Characteristic expression of Hck in human B-cell precursors

OBJECTIVE: To identify molecules involved in signaling for early B-cell development, we investigated the expression of signal transduction-related proteins in B-cell progenitors. Normal as well as leukemic B-cell progenitors were examined by immunoblotting and immunofluorescence study. In a survey of the expression of a broad range of signal transduction molecules, the Src-family protein tyrosine kinases were found to be differentially expressed in early B-cell differentiation. MATERIALS AND METHODS: Analysis of freshly prepared precursor-B acute lymphoblastic leukemia cells and B-lineage cell lines showed Hck and Lyn are major Src-family protein tyrosine kinases expressed in this type of leukemic blasts. RESULTS: However, heterogeneity of Hck and Lyn expression was found in these cells, and precursor-B acute lymphoblastic leukemia cells subsequently were classified according to the expression pattern of Hck and Lyn as Hck/Lyn dual-negative, Hck-predominant, Hck/Lyn dual-positive, and Lyn-predominant. Further studies on normal B-lineage cells indicated that the Src-family protein tyrosine kinases are expressed sequentially in a differentiation-dependent fashion during B-cell ontogeny and that the predominant expression of Hck is a common feature in B-cell progenitors, whereas Lyn expression is more significant in mature B cells. CONCLUSIONS: Although the biologic significance remains unknown, sequential expression of Src-family protein tyrosine kinases should play a role in regulation of early B-cell differentiation.     

Structural characterization of erythroid and megakaryocytic differentiation in Friend erythroleukemia cells

OBJECTIVE: The aim of this study was to examine the structural characterization of erythroid and megakaryocytic cell differentiation in Friend erythroleukemic cells using spectral imaging and electron microscopy. MATERIALS AND METHODS: Two variants of Friend erythroleukemia cells were treated with hexamethylene bisacetamide (HMBA) to induce differentiation: 1) MEL, which exhibit the normal phenotype and are susceptible to differentiation; and 2) the resistant R1 cells. The cells were analyzed by spectral imaging along with transmission and scanning electron microscopy. The expression of cell cycle regulatory proteins was analyzed by Western blotting. RESULTS: Spectral imaging of HMBA-treated MEL and R1 cells stained by May-Grünwald-Giemsa and subjected to spectral similarity mapping revealed five morphologic cell types: proerythroblast-like cells, normoblast-like cells, reticulocyte-like cells, megakaryocytes, and apoptotic cells. In MEL cells, both megakaryocytic differentiation characterized by nuclear lobes and erythroid differentiation characterized by accumulation of hemoglobin were detected; R1 cells were not committed to terminal differentiation. HMBA-induced cell cycle arrest at G(1) affected the expression of regulatory proteins in a similar manner in both types of cells. Expression of cyclin-dependent kinase 4 decreased and expression of p21(WAF1) increased. The level of the underphosphorylated form of phosphorylated retinoblastoma protein increased, inducing a decrease in the level of c-myc. In addition, we detected a decrease in the expression of the anti-apoptotic regulator, Bcl-2, and an increased expression of the pro-apoptotic regulator, Bax. CONCLUSIONS: Spectral imaging provides new insight for the morphologic characterization of erythroid and megakaryocytic cell differentiation as well as apoptosis. Image analysis was well correlated to cell cycle arrest and the expression of regulatory proteins.     

The proteomic study of sodium butyrate antiproliferative/cytodifferentiation effects on K562 cells

Employing methods of cell biology and proteome analysis tools, we examined effects of an inhibitor of histone deacetylases, sodium butyrate (SB), on the proliferation/differentiation characteristics of chronic myelogenous leukemia (CML)-derived cells K562. SB suppressed proliferation of K562 cells by inducing cell cycle arrest in G1 phase, which was followed by their transition to G0 phase (decrease of Ki-67 antigen-positive cells) and erythroid differentiation (increased glycophorin A expression and synthesis of hemoglobins). Neither terminal apoptosis (low counts of TUNEL-positive cells) nor necrosis (moderate counts of propidium iodide-positive cells) occurred. Importantly, SB attenuated protein expression of CML-related chimeric kinase BCR-ABL that is responsible for the deregulated proliferation of CML cells. The proteomic analysis (2-D electrophoresis combined with MALDI-TOF mass spectrometry and/or Western blotting) revealed several proteins that were differentially expressed or their mobility was altered due to butyrate treatment, namely, HSP90, HSP70, p23, cyclophilin A (CYPA), prefoldin2 (PFD2) and alpha-, gamma-, epsilon-human globin chains. Perturbation of HSP90 multichaperone complex of which BCR-ABL is the client protein is presumably a cause of BCR-ABL suppression. Changes in other proteins with chaperonic functions, CYPA and PFD2, may reflect SB antiproliferative and cytodifferentiation effects.     

