Irving M. London

Eukaryotic Translation Initiation Factor 4E Regulates Expression of Cyclin D1 at Transcriptional and Post-transcriptional Levels

Regulation of the cell cycle is orchestrated by cyclins and cyclin-dependent kinases. We have demonstrated previously that overexpression of eukaryotic translation initiation factor 4E (eIF-4E) in NIH 3T3 cells growing in 10% fetal calf serum leads to highly elevated levels of cyclin D1 protein without significant increase in cyclin D1 mRNA levels, suggesting that a post-transcriptional mechanism is involved. (Rosenwald, I. B., Lazaris-Karatzas, A., Sonenberg, N., and Schmidt, E. V.(1993) Mol. Cell. Biol. 13, 7358-7363). In the present reseach, we did not find any significant effect of eIF-4E on polysomal distribution of cyclin D1 mRNA. However, the total amount of cyclin D1 mRNA associated with polysomes was significantly increased by eIF-4E overexpression. Further, we determined that the levels of both cyclin D1 protein and mRNA are increased in serum-deprived cells overexpressing eIF-4E. Nuclear run-on experiments demonstrated that the rate of the cyclin D1 transcription is not down-regulated in serum-deprived cells overexpressing eIF-4E. Thus, elevated levels of eIF-4E may lead to increased transcription of the cyclin D1 gene, and this effect becomes visible when serum deprivation down-regulates the rate of cyclin D1 mRNA synthesis in control cells. However, artificial overexpression of cyclin D1 mRNA in serum-deprived cells in the absence of eIF-4E overexpression did not cause the elevation of cyclin D1 protein, and this overexpressed cyclin D1 mRNA accumulated in the nucleus, suggesting that one post-transcriptional role of eIF-4E is to transport cyclin D1 mRNA from the nucleus to cytoplasmic polysomes.

Regulation of protein synthesis by heme-regulated eIF-2α kinase

Abstract Protein synthesis is regulated by the phosphorylation of the α-subunit of eukaryotic initiation factor 2 (eIF-2α) in a variety of cells. At present, there are two distinct mammalian eIF-2α kinases that have been cloned, the double-stranded-RNA-dependent eIF-2α kinase (PKR) and the heme-regulated eIF-2α 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.

Permanent and panerythroid correction of murine β thalassemia by multiple lentiviral integration in hematopoietic stem cells

Achieving long-term pancellular expression of a transferred gene at therapeutic level in a given hematopoietic lineage remains an important goal of gene therapy. Advances have recently been made in the genetic correction of the hemoglobinopathies by means of lentiviral vectors and large locus control region (LCR) derivatives. However, panerythroid β globin gene expression has not yet been achieved in β thalassemic mice because of incomplete transduction of the hematopoietic stem cell compartment and position effect variegation of proviruses integrated at a single copy per genome. Here, we report the permanent, panerythroid correction of severe β thalassemia in mice, resulting from a homozygous deletion of the β major globin gene, by transplantation of syngeneic bone marrow transduced with an HIV-1-derived [β globin gene/LCR] lentiviral vector also containing the Rev responsive element and the central polypurine tract/DNA flap. The viral titers produced were high enough to achieve transduction of virtually all of the hematopoietic stem cells in the graft with an average of three integrated proviral copies per genome in all transplanted mice; the transduction was sustained for >7 months in both primary and secondary transplants, at which time ≈95% of the red blood cells in all mice contained human β globin contributing to 32 ± 4% of all β-like globin chains. Hematological parameters approached complete phenotypic correction, as assessed by hemoglobin levels and reticulocyte and red blood cell counts. All circulating red blood cells became and remained normocytic and normochromic, and their density was normalized. Free α globin chains were completely cleared from red blood cell membranes, splenomegaly abated, and iron deposit was almost eliminated in liver sections. These findings indicate that virtually complete transduction of the hematopoietic stem cell compartment can be achieved by high-titer lentiviral vectors and that position effect variegation can be mitigated by multiple events of proviral integration to yield balanced, panerythroid expression. These results provide a solid foundation for the initiation of human clinical trials in β thalassemia patients.

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 up-regulated 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/β-catenin/Tcf-4 pathway, and is strongly involved in colon tumorigenesis.

Mutagenesis of retroviral vectors transducing human beta-globin gene and beta-globin locus control region derivatives results in stable transmission of an active transcriptional structure.

