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Cell Growth & Differentiation Vol. 11, 315-324, June 2000
© 2000 American Association for Cancer Research

p53-mediated Differentiation of the Erythroleukemia Cell Line K5621

Kristina Chylicki2, Mats Ehinger, Helena Svedberg, Gösta Bergh, Inge Olsson and Urban Gullberg

Department of Hematology, Lund University, Sweden


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The tumor suppressor gene p53 can mediate both apoptosis and cell cycle arrest. In addition, p53 also influences differentiation. To further characterize the differentiation inducing properties of p53, we overexpressed a temperature-inducible p53 mutant (ptsp53Val135) in the erythroleukemia cell line K562. The results show that wild-type p53 and hemin synergistically induce erythroid differentiation of K562 cells, indicating that p53 plays a role in the molecular regulation of differentiation. However, wild-type p53 did not affect phorbol 12-myristate 13-acetate-dependent appearance of the megakaryocyte-related cell surface antigens CD9 and CD61, suggesting that p53 does not generally affect phenotypic modulation. The cyclin-dependent kinase inhibitor p21, a transcriptional target of p53, halts the cell cycle in G1 and has also been implicated in the regulation of differentiation and apoptosis. However, transiently overexpressed p21 did neither induce differentiation nor affect the cell cycle distribution or viability of K562 cells, suggesting that targets downstream of p53 other than p21 are critical for the p53-mediated differentiation response.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The tumor suppressor gene p53 is probably the most common target for genetic alterations in human cancer, indicating its importance for preserving a benign phenotype. p53 is known to induce either cell cycle arrest or apoptosis of potentially malignant cells (1) . Interestingly, p53 has also been shown to participate in the differentiation process of pancreatic carcinoma cells, muscle cells, keratinocytes, neurons, thyroid cells (2, 3, 4) , and various leukemic cell lines, such as leukemic L12 pre-B-cells, promyelocytic HL-60 cells, and erythroleukemic K562 cells (5, 6, 7, 8) . The molecular mechanisms behind p53-mediated differentiation remain elusive. In the majority of experimental models, constitutive or permanent expression of p53 has been studied. There are, however, certain disadvantages with the constitutive expression of p53; apoptosis-inducing and cell cycle-arresting features of p53 may cause a subclonal selection for compensatory mechanisms, promoting cell survival and proliferation. Selected cells may have either mutations in p53 itself or other changes that might affect the differentiation response. Moreover, because constitutive expression might select for cells with an inherent capacity for differentiation that does not involve the expression of p53 as such, it cannot from these studies be concluded with certainty that p53 plays a direct role in the molecular regulation of differentiation. Therefore, to determine the role of p53 per se in leukemic cell differentiation, it is important to establish models with an inducible expression of p53. Along these lines, inducible expression of p53 in Friend virus-transformed erythroleukemic cells and monoblastic U-937 cells has been shown to induce signs of differentiation (9 , 10) .

The cell cycle regulator p21 is a transcriptional target of p53 (1 , 11 , 12) . p21 arrests the cell cycle in the G1 phase, and expression of p21 correlates to the induction of differentiation in a variety of tissues, including muscle cells, nerve cells, and leukemic cells (13, 14, 15, 16) . Moreover, induction of antisense p21 expression results in inhibition of induced differentiation of monoblastic U-937 cells (17 , 18) , suggesting a causal connection between p21 and differentiation induction. However, whether p21 is necessary for p53-mediated differentiation has not been demonstrated, although it has been shown that p53-mediated differentiation correlates to expression of p21 both in a constitutive K562 model and in an inducible U-937 model. (16 , 19) .

To further explore p53-mediated differentiation in leukemic cells, we decided to express p53 in an inducible manner by transfecting the temperature-sensitive p53 mutant ptsp53Val135 into erythroleukemic K562 cells. Normally, K562 cells can be induced to differentiate along both the erythroid and the megakaryocytic pathways. The results show that although induced expression of wild-type p53 by itself causes very modest signs of differentiation, wild-type p53 indeed facilitates some aspects of the differentiation program. Hence, hemin-induced production of hemoglobin, but not phorbol ester-induced appearance of megakaryocytic markers of differentiation, was promoted by p53. Furthermore, a correlation between p53-mediated differentiation and high levels of p21 in the inducible K562 model was observed. However, transiently overexpressed p21 did neither induce differentiation nor affect the cell cycle distribution or viability of K562 cells, suggesting that targets downstream of p53 other than p21 are critical for the p53-mediated differentiation response.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Establishment and Characterization of K562 Clones Inducibly Overexpressing p53.
A temperature-sensitive form of p53 (ptsp53) was introduced into the erythroleukemia K562 cell line by electroporation. K562 cells lack endogenous expression of p53 (20) . At 32°C, the protein product from ptsp53 adopts a conformation permitting wild-type p53 activity (i.e., the permissive temperature), whereas at 37°C, the protein adopts a conformation restricting wild-type p53 activity (21 , 22) . Transfection of the K562 cells with ptsp53 resulted in six clones growing under the selective pressure of geneticin. When screened for expression of ptsp53 by Western blot, four clones were shown to express high and comparable amounts of ptsp53. These clones were designated K562/ptsp53 A2, A4, A5, and A10, respectively (Fig. 1)Citation . No p53 was detected in mock-transfected control clones (Fig. 1)Citation .



