Correction of sickle cell disease in transgenic mouse models by gene therapy.

Sickle cell disease (SCD) is caused by a single point mutation in the human betaA globin gene that results in the formation of an abnormal hemoglobin [HbS (alpha2betaS2)]. We designed a betaA globin gene variant that prevents HbS polymerization and introduced it into a lentiviral vector we optimized for transfer to hematopoietic stem cells and gene expression in the adult red blood cell lineage. Long-term expression (up to 10 months) was achieved, without preselection, in all transplanted mice with erythroid-specific accumulation of the antisickling protein in up to 52% of total hemoglobin and 99% of circulating red blood cells. In two mouse SCD models, Berkeley and SAD, inhibition of red blood cell dehydration and sickling was achieved with correction of hematological parameters, splenomegaly, and prevention of the characteristic urine concentration defect.

Regulation of protein synthesis by heme-regulated eIF-2 alpha kinase.

Protein synthesis is regulated by the phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF-2 alpha) in a variety of cells. At present, there are two distinct mammalian eIF-2 alpha kinases that have been cloned, the double-stranded-RNA-dependent eIF-2 alpha kinase (PKR) and the heme-regulated eIF-2 alpha kinase (HRI). HRI is activated under conditions of heme deficiency in immature erythroid cells, and its activity is inhibited by heme. The high levels of HRI in reticulocytes indicate that its major physiological role is the regulation of protein synthesis, particularly of hemoglobin, according to the concentration of heme in these cells.

Biosynthesis of the transferrin receptor in rabbit reticulocytes.

These studies were performed to determine whether the reticulocyte can synthesize its own transferrin receptor and, if so, whether synthesis is subject to translational control by intracellular heme. Reticulocytosis (20-35%) was produced by bleeding rabbits and the washed cells were incubated for 1-4 h at 37 degrees C in buffered nutritional medium containing L-[35S]methionine. After washing and detergent lysis in the presence of protease inhibitors, supernatant reticulocyte extracts were analyzed for transferrin receptors by immunoprecipitation with specific ovine receptor antibody raised against denatured rabbit transferrin receptor. Immunoprecipitates were analyzed by SDS-gel electrophoresis and fluorography. Antibody, but not preimmune sheep immunoglobin, consistently precipitated a 35S-labeled protein with an Mr of 90,000 (reduced), coincident with bona fide receptor subunits purified by ligand-affinity chromatography. Incorporation of radioactive methionine was exclusively associated with receptor in reticulocyte stroma, and nascent receptor was not detected on free polyribosomes. Incorporation of radioactivity in the receptor moiety accounted for 0.1-0.2% of total incorporation into TCA insoluble cell protein. Treatment of the cells with 40 micrograms/ml cycloheximide markedly inhibited amino acid incorporation into the receptor, thus indicating de novo synthesis of receptor protein. On treatment of reticulocytes with 4,6 dioxoheptanoate to induce heme deficiency by diminishing the formation of intracellular heme, synthesis of the receptor was inhibited by greater than 50%; synthesis was restored to control rates on addition of 50 microM exogenous hemin. These findings indicate that the reticulocyte retains receptor mRNA and that synthesis of the receptor in erythroid cells is subject to translational regulation by intracellular heme.

Erythroid expression of the heme-regulated eIF-2 alpha kinase.

