Activity In K562 Cells Research Articles

Modulatory Subdomains of the HS2 Enhancer Differentially Regulate Enhancer Activity in Erythroid Cells at Different Developmental Stages

ABSTRACT: The HS2 enhancer in the locus control region of human β-like globin genes displays developmental-stage-independent enhancer function. The mechanism by which it regulates the transcription of the globin genes in erythroid cells throughout development is not fully understood. In this paper we dissect the HS2 enhancer into an enhancer core and five modulatory subdomains M1 to M5. The enhancer core possesses developmental-stage-independent enhancer activity. The modulatory subdomains by themselves do not possess such enhancer activity, but they apparently respond to environmental signals and modulate enhancer core activity in a developmental-stage specific manner. M1 located 5' of the core strongly stimulates core activity in K562 cells at the embryonic stage. M2 and M3 located 3' of the core strongly stimulate core activity in MEL cells at the adult stage. Moreover, M3 suppresses core activity at the embryonic stage and exhibits an adult-stage-selector activity. These findings indicate that the apparent developmental-stage-independence of the HS2 enhancer is a result of multiple interactions between the core and the modulatory subdomains located both near and far from the core.

Stimulation of Interleukin-1β-Converting Enzyme Activity during Growth Inhibition by CPT-11 in the Human Myeloid Leukemia Cell Line K562

Camptothecin (CPT) was first extracted fromCamptotheca acuminataand has a strong antitumor effect. Its water-soluble derivative, CPT-11, has higher therapeutic efficacy and less toxicity than CPT. Recently, CPT-treated cells have been shown to undergo apoptosis. However, the mechanism of induction of apoptosis by CPT has not been characterized in detail in any type of cells. On the other hand, interleukin-1β-converting enzyme (ICE) is a mammalian homologue of CED3, a protein required for apoptosis in the nematodeCaenorhabditis elegans.To determine how CPT-11 brings about cell death by apoptosis, we investigated the effects of CPT-11 on the expression of ICE activity in K562 cells, which represent human myeloid leukemia cells. The proliferation of K562 cells was shown to be inhibited by the presence of CPT-11 in the culture medium. We also found that the levels of mRNA for ICE in the cells were increased in the presence of CPT-11. Furthermore, we demonstrated that when CPT-11 was added to the culture medium, apoptosis of K562 cells was clearly detectedin situ.These features suggested that CPT-11 enhances the apoptotic cell death in K562 cells and that a part of induction of apoptosis by CPT-11 may be correlated with the stimulation of the ICE activity.

Identification of a Major Positive Regulatory Element Located 5' to the Human ζ-Globin Gene

The function of the zeta-globin promoter was studied using a series of zeta-globin promoter deletion constructs to drive luciferase expression in transiently transfected human erythroleukemia cells. The promoters were used without enhancers, or with enhancers derived from the beta-globin locus control region and the alpha-globin HS-40 enhancer. When transfected into K562 cells, which express zeta-globin, comparable amounts of activity were obtained from the -557 and -417 zeta-luciferase constructs and the alpha-luciferase constructs when no enhancers or the alpha-globin locus enhancers were used. When the constructs were transfected into OCIM1 cells, which do not express zeta-globin, the zeta-globin promoters were at best 20% as active as the alpha-globin promoters. When sequences from -417 to -207 5' to the zeta-globin mRNA cap site were deleted, up to 95% of the zeta-globin promoter activity was lost in K562 cells. Reinsertion of these sequences into zeta-luciferase constructs missing the -417 to -207 region showed that the sequences lack classical enhancer activity. Point mutation of a GATA-1 site at -230 reduced promoter activity by 37%. Point mutation of a CCACC site at -240 had no effect. Electrophoretic mobility shift assays indicated that the -230 GATA-1 site has a relatively low affinity for GATA-1. These experiments show the presence of a strong positive-acting element, located between -417 and -207 bp 5' to the zeta-globin mRNA cap site, is necessary for high-level promoter activity in K562 cells. This element requires GATA-1 and additional unknown factors for maximal activity.