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Fig. 1. Expression of transfected p53 in K562 cells. p53-transfected K562 clones A2, A4, A5, and A10 and mock-transfected clones M1 and M2 were subjected to Western blot, as described in "Materials and Methods." Arrow to the right, position of the p53 protein at Mr 53,000 in the transfected clones. Left, positions of molecular weight standards.

 
Wild-Type p53 Inhibits Proliferation and Induces Cell Death 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 at the permissive temperature for 4 days. Each day, cells were counted, and viability was assessed by trypan blue exclusion (Fig. 2)Citation . As expected, the wild-type p53-expressing clones showed a retarded proliferation rate as compared with mock-transfected clones measured as total number of cells/ml (Fig. 2a)Citation . When incubated at 37°C (i.e., the temperature restrictive for wild-type p53 activity), no difference in proliferation rate was seen between mock-transfected and ptsp53-expressing clones (data not shown). Although all ptsp53-expressing clones showed extensive cell death at the permissive temperature as compared with mock-transfected control clones, clones A2 and A5 differed from clones A4 and A10 in their higher resistance to p53-induced cell death, reflected by the higher fraction of viable cells in these clones (Fig. 2b)Citation . To determine whether the cell death was attributable to apoptosis, the cells were analyzed for expression of Annexin V by FACS3 analysis concomitantly with propidium iodide staining. This is a selective method for early detection of apoptosis (Table 1Citation ; Ref. 23 ). As shown, the cells showed characteristics of apoptosis, as measured by expression of Annexin V. In accordance with the data on trypan blue exclusion, clones A4 and A10 showed higher rates of apoptosis than clones A2 and A5. Thus, expression of wild-type p53 in K562 cells results in reduced proliferation and apoptosis to varying degrees among the subclones.



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Fig. 2. Proliferation (A) and viability (B) of mock-transfected and p53-expressing K562 clones. Cells at an initial concentration of 0.2 x 106 cells/ml were grown in suspension culture at 32°C (i.e., the temperature permissive for the wild-type conformation of p53) for 4 days. Viability, as judged by trypan blue exclusion, as well as the total number of cells, was determined daily. Mean values are from four separate experiments. {circ}, Mock 1; {square}, Mock 2; •, ptsp53/A2; {blacksquare}, ptsp53/A4; {diamondsuit}, ptsp53/A5; {blacktriangleup}, ptsp53/A10.

 

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Table 1 Expression of the apoptosis-related cell surface antigen Annexin V in mock-transfected and wild-type p53-expressing K562 cells

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

 
Wild-Type p53 Promotes the Capacity for Hemoglobin Production Induced by Hemin.
When incubated with hemin, K562 cells show signs of erythroid differentiation, as judged by an increase of their hemoglobin content (24) . To study the role of p53 in erythroid differentiation without previous selection against wild-type p53-dependent activities, clones K562/ptsp53 A2 and K562/ptsp53 A5 were chosen for additional experiments. The two remaining clones were excluded from differentiation studies because of extensive p53-mediated cell death, which made it difficult to examine the differentiation response. To ascertain that the levels of p53 remained high during differentiation induction with hemin, a Western blot was performed after 24 h, showing unaffected ptsp53 levels after incubation with 20 µM hemin (data not shown).

After 4 days at the permissive temperature with or without 5 µM hemin, p53-expressing and mock-transfected cells were subjected to a benzidine oxidation test (Fig. 3)Citation . This test is performed to determine the peroxidase activity of the cells, which reflects their content of hemoglobin. At this concentration of hemin, the maturation response of the mock-transfected control clones was weak, measured both as fraction (Fig. 3a)Citation and as total number (Fig. 3b)Citation of cells oxidizing benzidine/ml. By contrast, the wild-type p53-expressing clones strongly responded to hemin, both measured as fraction (Fig. 3a)Citation and total number (Fig. 3b)Citation of benzidine-positive cells/ml. Regarded as fraction of cells, p53 increased the differentiation sensitivity of the K562 clones ~4-fold (Fig. 3a)Citation and as absolute/total number of cells ~2-fold (Fig. 3b)Citation . However, only a very modest differentiation inducing effect of p53 per se was observed (Fig. 3)Citation .



