Cyclosporin A induces erythroid differentiation of K562 cells through p38 MAPK and ERK pathways

We studied the effects of cyclosporin A (CsA) on the erythroid differentiation of human erythroid leukemia cell line K562. After K562 was treated with CsA for 4 days, the percentage of hemoglobinized cells was increased by 3.3 times. Because it was reported p38 MAPK (p38) and ERK are involved in erythropoietin-induced erythroid differentiation, we studied their roles using specific inhibitors. p38 inhibitor (SB203580) prevented CsA-induced hemoglobin synthesis in K562 cells, although MEK/ERK inhibitor (U0126) enhanced it by 3.3 times in K562 cells. These results indicate activation of p38 and inactivation of ERK are involved in CsA-induced erythroid differentiation of K562 cells.     

Apoptosis induced by erythroid differentiation of human leukemia cell lines is inhibited by Bcl-XL

The induction of tumor cell differentiation represents an attractive strategy for the treatment of a wide range of malignancies. Differentiation of HL-60 promyelocytic leukemia cells towards neutrophils or monocytes has been shown to induce apoptotic cell death, which is inhibited by bcl-2 over-expression. However, the role of the bcl-2 gene family during erythroid differentiation of human leukemia cells remains unknown. We found that human erythroleukemia (HEL) and K562, two leukemia cell lines that undergo erythroid differentiation do not express Bcl-2, but express Bcl-XL, a related protein that functions as an inhibitor of apoptosis. Differentiation of HEL or K562 cells with inducers of erythroid differentiation (hemin, retinoic acid, or transforming growth factor-beta) was accompanied by progressive cell death and degradation of genomic DNA into oligonucleosomal fragments. The loss of cellular viability was associated with downregulation of bcl-xL mRNA and protein. In contrast, the levels of Bax, another Bcl-2 family member implicated in apoptosis remained unaltered. Constitutive expression of Bcl-XL by gene transfer inhibited apoptosis triggered by erythroid differentiation of HEL K562 cells. Yet, Bcl-XL did not alter the expression of epsilon-globin, which is induced during erythoid differentiation of HEL and K562 cells, arguing that apoptosis and differentiation can be uncoupled by Bcl-XL. These results indicate that Bcl-XL acts as an antiapoptosis protein in leukemia cells that undergo erythroid differentiation and that downregulation of bcl-x is a component of the apoptotic response that is coupled to differentiation in human leukemia cells.     

Hormonal effects on cell proliferation in a human erythroleukemia cell line (K562)

We have investigated the hormonal responsiveness of K562 cells using a serum-substituted in vitro clonogenic assay. Dexamethasone inhibited colony formation by the K562 cells, and the inhibitory effect could be reversed by progesterone (10(-6) M). Fluoxymesterone caused a prominent enhancement of K562 colony growth, whereas estriol had no effect. Stimulation by triiodothyronine was maximal at 10(-7) M, and the thyroid effect could be abrogated by the beta 2-adrenergic antagonist butoxamine in equimolar concentrations. Using standard tissue culture conditions, the beta-adrenergic agent isoproterenol, but not the alpha catecholamine phenylephrine, enhanced the proliferation of K562 cells. When K562 cells were grown under hormone-depleted conditions, they developed responsiveness to phenylephrine and were no longer stimulated by isoproterenol. DbcAMP and prostaglandins of the E series also caused K562 colony enhancement. Prostaglandin F2 alpha had no effect on cell proliferation. Insulin was an effective stimulant of colony formation of K562 cells, as were human growth hormone and ovine prolacin. Bovine growth hormone had no effect. Our results are consistent with the identificaiton of K562 as an erythroid line, and they indicate that K562 cells respond to endocrine hormones in a manner analogous to normal erythroid progenitors.     

Accumulation of γ-globin mRNA and induction of erythroid differentiation after treatment of human leukaemic K562 cells with tallimustine

