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.     

N-myc modulates expression of p73 in neuroblastoma

The human p73 gene is a homolog of p53, which has been localized to chromosome 1p36 in a region that is frequently deleted in neuroblastoma. Transfection of the p73 gene into neuroblastoma cells that lack detectable p73 protein has been shown to result in growth suppression and to induce neuronal differentiation. In this study, we have identified by means of restriction landmark genome scanning (RLGS) a genomic fragment that was frequently reduced in intensity in neuroblastomas. The cloned fragment contained exon 1 of p73 as well as intronic and promoter sequences. We investigated the genomic and expression status of p73 and N-myc in 34 neuroblastoma tumors and 12 neuroblastoma cell lines. Approximately a third of neuroblastomas in our series exhibited deletion of p73. Most tumors analyzed exhibited reduced expression of p73, as determined by quantitative RT-PCR, in the absence of detectable p73 gene deletion. The reduced expression of p73 correlated with overexpression of N-myc in a statistically significant manner. The N-myc gene was transfected into two neuroblastoma cell lines that lacked N-myc amplification to determine its effect on p73 RNA levels. p73 was detectable at low level by RT-PCR in untransfected SK-N-AS cells and became undetectable following N-myc transfection, whereas in SH-EP1 cells, p73 levels were substantially reduced following transfection but remained detectable. Our data suggest that the N-myc gene modulates expression of p73, allowing neuroblastoma cells to escape the growth suppressing properties of p73.     

The N-myc and c-myc downstream pathways include the chromosome 17q genes nm23-H1 and nm23-H2

Gain of chromosome 17q material is the most frequent genetic abnormality in neuroblastomas. The common region of gain is at least 375 cR large, which has precluded the identification of genes with a role in neuroblastoma pathogenesis. Neuroblastoma also frequently show amplification of the N-myc oncogene, which correlates closely with 17q gain. Both events are strong predictors of unfavorable prognosis. To identify genes that are part of the N-myc downstream pathway, we constructed SAGE libraries of an N-myc transfected and a control cell line. This identified the chromosome 17q genes nm23-H1 and nm23-H2 as being 6-10 times induced in the N-myc expressing cells. Northern and Western blot analysis confirmed this up-regulation. Time-course experiment shows that both genes are induced within 4 h after N-myc is switched on. Furthermore, we demonstrate also that c-myc can up-regulate nm23-H1 and nm23-H2 expression. Neuroblastoma tumor and cell line panels reveal a striking correlation between N-myc amplification and mRNA and protein expression of both nm23 genes. We show that the nm23 genes are located at the edge of the common region of chromosome 17q gain previously described in neuroblastoma cell lines. Our findings suggest that nm23-H1 and nm23-H2 expression is increased by 17q gain in neuroblastoma and can be further up-regulated by myc overexpression. These observations suggest a major role for nm23-H1 and nm23-H2 in tumorigenesis of unfavorable neuroblastomas.     

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.     

Episome generated C-myc antisense RNA inhibits growth and tumorigenicity of a human neuroendocrine tumor-cell line

The neuroepithelioma cell line CHP100 expresses low but detectable amounts of N-myc protein together with large amounts of c-myc protein. We have recently demonstrated that antisense inhibition of N-myc expression in CHP100 cells leads to decreased in vitro growth and alterations in cellular morphology without affecting tumorigenicity in nude mice. In this study we report the construction of an episomally replicating vector designed to generate RNA antisense to part of the human c-myc gene. Such a Vector is able to inhibit c-myc expression in cell lines carrying multiple copies of the gene. Inhibition of c-myc expression leads to a decrease of in vitro growth and cloning efficiency and in vivo tumorigenicity of CHP100 cells. Our findings suggest that N-myc and c-myc subserve different functions in regulating the biology of CHP100 cells.     

Expression of myc-family genes in established human multiple myeloma cell lines: L-myc but not c-myc gene expression in the U-266 myeloma cell line.

Deregulated c-myc expression, as a consequence of translocation of the c-myc gene to one of the immunoglobulin loci, appears to play an important role in the pathogenesis of several B-cell tumors, including Burkitt's lymphoma, mouse plasmacytoma and rat immunocytoma. This study investigated the expression of c-myc and 2 other members of the myc gene family, L- and N-myc, at the mRNA and protein level, and analyzed for possible rearrangements of these genes in the human counterpart to the mouse plasmacytoma--multiple myeloma (MM). Nine well-characterized MM cell lines were examined by using Northern- and Southern-blot analysis and immunoprecipitation. The c-myc gene was found to be highly expressed in most MM cell lines. The level of expression was comparable to that observed in the COLO 320 and HL-60 cell lines, carrying amplified c-myc genes, and to that of B-cell lines with a higher proliferative activity than the MM cell lines. In the U-266 MM cell line, L-myc, but no c-myc mRNA or protein, was found. The L-myc gene was expressed in both early- and late-passage U-266 cells, suggesting that the L-myc expression was not the result of the in vitro cultivation. N-myc was not expressed in any of the MM cell lines. No rearrangements of c-myc or L-myc genes were found. We thus conclude that (a) in contrast to the corresponding mouse and rat B-cell tumors, c-myc is not frequently rearranged in MM; (b) c-myc is highly expressed in most MM lines; and (c) L-myc but not c-myc is expressed in the U-266 MM cell line.     