Retrovirus‐mediated gene transfer of the human beta‐globin gene into hematopoietic stem cells is an attractive approach to the therapy of human beta‐globin gene disorders. However, expression of the transduced beta‐globin gene linked to its proximal cis‐acting sequences (‐0.8 to +0.3 kb from the cap site) is considerably below the level required for a significant therapeutic effect. The discovery of the beta‐locus control region (beta‐LCR), organized in four major DNase I hypersensitive sites far upstream of the human beta‐like globin gene cluster, provided a potential means to achieve a high level of expression of a linked human beta‐globin gene, but initial attempts to incorporate beta‐LCR derivatives in retroviral vectors resulted in the production of low‐titer viruses with multiple rearrangements of the transmitted proviral structures. We now describe how extensive mutagenesis of the transduced beta‐globin gene, eliminating a 372 bp intronic segment and multiple reverse polyadenylation and splicing signals, increases viral titer significantly and restores stability of proviral transmission upon infection of cell lines and bone marrow‐repopulating cells. These optimized vectors have enabled us to analyze the expression properties of various retrovirally transduced beta‐LCR derivatives in dimethylsulfoxide‐induced murine erythroleukemia cells and to achieve ratios of human beta‐globin/murine beta maj‐globin mRNA, on a per gene basis, as high as 80%.

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.

12 Regulation of Protein Synthesis

Publisher Summary This chapter discusses the regulation of initiation by the phosphorylation of eIF-2 α and the effects of this phosphorylation on the exchange of guanosine triphosphate (GTP) for guanosine diphosphate (GDP) that is essential for the recycling of eIF-2. The chapter also discusses the activation, activity, and characteristics of the eIF-2a kinases and the distribution and function of these kinases in nonerythroid cells. The initiation of protein synthesis in reticulocytes and their lysates is inhibited by heme-deficiency, double-stranded RNA or oxidized glutathione. The dephosphorylation of eIF-2 α accompanies the recovery of protein synthesis upon the addition of hemin to inhibited heme-deficient lysates. The dephosphorylation of the RF eIF-2( α P) complex and the release of functional RF are responsible for the restoration of protein synthesis by GTP. The eIF-2 α kinases activated in the absence of heme (HRI) or by the addition of dsRNA (dsI) are different molecular species. The activation of HRI both in vitro and in the lysate is accompanied by the phosphorylation of HRI. In vitro , HRI is activated through an autokinase mechanism and is most active in the multiply phosphorylated state. The restoration of protein synthesis in lysates inhibited by heme deficiency requires glucose-6-P and hemin. In contrast to HRI, the dsRNA-activated eIF-2 α kinase (dsI) is associated with the ribosomal complement. The dsRNA-induced phosphorylation pattern of lysates is similar to that observed in dsRNA-treated extracts of interferon-treated cells.

Purification and characterization of a latent precursor of a double-stranded RNA dependent protein kinase from reticulocyte lysates

Abstract Double-stranded RNA (dsRNA) activates a cyclic 3′: 5′-AMP independent protein kinase (dsI) in reticulocyte lysates which inhibits protein synthesis by phosphorylating the 38, 000 dalton (38K) subunit of the initiation factor eIF-2 (eIF-2α). A latent precursor form of dsI (latent dsI) has been partially purified (1000–2000 fold) from lysates. Activation of dsI at all stages in the purification of latent dsI requires ATP and low levels of dsRNA (1–20 ng/ml), and is accompanied by the phosphorylation of a broad 67,000 dalton (67K) band. However, as purification proceeds the 67K band is resolved into two phosphorylated polypeptides of 68,500 and 67,000 daltons ( 68.5K 67K ). Although latent dsI and activated dsI have distinctly different chromatographic properties, both forms have similar molecular weights (∼120,000) and similar sedimentation coefficients (∼3.8S) in glycerol gradients. The data support the view that one or both components of the 68.5K 67K doublet are associated with the dsRNA-dependent protein kinase activity.

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(α‐32P). RF and eIF‐2(αP) 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(α‐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(αP) 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.

The storage of globin mRNA during the inhibition of protein synthesis by heme deprivation

The inhibition of protein synthesis in reticulocytes and their lysates caused by heme-deprivation is reversible on restoration of an optimal heme concentration. Inhibition is accompanied by the disaggregation of polyribosomes and the accumulation of components of the translational mechanism. By determining the fate of labeled globin 9S mRNA added to an unfractionated reticulocyte lysate cell-free system, we find that normal cellular mRNA accumulates during inhibition in 20S and 48S complexes and in a complex which sediments just ahead of the 80S ribosome dimer OD260 peak (designated as greater than or equal to 80S complex)1. The 20S and greater than 80S complexes are the major pools of stored mRNA which is readily translated if optimal heme conditions are restored. In the 48S complex, however, the mRNA remains non-functional, and the complex is abortive, probably as a result of deacylation of the Met.tRNAf.