N-myc translation is initiated via an internal ribosome entry segment that displays enhanced activity in neuronal cells.

Eukaryotic translation can be initiated either by a cap-dependent mechanism or by internal ribosome entry, a process by which ribosomes are directly recruited to structured regions of mRNA upstream of the initiation codon. We analysed the 5' untranslated region (UTR) of the proto-oncogene N-myc, and demonstrated by transfections in a dicistronic vector system that it contains a potent internal ribosome entry segment (IRES). The IRES is similar in length to the c-myc IRES and the activities of these IRESs are comparable in non-neuronal cells. Transfections were also carried out in cell lines derived from neuroblastomas, in which N-myc is expressed, and in a neuronal precursor cell line. In these cells the N-myc IRES is up to seven times more active than that of c-myc, suggesting that neuronal-specific non-canonical trans-acting factors are used by the N-myc but not the c-myc IRES. N-myc expression is increased by gene amplification in many neuroblastomas, but this is the first example of a translational mechanism by which N-myc expression could be further increased. The discovery of an IRES that displays enhanced activity in neuronal cell lines has important potential as a tool for protein expression in neural tissue.     

Translation of the human c-myc P0 tricistronic mRNA involves two independent internal ribosome entry sites.

The human c-myc proto-oncogene is transcribed from four alternative promoters (P0, P1, P2, and P3) giving rise to mRNAs having 5' leader sequences of various length. The c-myc P0 mRNA contains three open reading frames (ORFs), the last one encoding c-Myc1 and c-Myc2 proteins generated by alternative translation initiated at CUG and AUG codons. The middle ORF (MYCHEX1) and the 5' ORF (ORF1) code for proteins 188 and 114 amino acids in length, respectively. We and others previously identified an internal ribosome entry site (IRES) in P0 and P2 c-myc mRNAs, promoting the cap-independent translation of c-Myc1 and c-Myc2. Here, we report the presence of a second IRES (named IRES1) promoting the cap-independent translation of MYCHEX1 in c-myc P0 mRNA. Using deletion analysis, we mapped an 80-nt region essential for IRES1 activity. c-myc P0 mRNA is thus the first eukaryotic polycistronic mRNA described for which translation initiation of two different open reading frames (MYCHEX1 and c-Myc1/c-Myc2) involves internal ribosome entry.     

Initiation of Apaf-1 translation by internal ribosome entry

The apoptotic protease activating factor (Apaf-1) plays a central role in apoptosis: interaction of this protein with procaspase-9 leads to cleavage and activation of this initiator caspase. In common with other mRNAs whose protein products have a major regulatory function, the 5' untranslated region (UTR) of Apaf-1 is long, G-C rich and has the potential to form secondary structure. We have shown that the 5' UTR of Apaf-1 contains an internal ribosome entry segment, located in a 233 nucleotide region towards the 3' end of the leader, and that the translation initiation of this mRNA occurs only by internal ribosome entry. The Apaf-1 IRES is active in almost all human cell types tested, including Human cervical carcinoma (HeLa), Human liver carcinoma (HepG2), Human breast carcinoma (MCF7), Human embryonic kidney (HK293), African Green Monkey kidney (COS7) and Human lung (MRC5). The Apaf-1 IRES initiates translation as efficiently as the HRV IRES, but is less active than the c-myc IRES. We propose that the Apaf-1 IRES ensures that a constant cellular level of Apaf-1 protein is maintained even under conditions where cap-dependent translation is compromised. Oncogene (2000) 19, 899 - 905.     

Cellular internal ribosome entry segments: structures, trans-acting factors and regulation of gene expression

Initiation of translation in eukaryotic cells can occur by two distinct mechanisms, cap-dependent scanning and internal ribosome entry. The latter mechanism requires the formation of a complex RNA structural element termed an internal ribosome entry segment (IRES). IRESs are located in the 5' untranslated region of the message, and in the presence of trans-acting factors allow the ribosome to be recruited to a site that is a considerable distance from the cap structure. Many cellular mRNAs have now been shown to contain IRESs and it is likely that up to 10% of all mRNAs have the capability to initiate translation by this mechanism. The majority of IRESs that have been identified thus far are found in mRNAs whose protein products are associated with the control of cell growth and cell death, including many growth factors, proto-oncogenes and proteins required for apoptosis. In this review, we discuss the cellular situations when IRESs are required, the trans-acting factors that are necessary for IRES function and deregulation of IRES-mediated translation in tumorigenesis.     

