Inhibition of hemoglobin accumulation by monoclonal antibodies to the human transferrin receptor is reversed by hemin

We have studied the effects of hemin on the inhibition of K562 cell erythroid differentiation mediated by a monoclonal antibody to the human transferrin receptor. In the absence of hemin, the monoclonal antibody 42/6 suppresses the increase of hemoglobin accumulation induced by butyric acid or 5-azacytidine (5-azaCR). In contrast, in the presence of hemin, the hemin-resistant K562(h) cell line is induced to hemoglobin production by treatment with 5-azaCR even in the presence of the monoclonal antibody to human transferrin receptor. The results obtained suggest (1) that the human leukemic K562 cells treated with monoclonal antibodies to human transferrin receptor might retain molecular properties similar to those found in erythroid cells from subjects affected by alterations of iron metabolism, and (2) that hemin might allow hemoglobin synthesis in erythroid cells whose iron uptake is inhibited by monoclonal antibodies to human transferrin receptors.     

Effect of transforming growth factor-beta 1 on proliferation and induction of hemoglobin accumulation in K-562 cells

The effects of transforming growth factor-beta 1 (TGF-beta 1) on proliferation and hemoglobinization in K-562 cells, a human multipotential hematopoietic cell line, were studied. We found that TGF- beta 1 could induce hemoglobin accumulation in K-562 cells. Various clones were selected on the basis of the inducibility of hemoglobinization by TGF-beta 1. One high response clone (no. 1) and one low response clone (no. 8) were studied in detail. Hemoglobin accumulation peaked on day 5 of culture in the presence of TGF-beta 1 (0.5 ng/mL, 20 pmol/L), when 90% of clone 1 cells, 55% of parent line cells, and less than 10% of clone 8 cells contained hemoglobin. There was a concomitant reduction in proliferation of 60% for clone 1, 40% for the parent line, and 30% for the clone 8 on day 5 of culture. Quantitative analysis showed that the hemoglobin contents in clone 1 after 5-day induction by TGF-beta 1 and hemin were 1.0 pg/cell and 2.9 pg/cell, respectively. The hemoglobin induced by TGF-beta 1 showed the same electrophoretic characteristics as the hemoglobin induced by hemin. The expression of epsilon-globin mRNA was minimally detectable in control cells and was induced in both TGF-beta 1 and hemin treated cells. Other cytokines with potential effects on K-562 cell proliferation and differentiation were also studied. Interleukin-1, interleukin-3, interferon alpha, interferon gamma, and inhibin, tested as single agents, showed minimal effects on proliferation. None of these agents could induce hemoglobinization or inhibit the hemoglobinization induced by TGF-beta 1.     

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.     

Human leukemic K562 cells treated with cytosine arabinoside: enhancement of erythroid differentiation by retinoic acid and retinol

Abstract: Human leukemia K562 cells can be induced to erythroid differentiation when treated with a variety of compounds, including hemin, cytosine arabinoside and 5-azacytidine. Following erythroid induction, K562 cells express at high level γ-globin and accumulate both Hb Portland and Hb Gower 1. In this paper we determined whether a combination treatment of K562 cells with suboptimal concentrations of cytosine arabinoside and retinoids lead to full expression of differentiated functions. Cell growth kinetics studies, intracellular detection of hemoglobin by benzidine staining and hemoglobin analysis by cellulose acetate were performed. The results obtained show that (a) retinoic acid and retinol are not able to induce differentiaton of K562 cells and (b) cytosine arabinoside induces differentiation only when used at 100–300 nmol/l concentrations. In addition, our data demonstrate that erythroid differentiation of K562 occurs when 40 μmol/l of retinoic acid or retinol are added together with 75 nmol/l cytosine arabinoside.     