Human leukaemic K562 cells can be induced in vitro to erythroid differentiation by a variety of chemical compounds, including haemin, butyric acid, 5-azacytidine, cytosine arabinoside, mithramycin and chromomycin, cisplatin and cisplatin analogues. Differentiation of K562 cells is associated with an increase of expression of embryo-fetal globin genes, such as the zeta-, epsilon- and gamma-globin genes. The K562 cell line has been proposed as a very useful in vitro model system to determine the therapeutic potential of new differentiating compounds as well as to study the molecular mechanism(s) regulating changes in the expression of embryonic and fetal human globin genes. Inducers of erythroid differentiation stimulating gamma-globin synthesis could be considered for possible use in the therapy of haematological diseases associated with a failure in the expression of normal beta-globin genes. We have analysed the effects of tallimustine and distamycin on cell growth and differentiation of K562 cells. The results demonstrated that tallimustine is a potent inducer, while distamycin is a weak inducer, of K562 cell erythroid differentiation. Erythroid differentiation was associated with an increase of accumulation of gamma-globin mRNA and of production of both haemoglobin (Hb) Gower 1 and Hb Portland. In addition, tallimustine-mediated erythroid induction occurred in the presence of activation of the apoptotic pathway. The reasons for proposing tallimustine as an inducer of gamma-globin gene expression are strongly sustained by the finding that this compound stimulates fetal haemoglobin production in human erythroid precursor cells from normal subjects.     

K562 cells produce and respond to human erythroid-potentiating activity

Human erythroid-potentiating activity (EPA) is a 28,000 mol wt glycoprotein that stimulates the growth of erythroid progenitors in vitro and enhances colony formation by the K562 human erythroleukemia cell line. EPA has potent protease inhibitory activity, and is also referred to as tissue inhibitor of metalloproteinases (TIMP). We observed that colony formation by K562 cells in semi-solid medium containing reduced fetal calf serum (FCS) is not directly proportional to the number of cells plated, suggesting production of autostimulatory factors by K562 cells. Using radioimmunoprecipitation and a bioassay for EPA, medium conditioned by K562 cells was found to contain high levels of biologically active EPA; Northern hybridization analysis confirmed the expression of EPA mRNA. Radiolabeled EPA was used to identify cell surface receptors on K562 cells. Together, these results suggest that EPA may act as an autocrine growth factor for K562 cells.     

Tumor necrosis factor-alpha and hematopoietic progenitors: effects of tumor necrosis factor on the growth of erythroid progenitors CFU-E and BFU-E and the hematopoietic cell lines K562, HL60, and HEL cells

Macrophages can modulate the growth of hematopoietic progenitors. We have examined the effects of tumor necrosis factor-alpha, a product of activated macrophages, on human erythroid progenitors (CFU-E, BFU-E) and the hematopoietic cell lines K562, HL60, and HEL cells. Tumor necrosis factor (TNF) significantly inhibited CFU-E and BFU-E growth at concentrations as low as 10(-11)-10(-12) M (0.2 U/ml), although erythroid colony and burst formation were not totally ablated. Preincubation of marrow samples with TNF for 15 min was sufficient to suppress erythroid colony and burst formation. Addition of TNF after the start of culture inhibited CFU-E- and BFU-E-derived colony formation if TNF was added within the first 48 h of culture. Additionally, TNF inhibited the growth of highly purified erythroid progenitors harvested from day 5 BFU-E. The colonies which formed in cultures treated with TNF were significantly smaller than those formed in control cultures. TNF (10(-8)-10(-10) M) also suppressed the growth of the hematopoietic cell lines K562, HL60, and HEL cells, with 40%-60% of the cells being sensitive to TNF. Preincubation of HL60 cells with TNF for 15 min significantly inhibited their growth. K562, HL60, and HEL cells expressed high-affinity receptors for TNF in low numbers (6000-10,000 receptors per cell). Fluorescence-activated cell sorter analysis of TNF binding to HEL cells demonstrated that the majority of these cells expressed TNF receptors. These data suggest that: (1) TNF is a rapid irreversible and extremely potent inhibitor of CFU-E, BFU-E, and hematopoietic cell lines K562, HL60, and HEL cells; (2) TNF appears to be acting on a subpopulation of erythroid cells, predominantly CFU-E, BFU-E, and possibly proerythroblasts; (3) TNF appears not to require accessory cells such as lymphocytes or macrophages to inhibit erythroid progenitors; and (4) the presence of TNF receptors on hematopoietic cells is not sufficient to confer sensitivity to TNF since the majority (80%-95%) of HEL cells express TNF receptors while only 40%-60% are inhibited by TNF.     

