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.     

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.     

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.     

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.     

Demonstration of Epstein-Barr virus in malignant non-Hodgkin's lymphomas

Lymph nodes or tumor biopsies of 60 persons suspected of having a malignant lymphoma were examined for the presence of Epstein-Barr virus nuclear antigen (EBNA) by anticomplement immunofluorescence. In 8 cases the tissue specimens were also assayed for Epstein-Barr virus (EBV) DNA by nucleic acid hybridization. Serum samples of patients and controls were tested for EBV-related antibodies. The histological tests in 37 cases showed a malignant non-Hodgkin lymphoma, and in 23 cases a reactive lymphadenopathy. A Burkitt lymphoma of a European boy and a polymorphic centroblastoma contained EBNA and approximately 27 or 30 genome equivalents EBV DNA per cell, respectively. EBNA was also demonstrated in about 20% of the cells of a lymph node from a patient with recurrent reactive lymphadenopathy.     

Suppression of EBNA1 expression inhibits growth of EBV-positive NK/T cell lymphoma cells

Extranodal nasal-type NK cell lymphoma (ENKL) is a high-grade malignancy and associated with EBV latent infection. An optimal therapy has not been discovery. Here, we investigated whether cell proliferation was inhibited by silencing Epstein-Barr virus nuclear antigen 1 (EBNA1) using RNA interference (RNAi) method in an EBV-positive ENKL cell line, HANK1 cells. We found that silencing EBNA1 expression by RNAi strategy inhibited cell growth and increased the expression of p27 protein and caused cell cycle arrest at G(1)-phase in HANK1 cells. In conclusions, low-level expression of p27 protein may partially attribute to latent EBV infection in ENKL. EBNA1 may be a good target for the treatment of EBV associated ENKL.     

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.     

Somatic cell hybrids between human lymphoma cell lines. V. IdUrd inducibility and P3HR-1 superinfectability of Daudi/HeLa (DAD) and Daudi/P3HR-1 (DIP-1) cell lines.

We have studied two types of somatic cell hybrid with regard to expression of the Epstein-Barr virus (EBV) cycle and its regulation. The first, DIP-1, a hybrid formed between two human lymphoma EBV producers (Daudi and P3HR-1), contained EBV DNA, expressed the virus-determined nuclear antigen (EBNA), andwas a producer of the EBV-associated antigens EA (early antigen) and VCA (viral capsid antigen). The second, DAD, a hybrid series of clones formed between Daudi and a HeLa cell derivative (D98), differed with regard to the expression of EBNA, EA, VCA and the content of EBV DNA. EA was regularly induced in the EBV DNA-containing hybrids following treatment with iododeoxyuridine (IdUrd). This induction was greater in lines spontaneously expressing EA. In two hybrids, DIP-1 and DAD10, VCA and virus DNA synthesis were also induced in the presence of IdUrd, the latter being detected by in situ hybridization with P3HR-1 EBV complementary RNA. Finally, while DIP-1 was superinfectable by the P3HR-1 EBV strain, the DAD series of hybrids were refractory to P3HR-1 superinfection and lacked EBV receptors.     

Production by EBV infection of an EBNA-positive subline from an EBNA-negative human lymphoma cell line without detectable EBV DNA.

A continuous lymphoma cell line, BJAB, derived from the tumour of an exceptional African case of Burkitt's lymphoma, has previously been described. Unlike 97% of African BL cases studied, neither the original tumour cells nor the cell line contained detectable amounts of EBV (Epstein-Barr virus) DNA, nor did they express the EBV-determined nuclear antigen EBNA. The cells of the established line had the characteristics of B-type lymphocytes and they carried receptors for EBV. EBNA was induced in the majority of BJAB cells after EBV infection. Usually the cells died within 10 days of infection, but it was possible to establish a permanent EBNA-positive variant (GC-BJAB) of BJAB. The patient from whose tumour the original BJAB line was established was seropositive for EBV antigens, indicating previous exposure to and continuing presence of the virus; yet the tumour had not become infected by EBV. This evidence shows that EBV is not readily "picked up" by the lymphoma.     

