Identification of the erythropoietin receptors of erythroleukemic cells

Erythropoietin (epo) is the physiologic regulator of red blood cell development. We have demonstrated the presence of epo receptors on two erythroleukemia cell lines, IW32 and IW201. IW32 produces epo constitutively while IW201 is a nonproducing cell line. Neither cell lines respond to epo to differentiate. In this study, we have shown that these cells bound 125I-epo specifically. Binding could be displaced by the presence of unlabeled epo but not by insulin or epidermal growth factor. In contrast to that found in epo responsive cells isolated from Friend virus infected mouse spleen, which were reported to contain both "high" and "low" affinity receptors, our finding indicated only a single class of "low" affinity receptors with apparent dissociation constants of 1.7 nM and 2.0 nM for IW32 and IW201 cell lines, respectively. It is suggested that the absence of "high" affinity receptors may account for the lack of responsiveness of these cells to epo.     

Receptor binding of asialoerythropoietin.

The interaction of 125I-asialoerythropoietin (asialoepo) with receptors has been characterized both by binding assay and affinity cross-linking. Purified spleen cells from mice infected with the anemia strain of Friend virus (FVA cells) have receptors for 125I-asialoepo with two classes of affinity constant: one with Kd = 0.02-0.03 nM and 300-400 per cell, the other with lower affinity (Kd = 0.9-1.2 nM) and 1,000-1,200 per cell. The Kd value for the high affinity site is one-third of that for the binding of native 125I-erythropoietin (125I-epo) to the same FVA cells (Kd = 0.08-0.1 nM). Using 125I-asialoepo or 125I-epo affinity cross-linking methods, we find two components with apparent molecular weights of 88 kDa and 105 kDa in FVA cells, and in the transformed mouse cell lines, 201, IW32, and NN10, in agreement with earlier studies using 125I-epo. These results indicate that 125I-asialoepo binds to the same receptors as 125I-epo, but with greater affinity for the high affinity site. Since 201 cells contain only a single class of lower affinity receptors for erythropoietin (epo), finding the same two components as found for FVA cells by cross-linking experiment indicates that the two components do not represent the two classes of receptor.     

A minimal ankyrin promoter linked to a human gamma-globin gene demonstrates erythroid specific copy number dependent expression with minimal position or enhancer dependence in transgenic mice

In red blood cells ankyrin (ANK-1) provides the primary linkage between the erythrocyte membrane skeleton and the plasma membrane. We have previously demonstrated that a 271-bp 5'-flanking region of the ANK-1 gene has promoter activity in erythroid, but not non-erythroid, cell lines. To determine whether the ankyrin promoter could direct erythroid-specific expression in vivo, we analyzed transgenic mice containing the ankyrin promoter fused to the human (A)gamma-globin gene. Sixteen of 17 lines expressed the transgene in erythroid cells indicating nearly position-independent expression. We also observed a significant correlation between the level of Ank/(A)gamma-globin mRNA and transgene copy number. The level of Ank/(A)gamma mRNA averaged 11% of mouse alpha-globin mRNA per gene copy at all developmental stages. The addition of the HS2 enhancer from the beta-globin locus control region to the Ank/(A)gamma-globin transgene resulted in Ank/(A)gamma-globin mRNA expression in embryonic and fetal erythroid cells in six of eight lines but resulted in absent or dramatically reduced levels of Ank/(A)gamma-globin mRNA in adult erythroid cells in eight of eight transgenic lines. These data indicate that the minimal ankyrin promoter contains all sequences necessary and sufficient for erythroid-specific, copy number-dependent, position-independent expression of the human (A)gamma-globin gene.     

High-level production of human alpha- and beta-globins in insect cells.

High-level production of human alpha- and beta-globins in cultured Spodoptera frugiperda (Sf-9) cells infected with recombinant baculoviruses is described. The expressed globins are produced to 70-140 mg protein/liter of cell culture or 5-10% of the total cellular protein. Two recombinant baculoviruses for alpha-globin, H alpha and H beta alpha, differ in their construction in that the 5'-untranslated region of the beta-globin gene is inserted 5' to the alpha-globin mRNA coding region in H beta alpha. This insertion results in a 40% increase in yield of alpha-globin over that of H alpha. Consistent with previous observations of the processing of recombinant proteins in Sf-9 cells, both alpha- and beta-globins expressed in Sf-9 cells are correctly processed to remove the initiating methionine from the amino termini of the globins. Sequencing of the expressed globins in Sf-9 cells confirms their identity with globins purified from human normal adult hemoglobin.     