HEL and K562 cells: analysis of proteins by two-dimensional gel electrophoresis and comparison to erythroid and nonerythroid cells

We have examined the total protein composition of the erythroleukemia cell lines K562 and HEL using high-resolution two-dimensional gel electrophoresis and have compared them with the pattern obtained by normal erythroid and nonerythroid cells. The proteins from the two cell lines, K562 and HEL, gave two-dimensional patterns that were similar to each other and to that of normal lymphocytes or leukemic cell lines. In contrast, normal erythroid precursor cells (BFUe-derived normoblasts) and erythrocytes have a protein profile that is characteristic and significantly different from that of normal lymphocytes or the leukemic cell lines examined. These data suggest a common protein profile in hemopoietic cells at early stages of differentiation (K562, HEL, or other leukemic lines) and in normal lymphocytes. Erythroid cells, in contrast to lymphoid cells, appear to diverge significantly from this common protein profile when differentiation proceeds to the level of morphologically recognizable erythroid cells. Induction of K562 and HEL cells by hemin produces changes in the abundance of several proteins, but fails to change the overall protein profile of the two cell lines.     

Induction of erythropoietic colonies in a human chronic myelogenous leukemia cell line.

The ability of cells derived from the K562 cell line to generate erythropoietic colonies was studied. The K562 cell line was derived from a patient with chronic myelogenous leukemia 8 yr ago by Lozzio and Lozzio. Rare benzidine-positive colonies formed when these cells were cloned in plasma clots (3 +/- 1/10(4) cells), and their number was not substantially increased by the addition of erythropoietin (9.5 +/- 1/10(4) cells). Sodium butyrate was capable of markedly enhancing the number of benzidine-positive colonies (19.5 +/- 1/10(4) cells) formed, while the combination of sodium butyrate plus erythropoietin exerted a synergistic effect on erythropoietic colony formation (57 +/- 4/10(4) cells). The K562 cell line is a long-term culture system that contains human erythropoietic stem cells. This cell line should be useful in future studies on the cellular and molecular events associated with human erythroid cell differentiation.     

p16(INK4a) induces differentiation and apoptosis in erythroid lineage cells

OBJECTIVE: Hematopoiesis is regulated by proliferation, differentiation, and death. p16(INK4a) has been reported to regulate apoptosis and differentiation of diverse cells, as well as arresting the cell cycle at G1 phase. The aim of this study is to explore the properties of p16 in apoptosis and differentiation of erythroid cells. METHODS: We transfected the INK4a gene to K562 cells, which defect the INK4a gene, and compared the effect of enforced expression of p16(INK4a) with that of various additives, topoisomerase I inhibitor (SN 38), interferon-alpha, phosphatidyl-inositol-3 kinase inhibitor (LY294002), and serum deprivation, which arrest cell cycle at different phases. We also investigated the role of p16(INK4a) in normal day-6 human erythroid colony-forming cells by transfecting the INK4a gene. RESULTS: p16(INK4a) induced cell cycle arrest at the G0/G1 phase, and promoted erythroid differentiation in viable K562 cells, but induced apoptosis in K562 cells with incomplete differentiation. The apoptosis induced by p16 was accompanied with downregulation of bcl-x and nuclear NF-kappaB. These findings were not observed in K562 cells treated with various additives. p16(INK4a) decreased the cell viability and promoted apoptosis in day-9 ECFC. CONCLUSION: We propose that p16(INK4a) plays a role in maintaining homeostasis during erythroid differentiation, and that the mechanisms for this effect are not confined to those inducing cell cycle arrest.     

Hemin-induced erythroid differentiation changes the sensitivity of K562 cells to tumor necrosis factor-alpha

Tumor necrosis factor-alpha (TNF) can inhibit the growth of erythroid progenitors (erythroid colony-forming units [CFU-E] and erythroid burst-forming units [BFU-E]) at picomolar concentrations, but only if added within the first 48 h of culture. These data suggested that cells undergoing erythroid differentiation become resistant to TNF. To test this hypothesis, K562 cells were treated with hemin to induce erythroid differentiation and then tested for their sensitivity to TNF in terms of growth and TNF receptor expression. TNF inhibited the growth of untreated K562 cells, but not hemin-treated K562 cells. Untreated K562 cells expressed TNF receptors, whereas few hemin-treated K562 cells expressed TNF receptors within 24 h of exposure to hemin. These data show that K562 cells induced to differentiate along the erythroid pathway are resistant to TNF because they lack TNF receptors and suggest that the resistance of erythropoietin-treated human bone marrow cells to TNF added after 48 h of culture may also reflect loss of TNF receptors associated with erythroid differentiation.     