Ras transformation of RIE-1 cells activates cap-independent translation of ornithine decarboxylase: regulation by the Raf/MEK/ERK and phosphatidylinositol 3-kinase pathways

Ornithine decarboxylase (ODC) is the first and generally rate-limiting enzyme in polyamine biosynthesis. Deregulation of ODC is critical for oncogenic growth, and ODC is a target of Ras. These experiments examine translational regulation of ODC in RIE-1 cells, comparing untransformed cells with those transformed by an activated Ras12V mutant. Analysis of the ODC 5' untranslated region (5'UTR) revealed four splice variants with the presence or absence of two intronic sequences. All four 5'UTR species were found in both cell lines; however, variants containing intronic sequences were more abundant in Ras-transformed cells. All splice variants support internal ribosome entry site (IRES)-mediated translation, and IRES activity is markedly elevated in cells transformed by Ras. Inhibition of Ras effector targets indicated that the ODC IRES element is regulated by the phosphorylation status of the translation factor eIF4E. Dephosphorylation of eIF4E by inhibition of mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK) or the eIF4E kinase Mnk1/2 increases ODC IRES activity in both cell lines. When both the Raf/MEK/ERK and phosphatidylinositol 3-kinase/mammalian target of rapamycin pathways are inhibited in normal cells, ODC IRES activity is very low and cells arrest in G(1). When these pathways are inhibited in Ras-transformed cells, cell cycle arrest does not occur and ODC IRES activity increases, helping to maintain high ODC activity.     

Analysis of the tumorigenic potential of common marmoset lymphoblastoid cells expressing a constitutively activated c-myc gene.

The respective roles of Epstein-Barr virus (EBV) and c-myc in the pathogenesis of endemic Burkitt''s lymphoma (BL) are unclear. In order to help resolve the question whether constitutive expression of the c-myc gene in an EBV-immortalised B cell is sufficient to induce a tumorigenic phenotype, B cells from a common marmoset (Callithrix jacchus) were immortalised with EBV, transfected with a constitutively activated c-myc gene and inoculated into the host animals. Despite the cell line transfected with c-myc displaying enhanced growth characteristics, in vitro and in vivo experiments demonstrated that this was not sufficient to induce a tumorigenic phenotype. This supports our previous findings with EBV-immortalised human B cells transfected with an activated c-myc gene (Hotchin et al., 1990).     

Translocations involving anaplastic lymphoma kinase (ALK)

Anaplastic large-cell lymphoma (ALCL) comprises a group of non-Hodgkin's lymphomas (NHLs) that were first described in 1985 by Stein and co-workers and are characterized by the expression of the CD30/Ki-1 antigen (Stein et al., 1985). Approximately half of these lymphomas are associated with a typical chromosomal translocation, t(2;5)(p23;q35). Much confusion about the exact classification and clinicopathological features of this subgroup of NHL was clarified with the identification of NPM-ALK (nucleophosmin-anaplastic lymphoma kinase) as the oncogene created by the t(2;5) (Morris et al., 1994). With the discovery of NPM-ALK as the specific lymphoma gene mutation, this NHL subtype could be redefined on the molecular level. This achievement was enhanced by the availability of specific antibodies that recognize ALK fusion proteins in paraffin-embedded lymphoma tissues. Several excellent recent reviews have summarized the histopathological and molecular findings of ALCL and their use in the classification of this lymphoma entity (Anagnostopoulos and Stein, 2000; Benharroch et al., 1998; Drexler et al., 2000; Foss et al., 2000; Gogusev and Nezelof, 1998; Kadin and Morris, 1998; Ladanyi, 1997; Morris et al., 2001; Shiota and Mori, 1996; Skinnider et al., 1999; Stein et al., 2000). This review will focus on the molecular function and signal transduction pathways activated by ALK fusion oncogenes, with recent advances and possible clinical implications to be discussed.     