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Fig. 3. Effects of p53 and hemin on differentiation of K562 cells, assayed by the capacity to oxidize benzidine. Cells at an initial concentration of 0.2 x 106 cells/ml were incubated at 32°C (i.e., the temperature permissive for the wild-type conformation of p53) with or without hemin at 5 µM. After 4 days, cells were subjected to a benzidine oxidation test. The fraction (A) or total number per ml (B) of benzidine-oxidizing cells are shown. Mean values from nine separate experiments are shown. {square}, culture medium; {blacksquare}, 5 µM hemin; bars, SE.

 
The enhanced response to hemin was evident with concentrations of hemin ranging from 5 to 20 µM, as shown for clone A5 in Fig. 4Citation . The differentiation response of p53expressing clones reached with 5 µM hemin (Fig. 4)Citation was comparable with the level of hemoglobin production in non-p53 producer clones attained with 20 µM hemin, regarded both as fraction (Fig. 4a)Citation and as total number (Fig. 4b)Citation of benzidine-positive cells/ml. These data show that wild-type p53 promotes the capacity for hemoglobin production induced by hemin in K562 cells.



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

 
The Megakaryocyte-related Cell Surface Antigens CD9 and CD61 Were Not Affected by Wild-Type p53.
K562 cells can be induced to express markers associated with megakaryocytic differentiation when incubated with PMA (25) . To further explore the role of p53 along different lineages of differentiation, K562/ptsp53 clones and control clones were incubated with or without PMA at different concentrations at the permissive temperature. Because PMA has been shown to reduce the levels of p53 in some cells (26 , 27) , a Western blot was performed after 24 h in the presence of the highest concentration of PMA (i.e., 10 nM) showing reduced, but clearly visible, levels of transfected p53 (Fig. 5)Citation . After 4 days, the cells were subjected to a FACS analysis of the megakaryocyte-related cell surface antigens CD9 (28) and CD61 (25) , as described in "Materials and Methods." As shown in Fig. 6Citation , both control K562 clones and wild-type p53-expressing K562 clones responded to PMA with up-regulation of CD9 and CD61 at comparable levels. Thus, our results do not support that p53 influences the regulation of the megakaryocyte-related cell surface antigens CD9 and CD61. In parallel with the FACS analysis, cells were counted, and viability was assessed by trypan blue exclusion. The highest concentration of PMA (i.e., 10 nM) completely abolished proliferation of both mock-transfected and wild-type p53-expressing clones and also reduced viability to comparable levels in control clones and p53-expressing clones [the average viability among mock transfectants is 44% (SE is 8%), and among wild-type p53-expressing clones, 49% (SE is 12%)].



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

 


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Fig. 6. Expression of megakaryocyte-related cell surface antigens in control cells and p53-expressing cells upon incubation with PMA. Cells at an initial concentration of 0.2 x 106 cells/ml were incubated with or without PMA at different concentrations at 32°C (i.e., the temperature permissive for the wild-type conformation of p53). After 4 days, the cells were subjected to analysis of CD9 (A) and CD61 (B) by flow cytometry. Values shown are median fluorescence intensity for viable cells. Mean values from three to nine independent experiments are shown. {circ}, Mock 1; {square}, Mock 2; {diamond}, Mock 3; {triangleup}, Mock 4; •, ptsp53/A2; {diamondsuit}, ptsp53/A5; bars, SE.

 
p53-induced Cell Death Negatively Correlates to Expression of p21.
In addition to regulating the cell cycle, p21 has been implicated in the regulation of differentiation and apoptosis (17 , 29 , 30) . Because the sensitivity to p53-mediated apoptosis differed among the subclones (Fig. 2b)Citation , we wanted to determine whether this could be explained by p21. After incubation at the permissive temperature for 22 h, the different clones were analyzed for expression of p21 (Fig. 7)Citation . 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 presence of transcriptionally active p53. The amount of p21 did not correlate to the expression levels of p53 (compare Figs. 1Citation and 7Citation ). Interestingly, however, the p53-mediated cell death correlated inversely to the level of expression of p21, because clones A2 and A5 not only were partially protected against p53-mediated apoptosis (Fig. 2b)Citation but also on repeated Western blot analysis showed higher expression levels of p21 than did clones A4 and A10 (Fig. 7)Citation .