Depletion of the Shwachman-Diamond syndrome gene product,SBDS, leads to growth inhibition and increased expression of OPG and VEGF-A

Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by bone marrow failure and leukemia predisposition, pancreatic exocrine dysfunction, and skeletal abnormalities, manifesting as skeletal dysplasia and osteoporosis. Mutations in SBDS have been shown to cause SDS, but the function of the SBDS gene product is unclear. Accelerated angiogenesis has recently been described in bone marrow cells from SDS patients. To clarify the unknown function of SBDS, we performed experiments analyzing the cellular effects of depleting SBDS by RNA interference. The growth of HeLa cells constitutively depleted of SBDS was markedly hindered when compared to cells stably transfected with siRNA against an irrelevant control gene. Similarly, growth of HeLa cells induced to express siRNA against SBDS was specifically inhibited. Inducible SBDS knockdown was associated with modestly increased levels of apoptosis, suggesting a partial contribution of this process to growth inhibition. By microarray analysis of knockdown cells, we found marked differences in expression of genes in multiple pathways, and we chose to examine a selected subset more closely using quantitative PCR arrays. In constitutive and inducible SBDS-depleted HeLa cell clones, we found 3- to 6-fold elevated mRNA levels of osteoprotegerin (OPG or TNFRSF11B) and vascular endothelial growth factor-A (VEGF-A). We confirmed significant overexpression of both secreted proteins by ELISA from supernatants of SBDS-depleted HeLa cells. Osteoprotegerin and VEGF-A are known to have diverse effects on osteoclast differentiation, angiogenesis, and monocyte/macrophage migration, all processes that may be aberrant in SDS, and we propose that overexpression of these factors may contribute to its pathology.     

Butyrate-induced erythroid differentiation of human K562 leukemia cells involves inhibition of ERK and activation of p38 MAP kinase pathways

Butyrate induces cytodifferentiation in many tumor cells of different origin, suggesting that an as yet unidentified common mechanism inherent to malignant cells is the target of butyrate action. This study determined the role of different mitogen-activated protein (MAP) kinase signal transduction pathways in butyrate-induced erythroid differentiation of K562 human leukemia cells. Using a panel of anti-ERK, JNK, and p38 phosphospecific antibodies, the study showed that phosphorylation of ERK and JNK is decreased following treatment of cells with butyrate, whereas phosphorylation of p38 is increased. In contrast, a K562 subline defective in butyrate-mediated induction of erythroid differentiation did not reveal these changes in phosphorylation patterns. Inhibition of ERK activity by UO126 induces erythroid differentiation and acts synergistically with butyrate on hemoglobin synthesis and inhibition of cell proliferation, whereas inhibition of p38 activity by SB203580 completely abolished induction of hemoglobin expression by butyrate. Taken together, our data suggest a model in which butyrate induces erythroid differentiation of K562 cells by inhibition of ERK and activation of p38 signal transduction pathways.     

BMP4, SCF, and hypoxia cooperatively regulate the expansion of murine stress erythroid progenitors

The erythroid response to acute anemia relies on the rapid expansion in the spleen of a specialized population of erythroid progenitors termed stress BFU-E. This expansion requires BMP4/Madh5-dependent signaling in vivo; however, in vitro, BMP4 alone cannot recapitulate the expansion of stress BFU-E observed in vivo, which suggests that other signals are required. In this report we show that mutation of the Kit receptor results in a severe defect in the expansion of stress BFU-E, indicating a role for the Kit/SCF signaling pathway in stress erythropoiesis. In vitro analysis showed that BMP4 and SCF are necessary for the expansion of stress BFU-E, but only when spleen cells were cultured in BMP4 + SCF at low-oxygen concentrations did we recapitulate the expansion of stress BFU-E observed in vivo. Culturing spleen cells in BMP4, SCF under hypoxic conditions resulted in the preferential expansion of erythroid progenitors characterized by the expression of Kit, CD71, and TER119. This expression pattern is also seen in stress erythroid progenitors isolated from patients with sickle cell anemia and patients with beta-thalassemia. Taken together these data demonstrate that SCF and hypoxia synergize with BMP4 to promote the expansion and differentiation of stress BFU-E during the recovery from acute anemia.     