Functional Roles of in Vivo Footprinted DNA Motifs within an α-Globin Enhancer: ERYTHROID LINEAGE AND DEVELOPMENTAL STAGE SPECIFICITIES

Transcriptional regulation of the human alpha-like globin genes, embryonic zeta 2 and adult alpha, during erythroid development is mediated by a distal enhancer, HS-40. Previous protein-DNA binding studies have shown that HS-40 consists of multiple nuclear factor binding motifs that are occupied in vivo in an erythroid lineage- and developmental stage-specific manner. We have systematically analyzed the functional roles of these factor binding motifs of HS-40 by site-directed mutagenesis and transient expression assay in erythroid cell cultures. Three of these HS-40 enhancer motifs, 5'NF-E2/AP1, GT II, and GATA-1(c), positively regulate the zeta 2-globin promoter activity in embryonic/fetal erythroid K562 cells and the adult alpha-globin promoter activity in adult erythroid MEL cells. On the other hand, the 3'NF-E2/AP1 motif is able to exert both positive and negative regulatory effects on the zeta 2-globin promoter activity in K562 cells, and this dual function appears to be modulated through differential binding of the ubiquitous AP1 factors and the erythroid-enriched NF-E2 factor. Mutation in the GATA-1(d) motif, which exhibits an adult erythroid-specific genomic footprint, decreases the HS-40 enhancer function in dimethyl sulfoxide-induced MEL cells but not in K562 cells. These studies have defined the regulatory roles of the different HS-40 motifs. The remarkable correlation between genomic footprinting data and the mutagenesis results also suggests that the erythroid lineage- and developmental stage-specific regulation of human alpha-like globin promoters is indeed modulated by stable binding of specific nuclear factors in vivo.

Organization of the Gene Encoding the Human Kell Blood Group Protein

Kell is one of the major blood group systems in human erythrocytes. It is a complex system containing a large number of different antigens. Previously we cloned the Kell cDNA, which was predicted to encode an integral membrane protein with 731 amino acids. Now we have isolated overlapping genomic clones and determined the exon-intron structure of the KEL gene; it spans approximately 21.5 kb with its coding sequence being organized in 19 exons that range in size from 63 bp to 288 bp. The size of introns ranges from 93 bp to approximately 6 kb. The donor and acceptor splice sites all conform to the consensus splicing sequences. Exon 1 encodes only the initiation amino acid, methionine, and contains a consensus Sp1 binding site. The single membrane spanning region of Kell protein is encoded in exon 3 and the putative zinc endopeptidase active site is in exon 16. The amino acids encoded by the 19 exons are identical to those of a person with a common Kell phenotype, as determined by RNA polymerase chain reaction of peripheral blood. Amplification of cDNA 5' ends, derived from human fetal liver, indicated three transcription initiation sites located 30, 81, and 120 bp upstream of the initiation codon. The 5' flanking region of KEL from -176 does not contain a TATA sequence, but has possible GATA-1 binding sites and has significant promoter activity when determined by chloramphenicol acetyltransferase activity in K562 cells.

Characterization of Gly cophorin A Transcripts: Control by the Common Erythroid-Specific Promoter and Alternative Usage of Different Polyadenylation Signals1