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Fig. 7. Expression of the cell cycle regulator p21 in response to expression of wild-type p53. Mock-transfected and p53-expressing K562 clones were incubated at 32°C (i.e., the temperature permissive for the wild-type conformation of p53) for 22 h, after which the cells were subjected to Western blot, as described in "Materials and Methods." Arrow to the right, position of p21 protein at Mr 21,000. Left, positions of molecular weight standards.

 
Transient Overexpression of p21 Does Not Protect against p53-mediated Apoptosis of K562 Cells.
To determine whether the observed correlation between high levels of p53-induced p21 and protection against p53-mediated apoptosis actually reflected a causal relationship, transient transfection experiments with p21 were performed. K562 M1 cells were transfected either with p21 cDNA or with a control vector. The plasmid EGFP-N1 carrying the cDNA for EGFP was cotransfected with each of the plasmids, thus allowing cell sorting based on the green fluorescent light emitted by EGFP. Twenty-four h after transfection, cells were sorted for expression of EGFP by FACS analysis (for details, see "Materials and Methods"). After another 36 h, cells were analyzed for expression of p21 by Western blot (Fig. 8)Citation . As shown, no p21 was expressed in the mock-transfected cells, whereas high levels of p21 was seen in cells transiently transfected with p21, demonstrating the production of p21 protein.



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Fig. 8. Expression of p21 in p21 and control-transfected cells. The mock-transfected K562 clone M1 was transfected with control vector or the 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 weight standards.

 
Hypophosphorylation of the retinoblastoma protein pRb is an important mechanism for p21-mediated cell cycle arrest (31 , 32) . Moreover, wild-type p53 activity causes a prompt hypophosphorylation of pRb (16 , 33) . The phosphorylation status of pRb in control or p21-transfected K562 M3 cells was therefore investigated by IP-Western blot to assure that the transfected p21 was functionally active (Fig. 9)Citation . To achieve a positive control for induction of pRb hypophosphorylation, K562/ptsp53/A5 cells were transiently transfected with control vector 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. Expression of wild-type p53 induced a shift from hyper- to hypophosphorylated Rb, as demonstrated by the shift to faster migrating bands in the lane containing K562/ptsp53/A5 cells incubated at the permissive temperature (Fig. 9)Citation . Moreover, expression of p21 also caused a similar hypophosphorylation of pRb, an observation shown on repeated Western blots. This suggests that the transiently transfected p21 was indeed functionally active under the experimental conditions.



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Fig. 9. Degree of phosphorylation of pRb in p21 and control-transfected cells. The mock-transfected K562 clone M3 was transfected with control vector (C) or the cDNA for p21, respectively, as described in "Materials and Methods." K562/ptsp53/A5 cells transiently transfected with control vector (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 a positive control for induction of pRb hypophosphorylation. Seventy-two h after transfection, cells were subjected to analysis of pRb by IP-Western blot. Arrow to the right, position of pRb. Left, positions of molecular weight standards. One representative experiment of three performed is shown.

 
Because cell cycle arrest has been reported to protect against apoptosis (34, 35, 36) , we next asked whether expression of p21 would affect the cell cycle distribution of K562 cells. For this matter, mock-transfected K562 clones were transiently transfected with p21 or with control vector as described above. On days 1, 2, and 3 after cell sorting, the cell cycle distribution of transfected cells was investigated by FACS analysis. No difference was observed between p21-expressing cells and control-transfected cells (Table 2Citation and data not shown), indicating that expression of p21 does not affect the cell cycle distribution of K562 cells. Moreover, transient overexpression of p21 did not affect the proliferation rate of mock-transfected K562 cells (data not shown).


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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 and Methods." 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.

 
To examine whether transient expression of p21 in cells with low expression of p53-induced p21 would protect against p53-mediated apoptosis, K562/ptsp53/A4 and K562/ptsp53/A10 and mock-transfected control clones were transiently transfected with the cDNA for p21 or with a control vector. Cells were sorted for expression of EGFP by FACS, and 16 h after cell sorting, cell clones were incubated at the permissive (i.e., 32°C) temperature for 4 days. Each day, cells were counted, and viability was assessed by trypan blue exclusion. As expected, clones carrying ptsp53 showed reduced viability. However, no difference in viability was observed between clones transfected with p21 or with control vector (Fig. 10)Citation , suggesting that forced expression of p21 does not protect against p53-mediated apoptosis in K562 cells. Taken together, these data suggest that the p21-Rb axis is of minor importance for the regulation of cell cycle distribution and for the p53-induced cell death of K562 cells.