Induction to erythroid differentiation of K 562 cells by 1-β-D-arabinofuranosylcytosine is inhibited by iron chelators: reversion by treatment with hemin

Summary Erythroid differentiation of human leukemic K 562 cells is inhibited by the iron chelator desferrioxamine (DF). In addition, desferrioxamine induces an increase of uptake of hemin. When hemin is added to the culture medium, the DF-mediated inhibitory effects on erythroid induction are reversed. Briefly, hemin allows hemoglobin synthesis by K 562 cells induced to erythroid differentiation by 1--D-arabinofuranosylcytosine (ara-C) and treated with 12.5 g/ml DF. In addition, it was found that hemin treatment leads to a reversion of inhibition of K 562 cell proliferation mediated by 50–75 g/ml DF. This effect of hemin was also detected in other cultured human tumor cell lines (B-lymphoid, erythroleukemic and from breast carcinomas, melanomas and kidney carcinomas).     

Expression and modulation of specific, high affinity binding sites for erythropoietin on the human erythroleukemic cell line K562

Erythroid differentiation is mediated by several interacting factors which include the glycoprotein hormone erythropoietin (Epo), interleukin-3 (IL-3) in the mouse, and erythroid-potentiating activity (EPA) in humans. Each of these factors binds to specific cell surface receptors on responsive target cells, but the way in which these factors interact to modulate erythropoiesis is unknown. In the present study, we used the human erythroleukemic cell line K562 to examine expression and regulation of the receptor for Epo using 125I-labeled, bioactive recombinant human Epo. K562 cells expressed low numbers of a single class of high-affinity Epo receptors corresponding to 4 to 6 receptors per K562 cell (KD = 270 to 290 pmol/L). Treatment of K562 cell cultures with medium conditioned by the EPA-secreting cell line U937 (U937CM) increased receptor expression 2.6 to 3.5-fold to 13 to 17 receptors/cell (KD = 260 to 300 pmol/L). That all of the Epo receptor-potentiating activity in U937CM was accounted for by EPA was shown by a similar increase in Epo receptor expression on K562 cells with recombinant EPA. The effect of U937CM on Epo receptors was reversed by culturing cells in inducer-free medium for 3 days. Medium conditioned by the 5637 cell line had no effect on Epo receptors on K562 cells. In methylcellulose culture, U937CM and Epo acted synergistically to increase erythroid differentiation of K562. Similarly, U937CM stimulated human cord blood CFU-E growth under conditions in which Epo was limiting or in excess. Increases in Epo receptor expression on K562 cells and on CFU-E in response to EPA may mediate the effects of Epo on these cells.     

Cooperative effects of hemin and anthracyclines in promoting terminal erythroid maturation in K562 human erythroleukemia cells

Simultaneous exposure to 30 microM hemin and 3 x 10(-8) M aclacinomycin (ACL) or mussetamycin for 6 days led to terminal differentiation of K562 cells. The number of hemoglobinized cells and the total hemoglobin content of cells treated with both ACL and hemin exceeded the sum of the corresponding values induced in response to each of these two agents when used alone. Although neither ACL nor hemin alone induced substantial morphological maturation, 40%-45% of cells treated with both agents developed the morphological characteristics of orthochromatophilic normoblasts, a level of maturation not previously reported for this highly malignant cell line. Subcloning of K562 cells that had been treated with both ACL and hemin in inducer-free plasma clots revealed a 50% decrease in the clonogenic potential of these cells, even though the cells in the original cultures were still growing at only a moderately decreased rate. Despite the apparent terminal maturation of K562 cells induced with both ACL and hemin, with an advanced level of morphologic maturation and hemoglobin synthesis accompanied by a loss of proliferative capacity, these cells remained incapable of producing beta-globin chains and hence hemoglobin A.     

p38 MAP kinase activation mediates gamma-globin gene induction in erythroid progenitors

