immunoprecipi-tation with 2 mg of the mouse monoclonal anti-Rb antibody sc-102 (SantaCruz Biotechnology, Inc., Santa Cruz, CA), and immunocomplexes wereadsorbed to protein A-Sepharose (Pharmacia-Upjohn, Uppsala, Sweden)and protein G-Sepharose (Sigma Chemical Co., St. Louis, MO) underconstant gentle rocking at 4°C for 2–3 h. After washing in lysis buffer,immunoprecipitated proteins were separated by a precast 6% Tris-Glycingel electrophoresis (Novex, San Diego, CA). Separated proteins wereelectrophoretically transferred using a Graphite Electroblotter I (Milliblot;WEP Co., Seattle, WA) to Immobilon-P membranes (Millipore, Bedford,MA) in blotting buffer (39 mM glycin, 48 mM Tris, 1.3 mM SDS, and 20%methanol) at 25 V for 1 h. After incubation in coating buffer (10.6 mM

Na2CO3, 39.3 mM NaHCO3, and 0.02% NaN3) with 5% dry milk powder for30 min, the membrane was washed three times for 5 min each time withwash buffer (0.9% NaCl, 0.05% Tween 20). The membrane was thenincubated overnight with 0.1 mg/ml of the mouse monoclonal anti-Rb

antibody sc-102 in incubation buffer (0.137 M NaCl, 8 mM Na2HPO4 3 2H2O, 2.7 mM KCl, 1.5 mM KH2PO4, 0.02% NaN3, and 0.05% Tween 20).After washing as above, the membrane was probed with an alkaline-phosphatase conjugated secondary antibody diluted 1:500 in the sameincubation buffer for 1 h. After washing, proteins were visualized withchromogenic substrates [5-bromo-4-chloro-3-indolyl phosphate-p-tolui-dine salt (ICN) at 0.05 mg/ml and nitro blue tetrazolium (Sigma) at 0.1mg/ml] in 4 mM MgCl2 coating buffer without dry milk powder.

Western Blot. Expression of transfected p53 and of p21 was detectedwith the monoclonal mouse antibodies pAb 240 and 187, respectively(Santa Cruz Biotechnology, Inc.). The ECL-plus Western blot kit (Amer-sham Pharmacia Biotech, Buckinghamshire, United Kingdom) was usedaccording to the manufacturer’s instructions. Briefly, 5 3 106 cells werewashed once in PBS and then frozen at 280°C for at least 20 min. The cellpellet was diluted in 75 ml of lysis buffer (92 mM Tris, 12.1% glycerol, 2.4%SDS, 1.4% b-mercaptoethanol, and 2.9% bromphenol blue), after whichthe cells were lysed by sonication with a Dr. Hielsher sonicator (B. BraunBiotech International GmbH, Melsungen, Germany). Samples were boiledfor 5 min and subsequently spun down at 14,000 3 g for 10 min at 4°C.Cells (0.5 3 106) were loaded in each lane of a precast 10–20% Tris-glycine gel (Novex, San Diego, CA). The separated proteins were sub-jected to Western blot using Hybond-P polyvinylidene difluoride mem-branes (Amersham, Life Sciences International) and blotting buffer (39 mM

glycin, 48 mM Tris, 1.3 mM SDS, and 20% methanol) at 25 V for 1 h.Detection was performed according to the manufacturer’s instructions,and the membranes were exposed to ECL hyper film (Amersham, LifeSciences International) for 5–15 s.

Assessment of Differentiation by Benzidine Oxidation Test. Thebenzidine oxidation test was performed as described previously (47).Briefly, cells (0.2 3 106 cells/ml) were incubated with hemin (Sigma) for 4days, then washed twice in PBS, and finally resuspended in 0.9% NaCl.Benzidine reagent solution [to 1 ml of 0.2% tetramethylbenzidine (Sigma)in 0.5 M HAc, 20 ml of 30% H2O2 is added just prior to use] was added tostart the reaction. After incubation for 30 min in darkness at room tem-perature, 200 cells were counted in a Burker chamber, and the number ofcells containing oxidized tetramethylbenzidine (visualized as cells con-taining blue crystals), indicative of peroxidase activity and thus reflectinghemoglobin production, was determined.

Assessment of Cell Surface Antigens by Flow Cytometric Analysis.Cells (0.2 3 106 cells/ml) were incubated with PMA (Sigma) for 4 days,after which they were washed once in PBS and resuspended to 5 3 106

cells/ml. Fifty ml of the cell suspension were incubated for 10 min at roomtemperature under constant agitation with 5 ml of the following mono-clonal antibodies in microtiter wells: control IgG1-FITC/IgG1-PE, CD61-FITC (Becton Dickinson, San Jose, CA); CD9-FITC (DAKO A/S, Copen-hagen, Denmark); Annexin V-FITC (PharMingen, San Diego, CA); andpropidium iodide (Sigma). The cells were then washed three times andfixed in 1% paraformaldehyde before flow cytometric analysis (FACScan;Becton Dickinson). Ten thousand cells were collected for each antibody.Dead cell and debris were excluded from analysis by gating prior to thecalculation of the fraction of positive cells, using the control incubationwith IgG1-FITC/IgG1-PE for marker settings. Cells were analyzed forexpression of Annexin V in parallel with staining with propidium iodide,which makes it possible to exclude necrotic cells. This provides a selec-tive method for detection of apoptosis (23).

Cell Sorting. EGFP-expressing K562 cells were purified using a FACSVantage Cell sorter (Becton Dickinson Immunocytometry Systems, SanJose, CA) upgraded with a Turbo Sort unit and equipped with a 488-nmargon laser. Single, viable, and EGFP-expressing cells were selected bygating based on laser scatter profile and 515–545 nm fluorescence. TheK562 cells were sorted at a rate of 2000–4000 cells/s.

Cell Cycle Analysis. Staining of nuclei and flow cytometric analysiswere performed as follows. Cells were washed in Dulbecco’s PBS, afterwhich 0.2 ml of a nuclear isolation medium containing propidium iodidewas added (50 mg/ml propidium iodide, 0.6% NP40, 100 mg/ml RNase,DNase-free, in PBS; all reagents from Sigma). The cells were then incu-bated at room temperature in the dark for 60 min before the addition of 0.4ml PBS and taken to flow cytometric analysis in a FACScan flow cytom-eter (Becton Dickinson, San Jose, CA). Up to 20,000 nuclei were analyzedper sample. Using the electronic peak and area detectors, processorsignals from nuclei doublets were rejected. Cell cycle phase distribution,i.e., the percentages of G0 1 G1, S, and G2 nuclei of the analyzed cell


population, was determined by applying ModFit LT cell cycle analysissoftware (Verity Software House, Inc., Topsham, ME) on the DNA histo-grams. The DNA histogram was corrected for contribution of nucleicdebris.

AcknowledgmentsWe thank Dr. Tor Olofsson for valuable discussions and for thoughtfully

performing the analysis of cell cycle distribution and cell surface antigensby flow cytometry. We also thank Sverker Segren for cheerful and fluo-rescent cell sorting by flow cytometry.

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