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Fig. 10. Effect of p21 on the viability of wild-type p53-expressing and mock-transfected K562 clones. Cells transiently transfected with p21 or with control vector were seeded in culture medium at a concentration of 0.2 x 106 cells/ml and incubated at 32°C (i.e., the temperature permissive for the wild-type conformation of p53) for 4 days. Viability, as judged by trypan blue exclusion, as well as the total number of cells, was determined daily. Viability was always >90% on day 0. Mean values from three experiments are shown. {circ}, Mock 1/Control; •, Mock 1/p21; {square}, Mock 2/Control; {blacksquare}, Mock 2/p21; {square}, A4/Control; {blacksquare}, A4/p21; {triangleup}, A10/Control; {blacktriangleup}, A10/p21.

 
Transient Overexpression of p21 Does Not Induce Differentiation of K562 Cells.
p21 has been given a role in the differentiation process of a number of tissues, such as muscle cells, nerve cells, and a variety of hematopoietic cell lines (13, 14, 15 , 17 , 18 , 37 , 38) . As mentioned above, expression levels of p21 correlates to p53-mediated facilitation of hemin-induced differentiation of K562 cells, inasmuch as cell clones sensitive to p53-facilitated differentiation also show high levels of p21 (Figs. 3Citation and 7)Citation . To investigate whether p21 per se induces differentiation of K562 cells, mock-transfected K562 control clones were transiently transfected with p21 or with control vector. Sixteen h after cell sorting by FACS, cells were seeded with or without hemin at different concentrations in culture medium. After 4 days, a benzidine oxidation test was performed (Fig. 11)Citation . As shown, control-transfected and p21-transfected cells respond with oxidation of benzidine at comparable levels when incubated both with and without hemin [5 µM hemin does not induce expression of p21 in K562 cells (data not shown)]. Hence, these results suggest that expression of p21 per se does not induce or facilitate the hemin-induced differentiation of K562 cells.



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Fig. 11. Effects of p21 and hemin on the differentiation of K562 cells, assayed by the capacity to oxidize benzidine. The mock-transfected K562 clones M1 and M2 were transfected with control vector or the cDNA for p21, respectively, as described in "Materials and Methods." Forty-eight h after transfection, cells at an initial concentration of 0.2 x 106 cells/ml were incubated in culture medium with or without hemin at different concentrations. After 4 days, cells were subjected to a benzidine oxidation test. The fraction of benzidine-oxidizing cells are shown. Mean values from three separate experiments are shown. {square}, culture medium; , 1 µM hemin; , 5 µM hemin; {blacksquare}, 25 µM hemin; bars, SE.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Our results show that hemin and inducibly expressed wild-type p53 act in a cooperative manner to promote erythroid differentiation of K562 cells, in line with the previously observed cooperation of constitutively expressed wild-type p53 and tumor necrosis factor in K562 cells (8) .

Wild-type p53 sensitizes K562 cells to hemin-induced differentiation ~4-fold, regarded as fraction of cells. Moreover, a better differentiation response of the p53-expressing clones was obvious also when it was determined as total number of benzidine-oxidizing cells. This excludes the possibility that p53-dependent growth arrest or apoptosis of a subpopulation of cells not prone for differentiation were the cause of the increased fraction of cells showing signs of erythroid differentiation and indicates that wild-type p53 indeed has an inherent differentiation facilitating capacity. Furthermore, our results suggest that p53 and hemin have a cooperative effect on erythroid differentiation of K562 cells, as judged by the finding that the differentiation response between 1 and 10 µM hemin was higher than merely an additive effect of the two separate differentiation inducers. This suggests that hemin and p53 operate in at least partially separable pathways.