Expression of Epstein-Barr virus-encoded proteins in nasopharyngeal carcinoma

Expression of the Epstein-Barr virus (EBV) encoded nuclear antigens (EBNA 1 to 6) and membrane-associated protein (LMP) was investigated by immunoblotting in 83 nasopharyngeal carcinoma (NPC) biopsies and 25 other tumor and normal tissue specimens from the head and neck region. Fifty-eight of the 83 NPC biopsies were large enough to yield parallel data on virus DNA and viral expression. All 16 cases of clinically diagnosed and histologically confirmed NPCs from North Africa contained EBV DNA and expressed EBNA-1. Of 31 clinically diagnosed NPCs from China, 29 contained EBV DNA and 25 of these expressed EBNA-1. One control tissue biopsy from the oropharynx of NPC patients contained EBV DNA, but none expressed EBNA-1. The latent membrane protein (LMP) was detected in 22/31 of the Chinese and in 10/16 of the North African NPC biopsies. None of the NPC biopsies or control tissues expressed detectable amounts of EBNA 2 or any of the other 4 nuclear antigens which are invariably expressed in EBV-transformed B cells. A smaller number of tumors from Malaysia and East Africa exhibited a similar pattern of expression. EBV was rescued from a nude-mouse-passaged North African NPC tumor by co-cultivation of the tumor cells with umbilical cord blood lymphocytes. The tumor expressed EBNA 1 and LMP, but not EBNA 2 or the other 4 EBNAs. The resulting LCLs expressed all 6 nuclear antigens, EBNA 1 to 6 and LMP. Our data suggest that expression of the EBV genome is regulated in a tissue-specific fashion.     

Transient expression of the Epstein-Barr virus LMP1 gene in human primary B cells induces cellular activation and DNA synthesis.

The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) and Epstein-Barr virus nuclear antigen 2 (EBNA2) are expressed in EBV-immortalized human B cells. It has previously been shown that transfection of the LMP1 and EBNA2 genes into Burkitt's lymphoma cell lines results in the up-regulation of CD23, CD21, ICAM-1 and LFA-1 cell-surface proteins. In the present study, the effects of transient expression of the LMP1 and EBNA2 genes were studied in normal primary human B cells pretreated with UV-inactivated EBV particles. To identify and purify cells which express the transfected DNA we used a gene encoding a surface molecule, CD2, as a co-transfection marker. We show that transient expression of the LMP1 gene, from heterologous promoters, is sufficient to induce cellular enlargement and up-regulation of surface expression of the activation markers CD23, CD21, ICAM-1 and LFA-1 in primary B cells. Most importantly, we show that transient expression of the LMP1 gene is sufficient to induce DNA synthesis in human primary B cells. Transient EBNA2 expression enhanced the effect of transient LMP1 expression on CD21 and CD23 cell-surface expression but, under our experimental conditions, inhibited the induction of DNA synthesis by LMP1. We conclude that activation of primary B cells with inactivated EBV particles, followed by transient expression of only two viral genes, EBNA2 and LMP1, is sufficient to reconstitute some of the early events of B-cell immortalization by EBV.     

A mutation in the c-myc-IRES leads to enhanced internal ribosome entry in multiple myeloma: a novel mechanism of oncogene de-regulation

The 5' untranslated region of the proto-oncogene c-myc contains an internal ribosome entry segment (IRES) (Nanbru et al., 1997; Stoneley et al., 1998) and thus c-myc protein synthesis can be initiated by a cap-independent as well as a cap-dependent mechanism (Stoneley et al., 2000). In cell lines derived from patients with multiple myeloma (MM) there is aberrant translational regulation of c-myc and this correlates with a C-T mutation in the c-myc-IRES (Paulin et al., 1996). RNA derived from the mutant IRES displays enhanced binding of protein factors (Paulin et al., 1998). Here we show that the same mutation is present in 42% of bone marrow samples obtained from patients with MM, but was not present in any of 21 controls demonstrating a strong correlation between this mutation and the disease. In a tissue culture based assay, the mutant version of the c-myc-IRES was more active in all cell types tested, but showed the greatest activity in a cell line derived from a patient with MM. Our data demonstrate that a single mutation in the c-myc-IRES is sufficient to cause enhanced initiation of translation via internal ribosome entry and represents a novel mechanism of oncogenesis.     

Expression of the Epstein-Barr virus in lymphoproliferative diseases of the lung

There have been few studies regarding the etiology of lymphoproliferative disorders of the lung. To examine the possible involvement of the Epstein-Barr virus (EBV) in these diseases, EBV mRNAs, proteins and DNA, were detected. Two non-Hodgkin's lymphomas (NHL) originating in the lung, 5 mucosal-associated lymphoid tissue lymphomas (MALT lymphoma) of the lung, 1 lymphoid hyperplasia of the lung and 1 lymphoid interstitial pneumonia (LIP), were subjected to mRNA in situ hybridization, indirect immunofluorescence staining and PCR. mRNA in situ hybridization using BamHIW, BamHIY1Y2, the Epstein-Barr virus nuclear antigen (EBNA) and the EBV encoded small non-polyadenylated RNA (EBER1) probe revealed signals in all the examined samples, although some samples showed weak signals by using the EBER1 probe. Indirect immunofluorescence staining using the anti-leader protein, anti-EBNA2, the anti-latent member protein-1 and anti-BamHIZ coding leftward reading frame-1 antibodies showed definitive fluorescence. PCR also revealed EBV DNA in these specimens. EBV infected all the lymphoproliferative diseases of the lung irrespective of the histological or clinical stages. Furthermore, EBV infected not only the atypical lymphocytes but also the macrophages in the tissues of these diseases. These results suggest that the expression of EBV could be involved in the pathogenesis of many lymphoproliferative diseases of the lung.