Glycophorins A (GPA) and B (GPB) are specifically expressed in human erythrocytes and express MN and Ss blood group antigens. While the GPB gene produces one transcript, the GPA gene produces three or four transcripts of different sizes. To understand the mechanisms of production of the different transcripts and erythroid-specific expression, we characterized the transcripts and the transcriptional regulatory regions of the glycophorin A gene. The transcriptional start site was determined by primer extension and S1 nuclease protection analysis, and it was found that all of the transcripts start at one major site. The nucleotide sequence of the newly isolated longest GPA cDNA revealed several polyadenylation signal sequences. To determine which polyadenylation signals are utilized, cDNA clones encoding GPA were isolated from a cDNA library of human erythroleukemia cell line K562 and the 3'-regions of the cDNAs were specifically amplified by the polymerase chain reaction. These results showed that five different polyadenylation signals of the GPA gene are utilized and the size and abundance of the transcripts are consistent with those detected by Northern blot analysis of K562 mRNAs. The 5'-flanking sequence was found to contain several binding motifs for the transcription factors, which were also found in other erythroid-specific genes. These motifs include the binding sites for the GATA-1 and NF-E2 erythroid-specific transcription factors, in addition to Sp1 transcription factor. The 5'-sequence from -750 to +36 relative to the transcriptional start site could confer only a low basal transcriptional activity in K562 cells and Friend erythroleukemia cells. This activity was significantly increased after Friend erythroleukemia cells were differentiated by dimethylsulfoxide. These results suggest that the 5' region regulates the glycophorin gene expression in erythroid-specific fashion.

Transcriptional regulation of alpha IIb integrin gene expression during megakaryocytic differentiation of K562 cells. Role of a silencer element

A portion of the 5'-flanking region of the glycoprotein IIb (alpha IIb) integrin gene extending from -598 to +32 base pairs was isolated. This DNA segment is capable of driving low level base-line transcription in undifferentiated K562 cells. It also contains elements which direct the markedly increased expression observed following megakaryocytic differentiation of K562 cells with phorbol dibutyrate. Analysis of hybrid alpha IIb-chloramphenicol acetyltransferase reporter gene constructs indicates that at least three regions within the -598 to +32 region control differentiation-dependent alpha IIb transcription. Two enhancer elements as well as a silencer domain all regulate chloramphenicol acetyltransferase transcriptional activity in K562 cells. Gel mobility shift experiments revealed that nuclear binding proteins are able to interact with all three DNA regions. A small region lying between -124 and -99 bases is able to bind to nuclear proteins in undifferentiated cells but not in differentiated cells as evidenced by gel mobility shift and foot-printing studies and corresponds to the silencer element identified in the functional studies. Therefore, the tissue-specific expression of alpha IIb may be controlled transcriptionally by both positive and negative factors with the silencer element playing a major role in regulating differentiation-dependent expression.

Tyrphostin-Induced Inhibition of p210bcr-abl Tyrosine Kinase Activity Induces K562 to Differentiate

AbstractWe report on the potency of two Tyrphostin tyrosine kinase blockers, AG 1112 and AG 568, to inhibit p210bcr-abl tyrosine kinase activity in K562 cells, concomitant with the induction of erythroid differentiation. AG 568 and especially AG 1112 represent a specific group of nontoxic protein tyrosine kinase blockers among more than 1,400 tested. These compounds possess therapeutic potential for purging Philadelphia chromosome-positive cells in preparation for autologous bone marrow transplantation in chronic myelogenous leukemia.

Tyrphostin-induced inhibition of p210bcr-abl tyrosine kinase activity induces K562 to differentiate

We report on the potency of two Tyrphostin tyrosine kinase blockers, AG 1112 and AG 568, to inhibit p210bcr-abl tyrosine kinase activity in K562 cells, concomitant with the induction of erythroid differentiation. AG 568 and especially AG 1112 represent a specific group of nontoxic protein tyrosine kinase blockers among more than 1,400 tested. These compounds possess therapeutic potential for purging Philadelphia chromosome-positive cells in preparation for autologous bone marrow transplantation in chronic myelogenous leukemia.

Transcriptional activation of human zeta 2 globin promoter by the alpha globin regulatory element (HS-40): functional role of specific nuclear factor-DNA complexes.