羟基脲对人红白血病细胞株中球蛋白基因表达和细胞分化的影响

人红白血病细胞株（ＨＥＬ细胞）中珠蛋白基因表达具有自己的特点。即只表达胚胎型的γ－珠蛋白基因而不表达成人型的β－珠蛋白基因。羟基脲是一种抑制ＤＮＡ的小分子有机化合物。它在临床上被用来治疗地中海盆血和镰刀型贫血。我们的实验结果表明：胡羟基脲浓度增加ＨＥＬ细胞增殖速度减慢。用常规ＲＴ－ＰＣＲ方法和定量ＰＣＲ分析证明,用羟基脲诱导ＨＥＬ细胞后,β－珠蛋白基因表达增加,α－珠蛋白基因表达减少,而γ－珠蛋白     

Restriction endonuclease sensitivity of DNA containing globin genes in different murine erythroleukemia cell lines

The arrangement of the globin structural genes has been examined in murine erythroleukemia cells, strain DS19, and several related inducer-resistant variant cell lines. One fragment larger than 20 kilobases and six globin gene-containing fragments between 10 and 1.9 kilobases in size are detected in EcoRI-cleaved purified DNA prepared from strain DS19. By comparison, when isolated nuclei are digested with EcoRI, only two globin gene-containing fragments are detected, one greater than 20 kilobases and the other 1.9 kilobases. Of seven cell lines derived from DS19 and resistant to inducers, six had similar patterns to DS19 of globin gene-containing EcoRI-generated DNA fragments from nuclei and from purified DNA. One cell line, DR10, a DMSo-resistant cell line, lacks the 1.9 kilobase fragment after digestion of either nuclei or purified DNA. The 1.9 kilobase fragment hybridizes with alpha-globin cDNA but not with the beta-globin cDNA, suggesting either rearrangement or deletion of an alpha-globin gene-like fragment in DR10 DNA.     

A nucleolin-binding 3' untranslated region element stabilizes beta-globin mRNA in vivo

The normal expression of human beta globin is critically dependent upon the constitutively high stability of its encoding mRNA. Unlike with alpha-globin mRNA, the specific cis-acting determinants and trans-acting factors that participate in stabilizing beta-globin mRNA are poorly described. The current work uses a linker-scanning strategy to identify a previously unknown determinant of mRNA stability within the beta-globin 3' untranslated region (3'UTR). The new determinant is positioned on an mRNA half-stem opposite a pyrimidine-rich sequence targeted by alphaCP/hnRNP-E, a factor that plays a critical role in stabilizing human alpha-globin mRNA. Mutations within the new determinant destabilize beta-globin mRNA in intact cells while also ablating its 3'UTR-specific interaction with the polyfunctional RNA-binding factor nucleolin. We speculate that 3'UTR-bound nucleolin enhances mRNA stability by optimizing alphaCP access to its functional binding site. This model is favored by in vitro evidence that alphaCP binding is enhanced both by cis-acting stem-destabilizing mutations and by the trans-acting effects of supplemental nucleolin. These studies suggest a mechanism for beta-globin mRNA stability that is related to, but distinct from, the mechanism that stabilizes human alpha-globin mRNA.     

RNase H-mediated inhibition of translation by antisense oligodeoxyribonucleotides: use of backbone modification to improve specificity.