The role of heme-regulated eIF-2 alpha kinase (HRI) in the regulation of protein synthesis in rabbit reticulocytes is well documented. Inhibitors of protein synthesis with properties similar to those of HRI have been described in some nonerythroid cell types, but it has not yet been determined whether these eIF-2 alpha kinase activities are mediated by HRI or one or more as yet uncharacterized kinases. We have studied the expression of mRNA, polypeptide, and kinase activities of HRI in various tissues from both nonanemic and anemic rabbits. Our results indicate that HRI is expressed in an erythroid cell-specific manner. HRI is present in the bone marrow and peripheral blood of both nonanemic and anemic rabbits but not in any of the other tissues tested. HRI mRNA is present at low levels in uninduced mouse erythroleukemic (MEL) cells and human K562 cells and accumulates to higher levels upon induction. The accumulation of HRI mRNA in differentiating MEL cells is dependent upon the presence of heme. The addition of 3-amino-1,2,4-triazole (AT), an inhibitor of heme biosynthesis, to the induction medium markedly reduced HRI mRNA accumulation. Simultaneous addition of hemin and AT to the dimethyl sulfoxide induction medium largely prevented the inhibition of HRI mRNA induction by AT. These findings indicate that HRI is expressed in an erythroid cell-specific manner and that the major physiologic role of HRI is in adjusting the synthesis of globins to the availability of heme.

Upregulation of protein synthesis initiation factor eIF-4E is an early event during colon carcinogenesis.

A general increase in protein synthesis and a specific increase in the synthesis of growth-promoting proteins are necessary for mitogenesis. Regulation of protein synthesis, as well as preferential translation of some mRNAs coding for growth promoting proteins (e.g. cyclin D1), involves the essential protein synthesis initiation factor eIF-4E. This factor is induced by various oncoproteins, and, when overexpressed, it can transform cultured cells. In this report we explore the roles of eIF-4E in human neoplastic disorders of the colon and in the regulation of general and specific protein synthesis. We find that eIF-4E is increased in colon adenomas and carcinomas, and this increase is accompanied in most but not all cases by elevation of cyclin D1 levels. While general protein synthesis is increased by eIF-4E overexpression in cultured cells, only a small proportion of proteins is preferentially upregulated by eIF-4E, as revealed by two-dimensional gel electrophoresis. These results are consistent with the view that eIF-4E plays a role in carcinogenesis by increasing general protein synthesis and by preferentially upregulating a subset of putative growth promoting proteins. Our results, taken together with the recent findings that c-myc transcription is negatively regulated by APC and our earlier data on transcriptional activation of eIF-4E expression by c-Myc suggest that eIF-4E is a downstream target of the APC/beta-catenin/Tcf-4 pathway, and is strongly involved in colon tumorigenesis.

Transient inhibition of protein synthesis induces expression of proto-oncogenes and stimulates resting cells to enter the cell cycle.

There is evidence that resting cells are able to produce molecules with antiproliferative activity, some of which behave as short-lived repressor proteins. We suggest that transient inhibition of protein synthesis in resting cells would lead to a decrease in the levels of these negative growth regulators and might, therefore, promote mitogenic responses. We report that treatment of resting (serum-deprived) NIH 3T3 cells with cyclocheximide (CH) or puromycin induces expression of c-fos, c-jun and c-myc proto-oncogenes in a manner similar to that of platelet-derived growth factor (PDGF). Actinomycin D (Act D) abrogates the induction of proto-oncogene expression. Transient inhibition of protein synthesis by CH or puromycin also induces the resting NIH 3T3 and C3H 1OT1/2 cells to enter the cell cycle. Inhibition of new RNA or protein synthesis abolishes the proliferative response. These findings show that control mechanisms at both transcriptional and translational levels are operative in the resting cells treated with protein synthesis inhibitors. Cell fusion experiments with resting and serum-stimulated NIH 3T3 cells revealed that brief pre-incubation of resting cells with either PDGF, CH or puromycin abrogates their ability to suppress the onset of DNA synthesis in the nuclei of stimulated cells in heterodikaryons. However, the abrogative effect of PDGF disappeared in the presence of Act D, whereas the effects of protein synthesis inhibitors did not, indicating their independence of the induction of transcription. The data suggest that the observed effects of protein synthesis inhibitors are connected with elimination of some short-lived negative growth regulators, since a brief translational arrest is sufficient for the resumption of DNA synthesis in the nuclei of stimulated cells blocked by resting cells in heterodikaryons.