Expression of MRP and cMOAT in childhood neuroblastomas and malignant liver tumors and its relevance to clinical behavior.

Advanced neuroblastoma and malignant liver tumor are representative childhood cancers for which combined chemotherapy including cisplatin and doxorubicin is routinely performed. The prognosis of patients with tumors which develop multiple drug resistance (MDR) is unfavorable. To elucidate the role of multidrug resistance-associated protein (MRP) and canalicular multispecific organic anion transporter (cMOAT) in the clinical behavior of the tumors, we examined 42 neuroblastomas and 10 malignant liver tumors for the expressions of MRP and cMOAT by quantitative RNA-polymerase chain reaction (PCR). The amplification and expression of N-myc oncogene in the neuroblastomas were also investigated. We found a close association between MRP and N-myc expression in each neuroblastoma sample but no significant relationship between MRP expression and the patients' outcome. The forced expression of N-myc failed to enhance the expression of MRP in N-myc transfected neuroblastoma cell lines. cMOAT was rarely expressed in the neuroblastomas, but was frequently expressed in the malignant liver tumors. The expression of MRP and cMOAT in the childhood liver tumors was more common and higher, especially in advanced cases with a poor outcome, than that observed in normal liver or in 9 hepatocellular carcinomas from adult patients. The enhanced expression of these genes might be characteristic of childhood malignant liver tumors and related to their clinical chemoresistance.     

Efficient replication of plasmids containing the SV40 origin in N-myc overexpressing human neuroblastoma cells.

Using test plasmids containing the SV40 origin, we found a wide spectrum of permissiveness to their replication in different human cell lines. N-myc overexpressing neuroblastoma cells were highly permissive. LA-N-1 neuroblastoma cells were the most permissive of all the cell lines that we tested including the homologous CV-1 or COS-1 monkey kidney cells. Other human cell lines expressing various amounts of c-myc, and the 293 cell line expressing adenovirus E1A and E1B exhibited intermediate levels of permissiveness. T24 and EJ bladder carcinoma cells, which do not express the myc genes, were nonpermissive. Transient expression of c-myc or N-myc from plasmid vectors resulted in a modest stimulation of replication. Replication of test plasmids containing different configurations of the SV40 origin region was activated by the myc proteins. The high efficiency of replication in LA-N-1 cells is due to a combination of reasons including the overproduction of N-myc, high efficiency of expression of the SV40 replication initiator protein large T antigen from a cotransfected expression plasmid (containing the T antigen gene under the RSV LTR control), and other unknown host cell replication stimulatory factors. Replication of test plasmids was not detected in N-myc or c-myc overexpressing cells when the T antigen expression plasmid was not provided, showing that the myc proteins cannot substitute for T antigen in SV40 DNA replication.     

Expression of myc family oncoproteins in small-cell lung-cancer cell lines and xenografts

A number of genes have altered activity in small-cell lung cancer (SCLC), but especially genes of the myc family (c-myc, L-myc and N-myc) are expressed at high levels in SCLC. Most studies have explored expression at the mRNA level, whereas studies of myc family oncoprotein expression are sparse. We examined the expression of myc proto-oncogenes at the mRNA and protein level in 23 cell lines or xenografts. In the cell lines, the doubling time and the cell-cycle distribution, as determined by flow-cytometric DNA analysis, were examined to establish whether the level of myc-gene-family expression correlated with proliferative parameters. All tumours expressed at least one myc family member at the mRNA level. Exclusive c-myc mRNA expression was demonstrated in 8 tumours, L-myc in 7 and N-myc in I. Five tumours expressed both c-myc and L-myc, and 2 tumours expressed both c-myc and N-myc. In general, the level of expression of c-myc and N-myc was similar at the mRNA and the protein level. Expression of c-myc was positively correlated with the proliferative index (sum of S and G₂ + M phases) of cell lines, but not with the population doubling time. In general, L-myc-expressing cell lines had a low proliferative index. There was no systematic difference in myc expression between cell lines and xenografts of individual tumours.     

Expression of N-myc, c-myc, and MDR-1 proteins in newly established neuroblastoma cell lines: a study by immunofluorescence staining and flow cytometry.