A sequence of the rabbit alpha-globin mRNA is the primary target for ODN1, an unmodified 15-nucleotide (nt) antisense oligodeoxyribonucleotide (oligo). ODN1 prevented in vitro translation of both alpha- and beta-globin mRNAs in wheat germ extract. Nine secondary sites exhibiting more than 60% complementarity with ODN1 were present in the beta-globin message. The ODN1 inhibition of beta-globin synthesis was shown to be mediated by RNase H cleavage of the beta-globin mRNA at three partially complementary sites. Sandwich-type oligos consisting of a stretch of unmodified nt with a few methylphosphonate residues at both 5' and 3' ends were derived from ODN1. We have demonstrated that one such analogue (ODN2), with five phosphodiester linkages in the central region, exhibited improved specificity for alpha-globin mRNA compared with the unmodified parent 15-mer, due to a reduced ability of RNase H to cleave beta-mRNA/ODN2 mismatched duplexes.     

Frequency of abnormal human haemoglobins caused by C----T transitions in CpG dinucleotides

A large part of human genetic disease apparently arises from deamination of cytosines in methylated CpG dinucleotides. Their mutation rate is known to be high when C is present as 5-methylcytosine, but is believed to be normal when it is unmethylated. The beta-globin gene contains five, the gamma-globin gene two, and each of the alpha-globin genes contain 35 CpGs. The CpGs in the beta- and gamma-globin genes are methylated, while those in the alpha-globin genes are undermethylated. One would therefore have expected the CpGs to be a frequent source of mutations in the beta- and gamma-globin genes, but not in the alpha-globin genes. In fact, the evidence points to CpGs being a frequent source of mutations in both the alpha- and beta-globin genes. This suggests either that the mutation rates of both methylated and unmethylated CpGs are abnormally high, which conflicts with published evidence, or that there is a finite chance of some CpGs in the alpha-globin genes of certain individuals being methylated and therefore subject to mutation.     

Frequency of abnormal human haemoglobins caused by C----T transitions in CpG dinucleotides

A large part of human genetic disease apparently arises from deamination of cytosines in methylated CpG dinucleotides. Their mutation rate is known to be high when C is present as 5-methyl-cytosine, but is believed to be normal when it is unmethylated. The beta-globin gene contains five, the gamma-globin gene two, and each of the alpha-globin genes contain 35 CpG's. The CpG's in the beta-and gamma-globin genes are methylated, while those in the alpha-globin genes are undermethylated. One would therefore have expected the CpG's to be a frequent source of mutations in the beta- and gamma-globin genes, but not in the alpha-globin genes. In fact, the evidence points to CpG's being a frequent source of mutations in both the alpha- and beta-globin genes. This suggests either that the mutation rates of both methylated and unmethylated CpG's are abnormally high, which conflicts with published evidence, or that there is a finite chance of some CpG's in the alpha-globin genes of certain individuals being methylated and therefore subject to mutation.     

CACCC and GATA-1 sequences make the constitutively expressed alpha-globin gene erythroid-responsive in mouse erythroleukemia cells.

Although the human alpha-globin and beta-globin genes are co-regulated in adult life, they achieve the same end by very different mechanisms. For example, a transfected beta-globin gene is expressed in an inducible manner in mouse erythroleukemia (MEL) cells while a transfected alpha-globin gene is constitutively expressed at a high level in induced and uninduced MEL cells. Interestingly, when the alpha-globin gene is transferred into MEL cells as part of human chromosome 16, it is appropriately expressed in an inducible manner. We explored the basis for the lack of erythroid-responsiveness of the proximal regulatory elements of the human alpha-globin gene. Since the alpha-globin gene is the only functional human globin gene that lacks CACCC and GATA-1 motifs, we asked whether their addition to the alpha-globin promoter would make the gene erythroid-responsive in MEL cells. The addition of each of these binding sites to the alpha-globin promoter separately did not result in inducibility in MEL cells. However, when both sites were added together, the alpha-globin gene became inducible in MEL cells. This suggests that erythroid non-responsiveness of the alpha-globin gene results from the lack of erythroid binding sites and is not necessarily a function of the constitutively active, GC rich promoter.     