We studied the functional interaction between human embryonic zeta 2 globin promoter and the alpha globin regulatory element (HS-40) located 40 kb upstream of the zeta 2 globin gene. It was shown by transient expression assay that HS-40 behaved as an authentic enhancer for high-level zeta 2 globin promoter activity in K562 cells, an erythroid cell line of embryonic and/or fetal origin. Although sequences located between -559 and -88 of the zeta 2 globin gene were dispensable for its expression on enhancerless plasmids, they were required for the HS-40 enhancer-mediated activity of the zeta 2 globin promoter. Site-directed mutagenesis demonstrated that this HS-40 enhancer-zeta 2 globin promoter interaction is mediated by the two GATA-1 factor binding motifs located at -230 and -104, respectively. The functional domains of HS-40 were also mapped. Bal 31 deletion mapping data suggested that one GATA-1 motif, one GT motif, and two NF-E2/AP1 motifs together formed the functional core of HS-40 in the erythroid-specific activation of the zeta 2 globin promoter. Site-directed mutagenesis further demonstrated that the enhancer function of one of the two NF-E2/AP1 motifs of HS-40 is mediated through its binding to NF-E2 but not AP1 transcription factor. Finally, we did genomic footprinting of the HS-40 enhancer region in K562 cells, adult nucleated erythroblasts, and different nonerythroid cells. All sequence motifs within the functional core of HS-40, as mapped by transient expression analysis, appeared to bind a nuclear factor(s) in living K562 cells but not in nonerythroid cells. On the other hand, only one of the apparently nonfunctional sequence motifs was bound with factors in vivo. In comparison to K562, nucleated erythroblasts from adult human bone marrow exhibited a similar but nonidentical pattern of nuclear factor binding in vivo at the HS-40 region. These data suggest that transcriptional activation of human embryonic zeta 2 globin gene and the fetal/adult alpha globin genes is mediated by erythroid cell-specific and developmental stage-specific nuclear factor-DNA complexes which form at the enhancer (HS-40) and the globin promoters.

The function and distinctive regulation of the integrin VLA-3 in cell adhesion, spreading, and homotypic cell aggregation.

To assess directly the functional role of the integrin VLA-3 (alpha 3 beta 1), we transfected human alpha 3 cDNA into erythroleukemia (K562) cells and rhabdomyosarcoma (RD) cells. The resulting transfectants (KA3 and RA3) expressed alpha 3 beta 1 on the cell surface as confirmed using a panel of nine anti-alpha 3 monoclonal antibodies. Neither of the transfected cells exhibited increased adhesion to the extracellular matrix proteins fibronectin, laminin, and collagen. However, the KA3 transfectants did bind strongly to the extracellular matrix deposited by epidermal and carcinoma cell lines, allowing the cells to attach and spread. Binding to this cell-deposited ligand, probably containing epiligrin/kalinin, was specific to VLA-3 and could be inhibited by anti-alpha 3 antibodies and by EDTA, but not by RGD peptides. In marked contrast to other integrins (VLA-2 and VLA-4), VLA-3 showed high constitutive activity in K562 cells, but was minimally active in RD cells. Also contrasting with other beta 1 integrins, VLA-3 was minimally stimulated by the anti-beta 1 monoclonal antibody TS/216 under normal conditions. VLA-3-mediated adhesive function was well supported by either Mg2+ or Mn2+, but was almost completely abolished by the presence of 1 mM Ca2+. Surprisingly, this negative Ca2+ effect was completely overcome by the addition of the stimulatory anti-beta 1 monoclonal antibody TS2/16. Together, these results point to markedly distinct regulation for VLA-3 function compared to other beta 1 integrins. Also, all anti-VLA-3 antibodies were able to induce temperature-dependent homotypic cell aggregation of KA3 cells, but not K562 cells. However, this aggregation did not appear to be directly mediated by VLA-3 since it was not inhibited by EDTA. In addition, no enhancement of heterotypic cell-cell adhesion was observed in alpha 3-transfected cells.