The present modest differentiation-inducing activity of p53 alone in K562 cells is consistent with previous results on inducible expression of p53 in these cells (39) . However, K562 cells with constitutively expressed p53 can show as high as 40% benzidine positivity (7) . The divergence in differentiation response between cells exhibiting inducible or constitutive expression of p53 may be explained by a subclonal selection during establishment of cell clones constitutively expressing p53, which affects the differentiation response. Constitutive expression of p53 might select for cells with an inherent capacity for differentiation that is not directly connected to p53. For example, it has been demonstrated that cells engaged in a differentiation program can be protected against cell death (40) . Thus, although activation of p53 probably provokes apoptosis in the majority of the cells, a small differentiation-prone subpopulation could survive and proliferate. Another possible explanation is that differentiation depends on a certain pace of proliferation, and that the absence of cell cycle-arresting features of p53 during constitutive expression allows the differentiation-promoting effects of p53 to be more pronounced. This view is supported by the observation that differentiation synergistically induced by p53 and IFN-{gamma} in U-937 cells correlates to a decreased 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 surface markers on K562 cells. This suggests that p53 does not unspecifically influence phenotypic modulation. Rather, these data might suggest distinctive interactions with the differentiation response, perhaps reflecting specific molecular interactions pertaining to specific differentiation programs, because p53 facilitated differentiation induced by hemin but not by PMA of K562 cells. Although the levels of p53 declined in response to PMA in the K562 cells, the cells still expressed considerable amounts of p53 protein. Hence, it is very likely that the p53 levels present suffice for facilitation of differentiation but do not participate in the response to PMA, because low levels of p53 can induce differentiation (41) and that the transcriptional activity of p53 can increase concomitantly with decreasing levels of p53 protein during differentiation of mouse keratinocytes (42) .

The cell cycle regulator p21 is a transcriptional target of p53 and arrests the cell cycle in the G1 phase. Moreover, a number of studies demonstrate that p21 can protect against apoptosis of monoblastic U-937 cells as well as muscle cells (17 , 30) and also against p53-mediated apoptosis of human melanoma cells (29) . Furthermore, a correlative connection between high levels of p21 and differentiation has been shown in a number of tissues, such as muscle cells, nerve cells, and hematopoietic cells (13, 14, 15) .

In the present study, high levels of p53-induced p21 correlated to protection against p53-mediated apoptosis, allowing p53-mediated differentiation to proceed. Thus, levels of p21 seemed discriminative between p53-mediated differentiation and death. To examine whether this connection reflected a causal relationship or was merely correlative, p21 was transiently overexpressed in K562 cells expressing wild-type p53 and low levels of p53-induced p21 (i.e., clones A4 and A10). Overexpression of p21 did not affect the response to p53-mediated apoptosis, indicating that p21 does not protect against p53-mediated apoptosis of K562 cells. To analyze the role of p21 in the induced differentiation of K562 cells, p21 was transiently overexpressed in p53-null K562 cells with and without hemin. No p21-related effects on the differentiation response were demonstrated, suggesting that p21 alone does not induce or facilitate hemin-induced differentiation of K562 cells. However, a potential cooperation between p21 and p53 in the differentiation of p53-expressing cells proved impossible to analyze because of the low viability in the studied cell clones. Hence, because p21 was always expressed during the p53-mediated differentiation, it cannot be excluded that p21 and p53 cooperate for induction of differentiation.

Furthermore, despite the ability of the transfected p21 to activate the retinoblastoma protein by dephosphorylation, no p21-related effects on the cell cycle were observed. Taken together, these data suggest that the p21-Rb axis is of minor importance in the regulation of differentiation, cell cycle distribution and apoptosis of K562 cells. Interestingly, it was shown recently that inducible expression of pRb does not affect the growth of mouse lymphoid cells or human myeloid 32D cells. Instead, growth is inhibited by ectopic expression of p130 (43) , indicating distinct cell cycle regulatory functions for the members of the Rb family, possibly depending on the cellular context. Moreover, because p21 alone does not induce differentiation of K562 cells, other molecules than p21 are probably critical for mediating p53-induced differentiation.

In conclusion, our results show that even if p53 does not have a pronounced differentiation inducing capacity on its own, it facilitates hemin-induced erythroid differentiation of K562 cells. This indicates that p53 plays a role in the molecular regulation of specific differentiation programs. Moreover, other downstream targets than p21 are probably critical for p53-mediated differentiation of K562 cells.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cells and Culture Conditions.
The human erythroblastic leukemia cell line K562 (44) was cultured in RPMI 1640 (Gibco Ltd., Paisley, United Kingdom), supplemented with 10% heat-inactivated FCS (Gibco Ltd.) in a humidified CO2 atmosphere at 37°C. Exponentially growing cells were used for all experiments. The number of cells and viability, as judged by trypan blue exclusion, were determined by counting the cells in a Bürker chamber.