Somatic rearrangement of the c-myc oncogene in primary human diffuse large-cell lymphoma

Chromosome translocations involving 8q24, the band to which c-myc has been mapped (Dalla-Favera et al., 1982), are a uniform finding in Burkitt's lymphoma (Bernheim et al., 1981). However, in only a minority of the tumors is the rearrangement of the c-myc locus sufficiently close to the gene to be detected with currently available probes (Dalla-Favera et al., 1983). Approximately 25% of diffuse large-cell lymphomas have also been reported to have translocations involving 8q24 (Mitelman, 1985), but there have been no reports of c-myc rearrangements in this form of non-Hodgkin's lymphoma. We have examined the structure of the c-myc locus in primary tumor tissue of 10 cases of diffuse large-cell lymphoma. In one patient, Southern blot analysis revealed additional c-myc fragments in the tumor DNA but not in the germ-line DNA. Southern blot analysis using probes from both the heavy- and light-chain immunoglobin loci showed that the myc rearrangement was unlikely to involve the immunoglobulin loci in this patient.     

A high-efficiency translational control element with potential for cancer gene therapy

An active internal ribosome entry sequence (IRES) that efficiently mediates cap (m7pppGN)-independent translation in human carcinoma cells could be an effective device for gene co-transduction in cancer gene therapy. In this study using the cytomegalovirus (CMV) promoter, a remarkable internal translation activity was observed and mediated by the sequence localized to the 183-653 region of 5' NF-kappaB repressor mRNA (NFR183IRES). To test the potential of such sequence for therapeutic application, we carried out in vitro functional assays using the dicistronic constructs that internally expressed human PTEN tumor suppressor. The PTEN expression mediated by NFR183IRES was found to result in growth inhibition of carcinoma cells more effectively than the expression by NFR1IRES that contained the 1-653 region. When compared to the internal translation driven by the picornaviral IRES element of the encephalomyocarditis virus (EMCV) or the foot-and-mouth disease virus (FMDV), NFR183IRES consistently exhibited a higher activity in the human carcinoma cells, HeLa, LNCaP and MCF7. Such high-efficiency translational control element may prove useful for cancer gene therapy.     

Small molecule inhibitors of IRES-mediated translation

Many genes controlling cell proliferation and survival (those most important to cancer biology) are now known to be regulated specifically at the translational (RNA to protein) level. The internal ribosome entry site (IRES) provides a mechanism by which the translational efficiency of an individual or group of mRNAs can be regulated independently of the global controls on general protein synthesis. IRES-mediated translation has been implicated as a significant contributor to the malignant phenotype and chemoresistance, however there has been no effective means by which to interfere with this specialized mode of protein synthesis. A cell-based empirical high-throughput screen was performed in attempt to identify compounds capable of selectively inhibiting translation mediated through the IGF1R IRES. Results obtained using the bicistronic reporter system demonstrate selective inhibition of second cistron translation (IRES-dependent). The lead compound and its structural analogs completely block de novo IGF1R protein synthesis in genetically-unmodified cells, confirming activity against the endogenous IRES. Spectrum of activity extends beyond IGF1R to include the c-myc IRES. The small molecule IRES inhibitor differentially modulates synthesis of the oncogenic (p64) and growth-inhibitory (p67) isoforms of Myc, suggesting that the IRES controls not only translational efficiency, but also choice of initiation codon. Sustained IRES inhibition has profound, detrimental effects on human tumor cells, inducing massive (>99%) cell death and complete loss of clonogenic survival in models of triple-negative breast cancer. The results begin to reveal new insights into the inherent complexity of gene-specific translational regulation, and the importance of IRES-mediated translation to tumor cell biology.     

Regulation of BAG-1 IRES-mediated translation following chemotoxic stress

There are three major isoforms of BAG-1 in mammalian cells, termed BAG-1L (p50), BAG-1M (p46) and BAG-1S (p36) that function as pro-survival proteins and are associated with tumorigenesis and chemoresistance. Initiation of BAG-1 protein synthesis can occur by both cap-dependent and cap-independent mechanisms and it has been shown that synthesis of BAG-1S is dependent upon the presence of an internal ribosome entry segment (IRES) in the 5'-UTR of BAG-1 mRNA. We have shown previously that BAG-1 IRES-meditated initiation of translation requires two trans-acting factors poly (rC) binding protein 1 (PCBP1) and polypyrimidine tract binding protein (PTB) for function. The former protein allows BAG-1 IRES RNA to attain a structure that permits binding of the ribosome, while the latter protein appears to be involved in ribosome recruitment. Here, we show that the BAG-1 IRES maintains synthesis of BAG-1 protein following exposure of cells to the chemotoxic drug vincristine but not to cisplatin and that this is brought about, in part, by the relocalization of PTB and PCBP1 from the nucleus to the cytoplasm.