OBJECTIVE: Our goal was to determine the role of p38 mitogen-activated protein kinase (MAPK) signaling in fetal hemoglobin (HbF) induction. Two histone deacetylase inhibitors (HDAIs), sodium butyrate (NB), and trichostatin (TSA) and hemin were analyzed. In addition, the effect of direct activation of p38 MAPK on gamma-globin gene activity was studied. METHOD: Primary erythroid progenitors derived from peripheral blood mononuclear cell and K562 erythroleukemia cells were analyzed. Cells were grown in NB, TSA, hemin, or anisomycin either alone or in the presence of the p38 MAPK inhibitor SB203580. The effects of the various treatments on gamma-globin RNA, HbF, and phosphorylated p38 MAPK levels were measured by RNase protection assay, alkaline denaturation, and Western blot analysis, respectively. A K562 stable line overexpressing constitutively active p38 MAPK was established using MAPK kinase kinase 3 (MKK3) and MKK6, the immediate upstream activators of p38. The direct effect of p38 MAPK overexpression on gamma-globin mRNA synthesis was analyzed. RESULTS: NB and TSA activated p38 MAPK and increased gamma-globin mRNA levels in K562 cells and primary erythroid progenitors. Pretreatment with SB203580 blocked p38 MAPK and gamma-globin gene activation. In contrast, no change in p38 activity was observed with hemin inductions. Direct activation of p38 by anisomycin or constitutive overexpression also increased gamma-globin mRNA in the absence of HbF inducers in wild-type K562 cells and in the MKK stable lines. CONCLUSION: This study supports a novel role for p38 MAPK in gamma-globin regulation in human erythroid progenitors.     

Induction by hemin of proliferation and of differentiation of progenitor erythroid cells responsible for erythropoietin

Erythroid progenitor cells were obtained from rat fetal liver by immunolysis of the whole erythroid population with an antiserum directed against adult rat erythrocytes, followed by separation on a density gradient. Immediately after their isolation, these cells contained only minute amounts of globin mRNAs and their heme synthesis was negligible. In the absence of erythropoietin (Epo), they did not proliferate or differentiate. In the presence of Epo, they proliferated, synthetized heme and globins actively, accumulated large amounts of globin mRNAs, and developed hemoglobinized colonies in methylcellulose. Hemin, in concentrations of 5-100 microM, induced, in the absence of Epo, the proliferation and differentiation of these cells (e.g., accumulation of globin mRNAs, synthesis of heme and globins, and increased density of membrane antigens characteristic of the erythrocyte). Nevertheless, Epo and hemin actions were not superimposable: in methylcellulose, Epo induced the appearance of large (greater than or equal to 32 cells) hemoglobinized colonies in 48 h, whereas hemin induced smaller and fewer colonies in only 24 h. Succinylacetone (SA, inhibitor of heme synthesis) mostly prevented the effects of Epo on cell proliferation and differentiation; SA inhibition was relieved by hemin. Thus, hemin seems to intervene in erythroid differentiation as a factor of both proliferation and maturation.     

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.     

Loss of suppression of normal bone marrow colony formation by leukemic cell lines after differentiation is induced by chemical agents

The human leukemic cell lines K562 and HL-60 were cocultured with normal bone marrow (BM) cells. Coculture with 10(4) K562 or HL-60 cells results in 50% inhibition of normal CFU-E and BFU-E colony formation. However, when the same number of K562 and HL-60 cells is first treated for two to five days with agents that induce their differentiation, a gradual loss in their capacity to inhibit CFU-E and BFU-E colony formation is observed. The inhibitory material in K562 cells is soluble and present in conditioned medium from cultures of these cells. The degree to which leukemic cell suppression of CFU-E and BFU-E growth is reversed is correlated with the time of exposure to the inducing agent. Suppression is no longer evident after five days of prior treatment with inducers. In fact, up to a 90% stimulation of CFU-E growth is observed in cocultures with K562 cells that have been pretreated with 30 to 70 mumol/L hemin for five days. K562 cells treated with concentrations of hemin as low as 30 mumol/L demonstrate increased hemoglobin synthesis and grow normally, but no longer have an inhibitory effect on CFU-E growth. Hence, reversal of normal BM growth inhibition must be caused by the more differentiated state of the K562 cells and not by a decrease in the number of these cells with treatment. Thus, induction of differentiation in cultured leukemic cells not only alters the malignant cell phenotype but also permits improved growth of accompanying normal marrow progenitor cells. Both are desired effects of chemotherapy.     