Correlation between the distribution of the reversing factor and eukaryotic initiation factor 2 in heme-deficient or double-stranded RNA-inhibited reticulocyte lysates.

The recycling of eukaryotic initiation factor eIF-2 requires the exchange of GDP for GTP, in a reaction catalyzed by the reversing factor (RF). Recent studies have suggested that a 60 S ribosomal subunit-bound eIF-2.GDP complex is an intermediate in protein chain initiation. We have monitored the distribution of RF in heme-deficient and dsRNA-inhibited lysates by immunoblot analysis of sucrose gradient fractions and have compared the distribution with that of eIF-2(alpha-32P). RF and eIF-2(alpha P) were both found to be tightly associated with 60 S and 80 S ribosomes, as their distribution did not change in gradients containing up to 0.1 M K+. The association of eIF-2(alpha-32P) and RF with 60 S and 80 S ribosomes was enhanced in the presence of F-, indicating the presence of an endogenous ribosome-associated phosphatase activity which is capable of dephosphorylating eIF-2(alpha P) in the absence of F-. These observations are consistent with the hypothesis that under physiologic conditions, RF interacts with the 60 S-bound eIF-2.GDP complex to promote the dissociation of GDP from eIF-2 and the release of eIF-2 from the 60 S subunit as a complex with RF.

Regulation of heme-regulated eIF-2 alpha kinase and its expression in erythroid cells.

In this article we focus first on the molecular mechanisms controlling the activity of the heme-regulated translational inhibitor, HRI, in erythroid cells. Then we discuss the tissue-specific expression of HRI. The experimental evidence obtained to date indicates that the major physiological role of HRI is in adjusting the synthesis of globin to the availability of heme.

The effects of hemin and double-stranded RNA on alpha and beta globin synthesis in reticulocyte and Krebs II ascites cell-free systems and the relationship of these effects to an initiation factor preparation.

Protein synthesis in reticulocyte lysates ceases abruptly in the absence of added hemin or in the presence of double-stranded RNA. A similar effect of double-stranded RNA is observed in Krebs II ascites cell-free systems translating exogenous globin mRNA. The shut-off of protein synthesis is due to inhibition of initiation and can be prevented or reversed by addition of the initiation factor preparation M(3). Preparations of M(1), M(2), and dissociation factor are ineffective under these conditions. The effects of added hemin, M(3), and globin mRNA on the synthesis of alpha and beta globin chains have been studied in the reticulocyte and ascites cell extracts. When the concentration of M(3) is rate limiting, the synthesis of beta chains exceeds that of alpha chains. When the concentration of mRNA is rate limiting, synthesis of alpha and beta chains is more nearly equal.

On the mechanism of delayed inhibition of protein synthesis in heme-defecient rabbit reticulocyte lysates.

In the absence of added hemin, protein synthesis in a rabbit reticulocyte lysate declines abruptly (shuts off) after about 5 min at 30 degrees. In these studies we have examined the basis for the lag period preceding shut-off. The initiation factor that binds Met-tRNAf, previously shown to be rate-limiting in inhibited, heme-deficient lysates, is found to be used stoichiometrically in the presence of excess inhibitor. We suggest that a principal effect of the inhibitor is to impair the recycling of the Met-tRNAf-binding factor; the lag period is attributable largely to the presence of a pool of excess Met-tRNAf-binding factor, which, once used in initiation, cannot be recycled because of the action of the inhibitor.

Regulation of protein synthesis in rabbit reticulocyte lysates: purification and initial characterization of the cyclic 3':5'-AMP independent protein kinase of the heme-regulated translational inhibitor.