Transcriptional Activation of Human ζ2 Globin Promoter by the α Globin Regulatory Element (HS-40): Functional Role of Specific Nuclear Factor-DNA Complexes

We studied the functional interaction between human embryonic zeta 2 globin promoter and the alpha globin regulatory element (HS-40) located 40 kb upstream of the zeta 2 globin gene. It was shown by transient expression assay that HS-40 behaved as an authentic enhancer for high-level zeta 2 globin promoter activity in K562 cells, an erythroid cell line of embryonic and/or fetal origin. Although sequences located between -559 and -88 of the zeta 2 globin gene were dispensable for its expression on enhancerless plasmids, they were required for the HS-40 enhancer-mediated activity of the zeta 2 globin promoter. Site-directed mutagenesis demonstrated that this HS-40 enhancer-zeta 2 globin promoter interaction is mediated by the two GATA-1 factor binding motifs located at -230 and -104, respectively. The functional domains of HS-40 were also mapped. Bal 31 deletion mapping data suggested that one GATA-1 motif, one GT motif, and two NF-E2/AP1 motifs together formed the functional core of HS-40 in the erythroid-specific activation of the zeta 2 globin promoter. Site-directed mutagenesis further demonstrated that the enhancer function of one of the two NF-E2/AP1 motifs of HS-40 is mediated through its binding to NF-E2 but not AP1 transcription factor. Finally, we did genomic footprinting of the HS-40 enhancer region in K562 cells, adult nucleated erythroblasts, and different nonerythroid cells. All sequence motifs within the functional core of HS-40, as mapped by transient expression analysis, appeared to bind a nuclear factor(s) in living K562 cells but not in nonerythroid cells. On the other hand, only one of the apparently nonfunctional sequence motifs was bound with factors in vivo. In comparison to K562, nucleated erythroblasts from adult human bone marrow exhibited a similar but nonidentical pattern of nuclear factor binding in vivo at the HS-40 region. These data suggest that transcriptional activation of human embryonic zeta 2 globin gene and the fetal/adult alpha globin genes is mediated by erythroid cell-specific and developmental stage-specific nuclear factor-DNA complexes which form at the enhancer (HS-40) and the globin promoters.

Structure and function of the murine beta-globin locus control region 5' HS-3.

We previously identified the murine homologue of the human beta-globin Locus Control Region (LCR) 5' HS-2. The lambda clone containing murine 5' HS-2 extends approximately 12 kb upstream from this site; here, we report the sequence of this entire upstream region. The murine homologue of 5' HS-3 is located approximately 16.0 kb upstream from the mouse epsilon y-globin gene, but no region homologous to human 5' HS-4 was present in our clone. Using a reporter system consisting of a human gamma-globin promoter driving the neomycin phosphotransferase gene (gamma-neo), we tested murine LCR fragments extending from -21 to -9 kb (with respect to the epsilon y-globin gene cap site) for activity in classical enhancer and integration site assays in K562 and MEL cells. 5' HS-2 behaved as a powerful enhancer and increased the number of productive integration events (as measured by a colony assay) in both K562 and MEL cells. 5' HS-3 had no activity in K562 cells or in transiently transfected MEL cells, but was nearly as active as 5' HS-2 in the MEL cell colony assay. Two additional tests confirmed the identification of murine 5' HS-3: first, a DNA fragment containing 5' HS-3 confers copy number-dependent, integration-site independent inducibility on a linked beta-globin gene in the MEL cell environment. Secondly, a strong DNAseI hypersensitive site maps to the location of the 5' HS-3 functional core in chromatin derived from MEL cells. Collectively, these data suggest that we have identified the murine homologue of human 5' HS-3, and that this site is functional when integrated into the chromatin of MEL cells but not K562 cells. 5' HS-3 may therefore contain information that contributes to the development-specific expression of the beta-like globin genes.

In vivo protein-DNA interactions at the beta-globin gene locus.

We have investigated in vivo protein-DNA interactions in the beta-globin gene locus by dimethyl sulfate (DMS) footprinting in K562 cells, which express epsilon- and gamma-globin but not beta-globin. In the locus control region, hypersensitive site 2 (HS-2) exhibited footprints in several putative protein binding motifs. HS-3 was not footprinted. The beta promoter was also not footprinted, while extensive footprints were observed in the promoter of the active gamma-globin gene. No footprints were seen in the A gamma and beta 3' enhancers. With several motifs, additional protein interactions and alterations in binding patterns occurred with hemin induction. In HeLa cells, some footprints were observed in some of the motifs in HS-2, compatible with the finding that HS-2 has some enhancer function in HeLa cells, albeit much weaker than its activity in K562 cells. No footprint was seen in B lymphocytes. In vivo footprinting is a useful method for studying relevant protein-DNA interactions in erythroid cells.