The DNA-binding drugs mithramycin and chromomycin are powerful inducers of erythroid differentiation of human K562 cells

The human leukaemic K562 cell line can be induced in vitro to undergo erythroid differentiation by a variety of chemical compounds, including haemin, butyric acid, 5-azacytidine and cytosine arabinoside. Differentiation of K562 cells is associated with an increased expression of embryo-fetal globin genes, such as the zeta, epsilon and gamma globin genes. Therefore the K562 cell line has been proposed as a useful in vitro model system to determine the therapeutic potential of new differentiating compounds as well as to study the molecular mechanism(s) regulating changes in the expression of embryonic and fetal human globin genes. Inducers of erythroid differentiation which stimulate gamma-globin synthesis could be considered for possible use in the experimental therapy of those haematological diseases associated with a failure in the expression of adult beta-globin genes. In this paper we demonstrated that the G + C selective DNA-binding drugs chromomycin and mithramycin were powerful inducers of erythroid differentiation of K562 cells. Erythroid differentiation was associated with an increase in the accumulation of (a) Hb Gower 1 and Hb Portland and (b) gamma-globin mRNA.     

Hematologic abnormalities in Shwachman Diamond syndrome: lack of genotype-phenotype relationship

Shwachman-Diamond syndrome (SDS) is an autosomal-recessive disorder characterized by short stature, exocrine pancreatic insufficiency, and hematologic defects. The causative SBDS gene was sequenced in 20 of 23 unrelated patients with clinical SDS. Mutations in the SBDS gene were found in 75%, being identical in 11 patients. Hematologic parameters for all 3 lineages were determined over time such as absolute neutrophil counts (ANCs), granulocyte functions, and erythroid and myeloid colony formation (erythroid burst-forming unit [BFU-E] and granulocyte-monocyte colony-forming unit [CFU-GM]) from hematopoietic progenitor cells, percentage of fetal hemoglobin (HbF), and platelet counts. Persistent neutropenia was present in 43% in the absence of apoptosis and unrelated to chemotaxis defects (in 65%) or infection rate. Irrespective of the ANC in vivo, abnormal CFU-GM was observed in all patients with SDS tested (14 of 14), whereas BFU-E was less often affected (9 of 14). Cytogenetic aberrations occurred in 5 of 19 patients in the absence of myelodysplasia. One child died during allogeneic bone marrow transplantation. In conclusion, neutropenia and defective chemotaxis did not result in severe clinical infection in SDS. CFU-GMs were impaired in all patients tested. From the SBDS sequence data, we conclude that in patients with genetically proven SDS a genotype-phenotype relationship in SDS does not exist in clinical and hematologic terms.     

The α1-adrenergic receptor antagonists, benoxathian and prazosin, induce apoptosis and a switch towards megakaryocytic differentiation in human erythroleukemia cells

The erythroleukemia cell lines K562 and human erythroleukemia (HEL) are established models to study erythroid and megakaryocytic differentiation in vitro. In this study, we show that the α1-adrenergic antagonists, benoxathian and prazosin, inhibit the proliferation and induce apoptosis in K562 and HEL cells. Furthermore, both tested substances induced the expression of the megakaryocytic marker CD41a, whereas the expression of the erythroid marker glycophorin-a was decreased or unchanged. Even though the expression of differentiation markers was similar after benoxathian and prazosin treatment in both cell lines, endomitosis of erythroleukemia cells was observed only after prazosin treatment. So far, benoxathian and prazosin are the first described extracellular ligands, which cause megakaryocytic differentiation in K562 and HEL cells. In summary, these results indicate a possible role of α1-adrenergic receptor signaling in the regulation of erythroid and megakaryocytic differentiation, even though the receptor dependence of the observed effects needs further investigation. Konrad Schauenstein deceased at May 22, 2007.     

Anti-K562 cell monoclonal antibody RTF8X recognizes tumor-associated antigen of erythroid lineage

A new anti-K562 cell monoclonal antibody, RTF8X, a cytotoxic IgM, recognized a surface antigen on erythroblasts from patients with erythroleukemia and polycythemia vera. RTF8X, which is highly specific to K562 cells, did not react with the other 14 hematopoietic cell lines and the seven nonhematopoietic cell lines. RTF8X antigen was not detected in normal peripheral blood, but was found in less than 1% of normal marrow cells. RTF8X did not inhibit in vitro colony formation of CFU-E and BFU-E in a complement-dependent cytotoxicity assay. Cell- sorting analysis showed that, morphologically, the RTF8X-positive marrow cells from the patients and normal volunteers contained more than 60% erythroblasts and that CFU-E and BFU-E were not demonstrated in cells with RTF8X antigen. Enzyme treatment suggested that RTF8X antigen was a sialoglycolipid. These results indicate that RTF8X may recognize the surface antigen found increasingly in association with tumors of erythroid lineage. RTF8X should be useful for studies of erythroid differentiation and proliferation in patients.