The heme-regulated translational inhibitor (HRI) has been purified 4800-fold. On electrophoresis in sodium dodecyl sulfate/polyacrylamide gel, the purified HRI showed one major polypeptide band. The purified HRI inhibits protein synthesis in lysates containing optimal levels of hemin with inhibition kinetics which parallel those observed in heme-deficiency. Data are presented which are consistent with an enzymatic function of HRI in the inhibition of protein synthesis. The HRI is an adenosine 3':5'-cyclic monophosphate independent protein kinase which phosphorylates the small subunit (38,000) but not the large subunits (52,000 and 50,000) of the initiation factor which forms a ternary complex with Met-tRNAf and GTP. This evidence supports the hypothesis that inhibition of protein synthesis by HRI involves the phosphorylation of the initiation factor. These findings are discussed in relation to various models for the regulation of protein kinase activity by heme. (see article).

Regulation of protein synthesis: activation by double-stranded RNA of a protein kinase that phosphorylates eukaryotic initiation factor 2.

Incubation of reticulocyte lysates or isolated crude ribosomes with low levels of double-stranded RNA (0.1-10 ng/ml) induces the formation of an inhibitor of protein synthesis initiation similar to that observed in heme deficiency. The inhibitor is associated with a cyclic AMP-independent protein kinase activity (ATP:protein phosphotransferase, EC 2.7.1.37) that phosphorylates the small polypeptide (38,000 daltons) of the eukaryotic initiation factor eIF-2. Activation of the inhibitor requires ATP in addition to double-stranded RNA and is accompanied by the phosphorylation of a 67,000-dalton polypeptide of unknown function. The inhibitor remains associated with the ribosomes during high-speed sedimentation. Once formed, the ribosome-associated inhibitor phosphorylates eIF-2 and inhibits protein synthesis in the absence of double-stranded RNA. Inhibition is prevented by exogenous eIF-2. The bound inhibitor can be solubilized by extraction with 0.5 M KCl. The soluble inhibitor preparation retains the ability to phosphorylate the small polypeptide of eIF-2 and to inhibit protein synthesis. Untreated crude ribosomes also contain cyclic AMP-independent protein kinase activities that phosphorylate the middle polypeptide (49,000 daltons) of eIF-2 and several polypeptide subunits of eIF-3 (160,000, 125,000, and 65,000 daltons); these kinase activities are not affected by double-stranded RNA and do not inhibit protein synthesis.

Relationship between phosphorylation and activity of heme-regulated eukaryotic initiation factor 2 alpha kinase.

In heme-deficient reticulocytes and their lysates, a heme-regulated inhibitor of protein synthesis is activated; this inhibitor is a cyclic AMP-independent protein kinase that specifically phosphorylates the alpha subunit of the eukaryotic initiation factor 2 (eIF-2 alpha). Heme regulates this kinase by inhibiting its activation and activity. The purified heme-regulated kinase (HRI) undergoes autophosphorylation; at least 3 mol of phosphate can be incorporated per HRI subunit (Mr 80,000). The phosphorylation of HRI, its eIF-2 alpha kinase activity, and its ability to inhibit protein synthesis are diminished by hemin (5 microM) and increased by N-ethylmaleimide (MalNEt). Treatment of MalNEt-activated HRI with hemin reduces its autophosphorylation and its ability to inhibit protein synthesis . These findings demonstrate a correlation of the phosphorylation of HRI, its eIF-2 alpha kinase activity, and its inhibition of protein synthesis. The mechanism of hemin regulation of HRI activity was studied by examining the binding of hemin to purified HRI. Significant binding was demonstrable by difference spectroscopy which revealed a pronounced shift in the absorption spectrum of hemin with the appearance of a peak at 418 nm, a shift similar to that observed with proteins known to bind hemin. These findings are consistent with a direct effect of hemin on HRI.

Regulation of protein synthesis by phosphorylation of eukaryotic initiation factor 2 alpha in intact reticulocytes and reticulocyte lysates.