Vector Constructs.
The eukaryotic expression vector ptsp53 (pLTRp53cGval135) carrying the cDNA for a murine temperature-sensitive mutant of p53, driven by the long terminal repeat from Harvey murine sarcoma virus, was generously provided by Dr. Moshe Oren (Rehovot, Israel). At 32°C, the protein product from p53cGval135 cDNA adopts a conformation permitting wild-type p53 activity, whereas at 37°C, the protein adopts a conformation restricting wild-type p53 activity. The eukaryotic expression vector pRC-CMV was from InVitrogen (AMS Biotechnology, Oxon, United Kingdom). It provides a CMV promoter-driven expression of introduced cDNA and confers resistance to geneticin, allowing for selection of recombinant cells. Because ptsp53 lacks a selectable marker for eukaryotic cells, it was cotransfected with pRC-CMV to select for ptsp53-containing cells. Control clones were obtained by transfection with pRC-CMV alone. The p21 cDNA was cloned by reverse transcription-PCR on total RNA from vitamin D3-induced myelo-monoblastic U-937 cells as described (45) . After control sequencing, the p21 cDNA was cloned into the eukaryotic expression vector pcDNA3, providing a CMV promoter-driven expression of p21. The pEGFP-N1 plasmid expressing EGFP under the control of a CMV promoter was from Clontech Laboratories, Inc. (Palo Alto, CA).

Transfection Procedure.
The transfection was performed as described previously (46) . Briefly, for constitutive expression cells were resuspended in 37°C culture medium (RPMI 1640 + 10% FCS) to a concentration of 10 x 106 cells/ml. The plasmid was introduced into the cells by electroporation using the Bio-Rad gene-pulser (Bio-Rad, Melville, NY) with electrical settings of 260 V and 960 µF. After 2 days, cells were seeded together with Geneticin (Boehringer Mannheim, Mannheim, Germany) at a concentration of 1.5 mg/ml in 96-well plates to allow for selection of transfected cells. After 2–3 weeks, individual cell clones were expanded to mass cultures and assayed for expression of p53. For transient transfections, electroporation was performed as above but with electrical settings of 300 V and 960 µF. p21/pcDNA3 and pcDNA3 (control vector), respectively, were cotransfected with pEGFP-N1. After 24 h, transfected cells were separated from nontransfected cells by a FACS sorting based on the fluorescent light emitted by EGFP. The transfection efficiency (i.e., the percentage fluorescent cells) varied between 32 and 48% among the individual cell sortings. For viability experiments, FACS-sorted cells were incubated 16 h in culture medium at 37°C prior to incubation at 32°C. For cell cycle experiments, FACS-sorted cells were incubated 16 h in culture medium at 37°C before FACS analysis. For differentiation and proliferation experiments, FACS-sorted cells were incubated 16 h in culture medium at 37°C prior to seeding at 0.2 million cells/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 to Western and IP-Western blot.

IP-Western Blot.
Expression and phosphorylation status of the retinoblastoma protein pRb was detected by immunoprecipitation followed by 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 (Complete; Boehringer Mannheim). Lysates were vortexed for 10 s, after which they were incubated on ice for 1 h. DNA was then removed by centrifugation at 14,000 x g for 1 h. Lysates were subjected to immunoprecipitation with 2 µg of the mouse monoclonal anti-Rb antibody sc-102 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and immunocomplexes were adsorbed to protein A-Sepharose (Pharmacia-Upjohn, Uppsala, Sweden) and protein G-Sepharose (Sigma Chemical Co., St. Louis, MO) under constant gentle rocking at 4°C for 2–3 h. After washing in lysis buffer, immunoprecipitated proteins were separated by a precast 6% Tris-Glycin gel electrophoresis (Novex, San Diego, CA). Separated proteins were electrophoretically 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 for 30 min, the membrane was washed three times for 5 min each time with wash buffer (0.9% NaCl, 0.05% Tween 20). The membrane was then incubated overnight with 0.1 µg/ml of the mouse monoclonal anti-Rb antibody sc-102 in incubation buffer (0.137 M NaCl, 8 mM Na2HPO4 x 2 H2O, 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 same incubation buffer for 1 h. After washing, proteins were visualized with chromogenic substrates [5-bromo-4-chloro-3-indolyl phosphate-p-toluidine salt (ICN) at 0.05 mg/ml and nitro blue tetrazolium (Sigma) at 0.1 mg/ml] in 4 mM MgCl2 coating buffer without dry milk powder.