Oxidative stress is involved in hydroxyurea-induced erythroid differentiation

Hydroxyurea (HU), an inhibitor of DNA synthesis, can also induce haemoglobinization in certain erythroid cell lines. In this study, we report that intracellular peroxides levels were increased in HU-treated murine erythroleukaemia (MEL) cells and that l-acetyl-N-cysteine (LNAC), a potent reducing reagent, had a significant inhibitory effect on the HU-mediated induction of beta-globin, delta-aminolaevulinate synthase mRNA expression and haemoglobinization of MEL cells. In contrast, the addition of LNAC to dimethyl sulphoxide (DMSO)-treated MEL cells had a much smaller effect on the number of haemoglobinized cells. These findings suggest that oxidative stress is involved in HU-mediated induction of erythroid differentiation and that HU induces MEL cell differentiation by a mechanism different to that involved in DMSO-mediated differentiation. Our findings also suggest that the induction of MEL cell differentiation by HU does not involve RAS-MAP (mitogen-activated protein) kinase signalling.     

A novel system to identify Myb target promoters in friend murine erythroleukemia cells

Friend murine erythroleukemia (MEL) cells provide an early erythroid precursor model that can be induced to terminally differentiate in cell culture and has been used to study erythroid differentiation as well as multistage tumorigenesis. During the chemically induced differentiation of MEL cells, expression of the c-myb protooncogene is downregulated in a biphasic fashion and forced expression of c-myb is able to block the differentiation process, suggesting that c-myb activity may be limiting for differentiation in MEL cells. We have recently produced stable transfectants in the C19 MEL cell line that carry a dominant interfering myb allele (MEnT) under the control of an inducible mouse metallothionein I (MTH) promoter. Upon inducing expression of MEnT, transfected cells enter a differentiation program and begin to produce alpha-globin mRNA, assemble hemoglobin, and stop proliferating. Differential display was used to compare mRNA expression between parental C19 MEL cells induced to differentiate with hexamethylene bisacetamide (HMBA) and stable transfectants induced to differentiate via expression of MEnT to identify potential Myb target promoters. We identified six candidate cDNAs in this fashion and present evidence that two of these represent genes that are dependent on c-Myb activity for maximal expression in MEL cells.     

Increase in γ-globin mRNA content in human erythroid cells treated with angelicin analogs

The aim of the present study was to identify molecular analogs of angelicin (ANG) able to increase erythroid differentiation of K562 cells and expression of γ-globin genes in human erythroid precursor cells, with low effects on apoptosis. ANG-like molecules are well-known photosensitizers largely used for their antiproliferative activity in the treatment of different skin diseases (i.e., psoriasis, vitiligo, eczema, and mycosis fungoides). To verify the activity of these derivatives, we employed three experimental cell systems: (1) the human leukemic K562 cell line, (2) K562 cell clones stably transfected with a pCCL construct carrying green-EGFP under the γ-globin gene promoter, and (3) the two-phase liquid culture of human erythroid progenitors isolated from normal donors and β-thalassemia patients. The results of our study suggest that trimethyl ANG is a powerful inducer of erythroid differentiation, compared with known inducers, such as ANG, cytosine arabinoside, mithramycin, and cisplatin. These data could have practical relevance, because pharmacologically mediated regulation of human γ-globin gene expression, with the consequent induction of fetal hemoglobin, is considered a potential therapeutic approach in hematological disorders including β-thalassemia and sickle cell anemia.     

Regulation of hemoglobin synthesis and proliferation of differentiating erythroid cells by heme-regulated eIF-2alpha kinase

Protein synthesis in reticulocytes depends on the availability of heme. In heme deficiency, inhibition of protein synthesis correlates with the activation of heme-regulated eIF-2alpha kinase (HRI), which blocks the initiation of protein synthesis by phosphorylating eIF-2alpha. HRI is a hemoprotein with 2 distinct heme-binding domains. Heme negatively regulates HRI activity by binding directly to HRI. To further study the physiological function of HRI, the wild-type (Wt) HRI and dominant-negative inactive mutants of HRI were expressed by retrovirus-mediated transfer in both non-erythroid NIH 3T3 and mouse erythroleukemic (MEL) cells. Expression of Wt HRI in 3T3 cells resulted in the inhibition of protein synthesis, a loss of proliferation, and eventually cell death. Expression of the inactive HRI mutants had no apparent effect on the growth characteristics or morphology of NIH 3T3 cells. In contrast, expression of 3 dominant-negative inactive mutants of HRI in MEL cells resulted in increased hemoglobin production and increased proliferative capacity of these cells upon dimethyl-sulfoxide induction of erythroid differentiation. These results directly demonstrate the importance of HRI in the regulation of protein synthesis in immature erythroid cells and suggest a role of HRI in the regulation of the numbers of matured erythroid cells.     