Studies in intact rabbit reticulocytes and reticulocyte lysates provide further evidence of a functional role for the phosphorylation of eukaryotic initiation factor 2 alpha (eIF-2 alpha) in the regulation of initiation of protein synthesis in eukaryotic cells. In intact reticulocytes treated with isonicotinic acid hydrazide to inhibit heme synthesis, the phosphorylation of eIF-2 alpha was significantly greater than in control cells. In heme-deficient reticulocyte lysates and in lysates treated with double-stranded RNA, significant phosphorylation of eIF-2 alpha occurred prior to the onset of inhibition of protein synthesis; a large proportion, however, of the total eIF-2 alpha remained unphosphorylated. These findings indicate that a modest concentration of phosphorylated eIF-2 alpha can suffice to inhibit initiation, and they suggest that one of the factors with which eIF-2 must interact may be rate limiting, especially when eIF-2 alpha is phosphorylated.

Hemin enhances the differentiation of mouse 3T3 cells to adipocytes.

Hemin enhances the adipose differentiation of mouse 3T3-F442A cells in a medium containing high levels of adipogenic factor that is present in fetal calf serum or calf serum. This enhancement is more prominent in the presence of insulin. Hemin also promotes adipose differentiation in a medium containing 10% cat serum, which has very little adipogenic factor; this effect is in contrast to that of insulin, which is relatively ineffective in promoting adipose differentiation in this medium. Adipose differentiation of 3T3-F442A cells is inhibited by the addition of aminotriazole, an inhibitor of heme biosynthesis. The inhibition of adipose differentiation by aminotriazole is prevented by the simultaneous addition of hemin and aminotriazole. These results indicate that treatment with hemin can promote the differentiation of fibroblasts to adipocytes in vitro, and they raise the possibility that endogenous heme may have a physiological role in the differentiation of fibroblasts to adipocytes in vivo.

Regulation of protein synthesis in rabbit reticulocyte lysates: additional initiation factor required for formation of ternary complex (eIF-2.GTP.Met-tRNAf) and demonstration of inhibitory effect of heme-regulated protein kinase.

Heme deficiency in rabbit reticulocytes and their lysates leads to the activation of a heme-regulated translational inhibitor (HRI) which causes the cessation of polypeptide initiation. HRI is a protein kinase that specifically phosphorylates the 38,000-dalton subunit of eukaryotic initiation factor 2 (eIF-2). eIF-2 binds Met-tRNA(f) and GTP in ternary complex. As a continuation of the studies on the molecular basis of the inhibition of the formation of 40S ribosomal subunit-Met-tRNA(f) complexes by HRI [Ranu, R. S., London, I. M., Das, A., Dasgupta, A., Majumdar, A., Ralston, R., Roy, R. & Gupta, N. K. (1978) Proc. Natl. Acad. Sci. USA 75, 745-749], we describe here the isolation and some characteristics of a factor that is required for the HRI-catalyzed inhibition of eIF-2-promoted ternary complex formation. In the presence of 1 mM Mg(2+), ternary complex formation by eIF-2 is dependent on the presence of this stabilization factor (SF). Under these conditions, SF increases the rate and the extent of ternary complex formation. This finding suggests that the interaction of SF with eIF-2 causes a conformational change that stabilizes eIF-2 and promotes efficient ternary complex formation by increasing the affinity of eIF-2 for GTP and Met-tRNA(f). In the absence of Mg(2+), however, eIF-2 efficiently forms the ternary complex and SF has little effect on its ternary complex formation capacity-hence, the name eIF-2 stabilization factor (SF). In the presence of SF, HRI markedly inhibits (70-80%) the ternary complex formation capacity of eIF-2. The inhibitory effect requires both HRI and ATP. Under these conditions, HRI phosphorylates only the 38,000-dalton subunit of eIF-2. Both the rate and the extent of the SF-dependent ternary complex formation are inhibited. These findings are consistent with the idea that phosphorylation causes a conformational change in eIF-2 such that its interactions with other initiation factors in the formation and the binding of ternary complex to 40S ribosomal subunits are inhibited.