Differences in human alpha-, beta- and delta-globin gene expression in monkey kidney cells

We have compared the function of the human alpha-, beta- and delta-globin genes using various plasmid expression vectors derived from pBR322. Amplification of recombinants occurred after their introduction, by calcium-phosphate-mediated DNA transfer, into monkey kidney cells that constitutively produce T antigen (COS cells). The human alpha-globin gene promoter functioned independently, but the beta-globin gene promoter was nearly totally dependent on the enhancing activity of the 72 bp direct repeats from the SV40 genome. Furthermore, when the human alpha- and beta-globin genes were linked in the same vector, the alpha promoter was active but the beta promoter was not. Function of the delta-globin gene promoter also depended on the enhancer element. In vectors containing the 72 bp repeats and the beta- or delta-globin gene, the activity of the beta-globin gene was approximately 50 times greater than that of the delta-globin gene, approximating the ratio of beta and delta mRNA observed in normal human bone marrow cells.     

Perforin mRNA in primary peritoneal exudate cytotoxic T lymphocytes

Considerable evidence indicates that cloned CTL cell lines kill target cells by releasing toxic granules that contain a cytolytic protein, called perforin, and several serine esterases (granzymes A to F). However, primary CTL, such as the highly cytolytic peritoneal exudate lymphocyte (PEL) cell population, have been found by a hemolytic assay to have no perforin, or perhaps only borderline levels of that protein, suggesting that these cells use a different lytic mechanism. To determine whether or not primary CTL express the perforin gene, we have here compared mRNA from PEL CTL and from a cloned CTL cell line, 2C, by Northern blot analysis using a perforin cDNA probe. CD8+ PEL CTL contain approximately 30% of the amount of perforin message present in 2C. Moreover, depletion of CD8+ T cells from the total peritoneal exudate cell population removes both cytolytic activity and perforin message. We have previously shown that PEL CTL elicit the same changes in target cells as cloned CTL cell lines and are resistant to lysis by the toxic granules purified from these cells lines. Taken together these results are consistent with the view that primary CTL, as well as long term cloned CTL cell lines, exercise their cytolytic activity by means of perforin.     

Altered translatability of messenger RNA from suspended anchorage-dependent fibroblasts: Reversal upon cell attachment to a surface

Anchorage-dependent cells, when forced into suspension culture, display a repertoire of dramatic, coordinated regulatory phenomena. Message production promptly decreases 5 fold but the cells maintain a constant amount of poly(A)⁺ by means of a concomitant stabilization of mRNA against decay. Protein synthesis shuts down much later and the mRNA is stored in a nonfunctioning state. In this study, the inactive mRNA is extracted from suspended cells and shown to have aberrant translation properties. Well defined polypeptides are apparently no longer synthesized when this mRNA directs protein formation in either reticulocyte or wheat germ-derived heterologous translation systems. Rather, shortened peptides are formed by this mRNA and these become smaller as mRNA is used from cells suspended for longer periods of time. Very few focused spots are formed when the aberrant polypeptides are analyzed in two-dimensional electrophoresis.The sedimentation properties of suspended cell mRNA and the size of poly(A) are unchanged from control monolayer cells. Cross-hybridization of cDNA transcribed from a control cell message population with suspended cell mRNA shows that all sequences are present in normal concentrations. While most identifiable spots disappear from the two-dimensional gel electropherograms of the protein products produced by suspended cell mRNA, a few polypeptides are still synthesized in relatively normal amounts. Conserved polypeptides are found in products of both the reticulocyte and wheat germ systems, but they are different products in each case. The lesion in the suspended cell mRNA does not seem to be at the 5′ termini, since synthesis of the shortened peptides is fully sensitive to inhibition by pm⁷G.Cells that contain extensively modified message can resume protein synthesis when allowed to reattach to a solid substrate. There is an apparent remodification of mRNA to normal translatability within a few hours of cell reattachment, since mRNA from recovering cells quickly resumes directing relatively normal patterns of polypeptide synthesis in vitro. The restoration of normal message function occurs even when new message formation is blocked with actinomycin.Cells recovering after reattachment synthesize supranormal amounts of a few major proteins involved with cell structure, as shown in these studies by an increased amount of translatable sequences which encode these proteins. The most apparent enhanced message is that coding for actin. mRNA from recovering cells produces in vitro several times more actin relative to other proteins than does control cell mRNA. The enhancement of actin mRNA is not seen in the message population of cells that reattach in the presence of actinomycin. The results suggest a morphologically related induction of gene expression.