A methodology for rapid isolation of neuroblastoma cells from marrow with metastatic neuroblastoma cells was developed using a cocktail of five antibodies and magnetic microspheres coated with secondary antibodies. Cells bound to microspheres were released by brief exposure to chymopapain, followed by repeated culture of released cells in serum-supplemented Dulbecco's modified Eagle's medium and selection for adherent cells. Using this methodology, over 35 primary cell lines were obtained free of contaminating normal cells. Detailed analyses of over 14 cell lines revealed gross differences in cell phenotype, size, morphology development of neurite processes, and doubling time (40 to 80 h). All cell lines expressed the M(r) 145,000 neurofilament, and a few expressed the M(r) 200,000 neurofilament, with very little or no expression of the M(r) 68,000 neurofilament. Eight % of all cells lines had near-diploid DNA content. High expression of the MDR-1 protein was detected in six of the 22 cell lines tested. Great heterogeneity was observed in the expression of N-myc oncoprotein, with ten of 13 patients overexpressing the protein. c-myc oncoprotein was also expressed in all cell lines; however, the level of expression was 4- to 10-fold lower than the N-myc oncoprotein. Localization studies of c-myc and N-myc oncoproteins on the level of light microscopy and electron microscopy revealed exclusive nuclear localization of c-myc, whereas N-myc was localized to the nucleus and to the cytoplasm.     

Beta 2-microglobulin expression in human embryonal neuroblastoma reflects its developmental regulation

Class I major histocompatibility complex (MHC) antigen expression in neuroblastoma may play a role in the oncogenicity of this embryonal tumor of childhood. Since N-myc amplification in neuroblastoma tumors is associated with rapid tumor progression (33) and N-myc decreases Class I MHC antigen expression in rat neuroblastoma cells (21), we quantitated levels of N-myc mRNA and Class I MHC cell surface antigens in a panel of 24 human neuroblastoma cell lines. We found that N-myc expression is not invariably associated with low levels of beta 2-microglobulin (B2M) and Class I MHC antigen expression. As we considered that Class I MHC antigens may be regulated in association with the differentiation stage of the neuroblastoma tumor, we examined the expression of B2M during development of the human adrenal medulla, the tissue of origin of most neuroblastomas. We found that B2M is a marker of differentiated adrenal medullary cells, expressed late during the third trimester of development. Moreover, using morphological and immunological criteria, we found that B2M is expressed in differentiated tumor cells. These data suggest that the expression of B2M in neuroblastoma is associated with the stage of differentiation of the tumor cell and not N-myc expression. Furthermore, these findings suggest that neuroblastomas may correspond to the arrested differentiation of adrenal neuroblasts at different stages of development.     

Direct recruitment of N-myc to target gene promoters

The N-myc gene is amplified in 20-25% of human neuroblastomas, and this amplification serves as a poor prognostic factor. However, few genes have been determined to be direct targets of N-myc. Our current studies focused on identifying N-myc target genes, especially those affected in cells such as neuroblastomas that have high levels of N-myc protein. To pursue this goal, we performed differential expression screens with cell-culture systems containing high versus low levels of N-myc. The design of our experiments was such that we should identify genes both upregulated and downregulated by N-myc. Accordingly, we identified 22 genes upregulated by N-myc and one gene downregulated by N-myc. However, only five of these genes responded to increased N-myc levels in more than one system. Further analysis of the regulation of these genes required determining whether they were direct or indirect targets of N-myc. Therefore, we used a formaldehyde crosslinking and immunoprecipitation procedure to determine whether N-myc was bound to the promoters of these putative target genes in living cells. We found that low levels of N-myc were bound to the promoters of the telomerase and prothymosin genes in neuroblastoma cells having low amounts of N-myc but that the amounts of N-myc bound to these promoters greatly increased with overexpression of N-myc. However, the amount of max bound to the promoters was high before and after induction of N-myc. Therefore, our studies suggest that N-myc competes with other max partners for binding to target promoters. Our use of the chromatin immunoprecipitation assay suggests a molecular explanation for the consequences of amplification of the N-myc gene in neuroblastomas.     

Amplification of the c-myc oncogene in a subpopulation of human small cell lung cancer

We have examined a panel of human lung cancer cell lines for amplification and expression of the c-myc, N-myc, and c-myb oncogenes. The cell lines analyzed represent various histopathological types of lung cancer: small cell carcinoma with neuroendocrine properties; squamous cell carcinoma with epithelial markers; and large cell carcinoma with a mixed neuroendocrine-epithelial phenotype. Two of six cell lines, both of which were small cell carcinomas, showed about a 20-fold amplification of the c-myc oncogene. In both cell lines, the amplification is accompanied by an enhanced expression of c-myc. The N-myc or c-myb genes were not amplified in any of the cell lines, nor were they expressed in detectable amounts. The results confirm and extend earlier findings on c-myc amplification in small cell lung cancer.