Western Blot.
Expression of transfected p53 and of p21 was detected with the monoclonal mouse antibodies pAb 240 and 187, respectively (Santa Cruz Biotechnology, Inc.). The ECL-plus Western blot kit (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom) was used according to the manufacturer’s instructions. Briefly, 5 x 106 cells were washed once in PBS and then frozen at -80°C for at least 20 min. The cell pellet was diluted in 75 µl of lysis buffer (92 mM Tris, 12.1% glycerol, 2.4% SDS, 1.4% ß-mercaptoethanol, and 2.9% bromphenol blue), after which the cells were lysed by sonication with a Dr. Hielsher sonicator (B. Braun Biotech International GmbH, Melsungen, Germany). Samples were boiled for 5 min and subsequently spun down at 14,000 x g for 10 min at 4°C. Cells (0.5 x 106) were loaded in each lane of a precast 10–20% Tris-glycine gel (Novex, San Diego, CA). The separated proteins were subjected to Western blot using Hybond-P polyvinylidene difluoride membranes (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, Life Sciences International) for 5–15 s.

Assessment of Differentiation by Benzidine Oxidation Test.
The benzidine oxidation test was performed as described previously (47) . Briefly, cells (0.2 x 106 cells/ml) were incubated with hemin (Sigma) for 4 days, 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 µl of 30% H2O2 is added just prior to use] was added to start the reaction. After incubation for 30 min in darkness at room temperature, 200 cells were counted in a Bürker chamber, and the number of cells containing oxidized tetramethylbenzidine (visualized as cells containing blue crystals), indicative of peroxidase activity and thus reflecting hemoglobin production, was determined.

Assessment of Cell Surface Antigens by Flow Cytometric Analysis.
Cells (0.2 x 106 cells/ml) were incubated with PMA (Sigma) for 4 days, after which they were washed once in PBS and resuspended to 5 x 106 cells/ml. Fifty µl of the cell suspension were incubated for 10 min at room temperature under constant agitation with 5 µl of the following monoclonal antibodies in microtiter wells: control IgG1-FITC/IgG1-PE, CD61-FITC (Becton Dickinson, San Jose, CA); CD9-FITC (DAKO A/S, Copenhagen, Denmark); Annexin V-FITC (PharMingen, San Diego, CA); and propidium iodide (Sigma). The cells were then washed three times and fixed 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 the calculation of the fraction of positive cells, using the control incubation with IgG1-FITC/IgG1-PE for marker settings. Cells were analyzed for expression of Annexin V in parallel with staining with propidium iodide, which makes it possible to exclude necrotic cells. This provides a selective method for detection of apoptosis (23) .

Cell Sorting.
EGFP-expressing K562 cells were purified using a FACS Vantage Cell sorter (Becton Dickinson Immunocytometry Systems, San Jose, CA) upgraded with a Turbo Sort unit and equipped with a 488-nm argon laser. Single, viable, and EGFP-expressing cells were selected by gating based on laser scatter profile and 515–545 nm fluorescence. The K562 cells were sorted at a rate of 2000–4000 cells/s.

Cell Cycle Analysis.
Staining of nuclei and flow cytometric analysis were performed as follows. Cells were washed in Dulbecco’s PBS, after which 0.2 ml of a nuclear isolation medium containing propidium iodide was added (50 µg/ml propidium iodide, 0.6% NP40, 100 µg/ml RNase, DNase-free, in PBS; all reagents from Sigma). The cells were then incubated at room temperature in the dark for 60 min before the addition of 0.4 ml PBS and taken to flow cytometric analysis in a FACScan flow cytometer (Becton Dickinson, San Jose, CA). Up to 20,000 nuclei were analyzed per sample. Using the electronic peak and area detectors, processor signals from nuclei doublets were rejected. Cell cycle phase distribution, i.e., the percentages of G0 + G1, S, and G2 nuclei of the analyzed cell population, was determined by applying ModFit LT cell cycle analysis software (Verity Software House, Inc., Topsham, ME) on the DNA histograms. The DNA histogram was corrected for contribution of nucleic debris.


    Acknowledgments
 
We thank Dr. Tor Olofsson for valuable discussions and for thoughtfully performing the analysis of cell cycle distribution and cell surface antigens by flow cytometry. We also thank Sverker Segrén for cheerful and fluorescent cell sorting by flow cytometry.


    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 This work was supported by the Swedish Cancer Foundation; the Swedish Childhood Cancer Foundation; The Tobias Foundation; The Swedish Medical Research Council (Project 11546); Funds of Lunds Sjukvårdsdistrikt; The Gunnar, Arvid, and Elisabeth Nilsson Foundation; and The Royal Physiographic Society of Lund. Back

2 To whom requests for reprints should be addressed, at Research Department 2, EB-block, University Hospital, S-221 85 Lund, Sweden. Phone: 46-46-173556; Fax: 46-46-184493; E-mail: Kristina.Chylicki{at}hematologi.lu.se Back

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

Received for publication 1/24/00. Revision received 4/13/00. Accepted for publication 4/24/00.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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