Differing responses of globin and glycophorin gene expression to hemin in the human leukemia cell line K562

The human leukemia cell line, K562, produces embryonic and fetal hemoglobins and glycophorin A, proteins normally associated only with erythroid cells. Hemoglobin accumulation is enhanced by exposure of the cells to 0.05 mM hemin. We have examined K562 cells before and after exposure to hemin to determine whether expression of these erythroid proteins was shared by all cells or confined to specific subpopulations. Globin gene expression was examined by quantitation of globin mRNA sequences, using a 3H-globin cDNA molecular hybridization probe. Constitutive cells produced globin mRNA, the content of which was increased 3-4-fold by hemin. Cell-to-cell distribution of globin mRNA was determined by in situ hybridization of 3H-globin cDNA to constitutive and hemin-treated K562 cells. Virtually all cells in the culture exhibited grain counts above background, indicating globin gene expression by all cells, rather than a confined subpopulation. Virtually all hemin-treated cells had 3-5-fold higher grain counts, indicating uniformly increased globin gene expression. The glycophorin content of K562 cells was estimated by fluorescence-activated cell sorting (FACS) of cells labeled with fluorescein-labeled antiglycophorin antiserum. The vast majority of constitutive cells contained glycophorin, but exhibited to apparent increase in glycophorin accumulation after hemin exposure. Thus, glycophorin and globin genes exhibited differential responses to hemin. These differences could reflect normal differences in the patterns of specialized gene expression in stem cells. Alternatively, different aberrations of gene expression could be occurring in response to the determinants of the neoplastic properties of K562.     

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.     

Expression of the transcription factor Evi-1 in human erythroleukemia cell lines and in leukemias

The Evi-1 proto-oncogene is a zinc finger DNA binding protein. Although activation of the Evi-1 gene has been associated with chromosomal rearrangements of the 3q25-q28 region, ectopic expression of Evi-1 could also be observed in acute myelogenous leukemias and myelodysplastic syndromes without cytogenetic abnormalities of the 3q26 locus. In this study, human erythroleukemic cell lines were screened for the expression of Evi-1 mRNA by northern blotting. Evi-1 was expressed in all the erythroid cell lines, whether undifferentiated (K 562, HEL, LAMA 84) or exhibiting spontaneous terminal erythroid differentiation (KU 812, JK-1). Evi-1 mRNA levels were constant or elevated in hemoglobin-synthesizing KU 812 or K 562 cells in response to erythropoietin or hemin treatment, respectively. In human acute myeloblastic leukemias (AML), 11/30 expressed Evi-1 by RT-PCR. Among these cases, 4/6 erythroleukemias without abnormalities of the 3q25-q28 region were found positive. The presence of acidophilic erythroblasts (15–47% of bone marrow cells) accounted for the existence of a terminal erythroid differentiation in all Evi-1-positive AML M6, whereas one negative case was poorly differentiated and referred to as AML M6 variant. These results suggest that Evi-1 mRNA expression can coexist with erythroid differentiation.     

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.     

In vitro effect on human leukemic K562 cells of co-administration of liposome-associated retinoids and cytosine arabinoside (Ara-C).

The administration of retinoids has been demonstrated to be of potential utility in the therapy of a wide spectrum of neoplastic pathologies due to the ability to induce differentiation in a large variety of primary tumor cells as well as in vitro cultured cell lines. Moreover, a number of compounds, including hemin, cytosine arabinoside, and 5-azacytidine are able to induce erythroid differentiation of the erythroleukemic cell line K562. In this paper we determined whether a combined treatment of K562 cells with suboptimal concentrations of cytosine arabinoside and retinoids containing liposomes lead to a full expression of differentiated functions. Liposomes were prepared by reverse phase evaporation technique followed by extrusion through polycarbonate filters. Cell growth kinetics studies and intracellular detection of hemoglobin by benzidine staining were performed. The results obtained showed that the combined treatment with liposomes containing retinoids and sub-optimal concentration of ara-C is an effective strategy to induce K562 cell differentiation, minimizing at the same time toxic effects. Control experiments aimed to determine possible selection of subpopulations of K562 cells suggest that the observed results are not related to toxicity and/or potential selection of induced cells. In conclusion, liposomally delivered retinoids could be proposed for differentiation therapy as an effective strategy in the treatment and management of malignancy. In addition, the finding that liposomally delivered retinoids increase the capacity of cytosine arabinoside to induce erythroid differentiation, could be of interest in studies aimed at the development of treatment able to reactivate fetal globin genes in beta-thalassemia patients.