Accumulation of gamma-globin mRNA in human erythroid cells treated with angelicin

The aim of the present study was to determine whether angelicin is able to increase the expression of gamma-globin genes in human erythroid cells. Angelicin is structurally related to psoralens, a well-known chemical class of photosensitizers used for their antiproliferative activity in treatment of different skin diseases (i.e., psoriasis and vitiligo). To verify the activity of angelicin, we employed two experimental cell systems, the human leukemic K562 cell line and the two-phase liquid culture of human erythroid progenitors isolated from normal donors. The results of our investigation suggest that angelicin, compared with cytosine arabinoside, mithramycin and cisplatin, is a powerful inducer of erythroid differentiation and gamma-globin mRNA accumulation of human leukemia K562 cells. In addition, when normal human erythroid precursors were cultured in the presence of angelicin, increases of gamma-globin mRNA accumulation and fetal hemoglobin (HbF) production, even higher than those obtained using hydroxyurea, were detected. These results could have practical relevance, as pharmacologically-mediated regulation of the expression of human gamma-globin genes, leading to HbF induction, is considered a potential therapeutic approach in hematological disorders, including beta-thalassemia and sickle cell anemia.     

Induction of an embryonic globin gene promoter by short-chain fatty acids

Short-chain fatty acids (SCFAs) and dimethyl sulfoxide (DMSO) induce adult erythroid differentiation in murine erythroleukemia (MEL) cells, but only SCFAs concurrently up-regulate expression from the endogenous embryonic globin gene epsilony. The epsilony promoter, linked to a reporter gene and stably transfected into MEL cells, was tested during adult erythroid differentiation. Both the epsilony-CACCC site at -114 bp and enhancer sequences (hypersensitive site 2 [HS2]) from the beta-globin locus control region (LCR) were essential to maximal SCFA-mediated induction of expression from these constructs in MEL cells. Gel-shift analyses of binding activity from SCFA-induced MEL cell nuclear extracts showed in vitro binding by specificity proteins 1 and 3 (SP1, SP3) and basic or erythroid Krüppel-like factors (BKLF, EKLF) at the epsilony-CACCC site. In a functional analysis, transient cotransfections in nonerythroid NIH/3T3 cells of SP1, SP3, BKLF, or EKLF and HS2 epsilony promoter-luciferase constructs, with or without coactivators (p300, CREB-binding protein [CBP], or p300/CBP-associated factor [PCAF]) and SCFAs, were performed. SP1, SP3, and EKLF further increased expression from HS2 epsilony promoter constructs following exposure to SCFAs. This effect was variably augmented by coactivators and was diminished in EKLF mutants that were unable to undergo histone/factor-acetyl transferase (H/FAT)-mediated acetylation. In addition, acetylation of SP1 was detectable in NIH/3T3 cells following exposure to SCFAs. In sum, LCR sequence and an embryonic globin gene promoter CACCC site were essential to that promoter's up-regulation during SCFA-mediated induction of adult erythroid differentiation in vitro. Of factors that interact at the CACCC site, SCFA-mediated acetylation is implicated in SP1 and EKLF, and may be a mechanism through which SCFAs induce embryonic/fetal globin gene promoters during adult erythroid differentiation.     

Erythroid blast crisis in chronic myelogenous leukemia

Blood or bone marrow specimens from 22 patients with chronic myelogenous leukemia in blast crisis were studied for the surface expression of glycophorin-A, a marker for early erythroid differentiation. The leukemic blasts were stained with rabbit anti- glycophorin-A antiserum. The glycophorin-A molecules detected by the rabbit antiserum were identified by polyacrylamide slab gel electrophoresis of the immunoprecipitates from the membrane lysates of surface-labeled blasts. Blasts expressing surface glycophorin-A were found in 9 of the 22 patients. In 4 patients, almost all blasts were glycophorin-A positive, and in 5 patients, less than half of the blast population expressed glycophorin-A. The present study shows that when glycophorin-A is used as a marker for erythroid blasts, involvement of the erythroid lineage during blast crisis of chronic myelogenous leukemia seems to occur more frequently than previously recognized.     

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.