Cytotoxicity Evaluation of Mycotoxins by an MTT-Bioassay

A colorimetric tetrazolium (MTT) cleavage test was modified and established as a bioassay for the cytotoxicity of mycotoxins. Using the human erythroleukemia cell line K562 and porcine white blood cells (lymphocytes and granulocytes) we evaluated the influence of deoxynivalenol, ochratoxin A, and zearalenone on cellular MTT cleavage activity. The yellow MTT is reduced by mitochondrial enzymes of metabolically active cells into a dark blue formazan product, the optical density (OD) of which can be measured by an ELISA reader. After an exposure time of 24hours, concentrations of deoxynivalenol and ochratoxin A as low as 0.4 \gmg/mL were found to inhibit significantly the cleavage activity in K562 cells. Cytotoxicity in lymphocytes and granulocytes was observed at concentrations of 0.8 up to 0.4 \gmg/mL for deoxynivalenol and 3.1 and 0.8 \gmg/mL for ochratoxin A, respectively. Zearalenone concentrations of 25.0 to 12.5 \gm/mL inhibited the mitochondrial cleavage activity of lymphocytes and of K562 cells significantly, whereas in granulocytes none of the concentrations tested was proved to be toxic. Morphological findings on the ultrastractural level showed that toxin incubation (28 hours) resulted in massive cell damage. Similar alterations were observed in about 15% of control cells. This indicates, that the massive cytotoxic effect of the mycotoxin deoxynivalenol is more likely to be an unspecific than a specific one. The modified MTT cleavage assay was found to be a quick (28 hours) and efficient colorimetric test for examining the cytotoxicity of three mycotoxins. The simplicity and speed of the procedure, which allows the simultaneous testing of various parameters and the possibility of objective data analysis could establish this test as an additional bioassay for the evaluation of cytotoxicity of mycotoxins.

Hemin Control of Heme Biosynthesis and Catabolism in a Human Leukemia Cell Line

Hemin treatment of the Philadelphia chromosome positive leukemia cell line, K562, accentuates a number of erythroid phenotypic characteristics. The nature of this hemin effect was investigated by examining heme production and heme biosynthetic and catabolic enzyme activity in untreated and 0.05 mM hemin-treated cells. Activities of δ-aminolevulinic acid synthetase (ALAS), the rate limiting heme synthetic enzyme, and δ-aminolevulinic dehydratase (ALAD) were detectable in both uninduced and induced K562 cells. However, cells treated with hemin showed a significant increase in ALAS and ALAD activity by 2.5 days of treatment compared to untreated cells, and both enzyme activities further increased over the remainder of the incubation period. Incorporation of 3H-ALA into heme was also monitored. 3H-ALA was incorporated into heme in both untreated and treated cultures, but this incorporation was significantly greater in hemin-treated cells. Incorporation of 3H-ALA into heme indicates that cellular heme biosynthesis accounts for a portion of the heme utilized for hemoglobin production and that hemin stimulates heme synthesis by increasing enzyme activities distal to ALAS in the heme biosynthetic pathway. Heme oxygenase, the rate-limiting enzyme in mammalian heme degradation, was also studied and found to be present in large amounts in treated and untreated K562 cells. However, heme oxygenase activity was significantly decreased following hemin treatment. The demonstration of heme oxygenase activity in K562 cells documents the presence of this heme catabolic enzyme in human erythropoietic stem cells. The fall in heme oxygenase activity that accompanies hemin treatment might reflect an effort by erythroid cells to conserve heme during erythroid differentiation. These studies indicate the central role that heme synthesis and catabolism play during human erythroid differentiation.