Antisense inhibition of single copy N-myc expression results in decreased cell growth without reduction of c-myc protein in a neuroepithelioma cell line

The N-myc gene is transiently expressed during normal embryonic development and abnormally expressed in several tumors of neuroendocrine origin. Little is known of the function of the N-myc gene product in either normal or neoplastic tissue. We utilized synthetic antisense oligodeoxynucleotides to specifically inhibit N-myc gene expression in the neuroepithelioma cell line CHP100. These cells contain single copy N-myc alleles but overexpress c-myc. N-myc antisense oligomer treatment was found to be growth inhibitory without affecting levels of c-myc protein. N-myc antisense oligomer-treated cells also lost the characteristic cellular heterogeneity displayed by CHP100 in vitro.     

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.     

Modification of myc gene amplification in human somatic cell hybrids

In order to study whether cell fusion would modify the DNA copy number of an amplified oncogene, somatic cell hybrids were made between the human neuroepithelioma cell line MCIXC and HeLaCOT human adenocarcinoma cells. MCIXC contains approximately 21 copies of the c-myc oncogene and HeLaCOT contains approximately 5 copies relative to the control. All hybrid clones investigated displayed a marked decrease in the number of copies of c-myc DNA (an average of 5 copies), while the level of c-myc RNA in the hybrids was similar to that found in both parents. All eight hybrid clones were found to be completely nontumorigenic even though both parent cells formed tumors in 100% of the nude mice treated by injection. This loss of oncogene amplification in the hybrids was shown not to be due to either heterogeneity of c-myc amplification in the MCIXC parent or segregation of a copy of the chromosome 22 from the hybrids. This loss most likely resulted from the breakdown of a homogeneously staining region (containing the amplified gene copies) into double minutes, which were subsequently lost from the cells. The HeLaCOT cell line was also fused to the human neuroblastoma BE(2)C, which contains approximately 123 copies of the N-myc oncogene relative to control. Ten hybrid clones were found to contain an average of 47 copies of N-myc DNA, significantly less than the 91 copies predicted had no loss occurred. These BE(2)C x HeLaCOT hybrids expressed on average about 15% the N-myc RNA seen in the BE(2)C parent and, as with the MCIXC x HeLaCOT hybrids, were found to be completely nontumorigenic. However, upon passage in culture, one BE(2)C x HeLaCOT hybrid eventually became tumorigenic. This hybrid also displayed reduced copies of N-myc DNA in comparison to its parent hybrid but surprisingly showed a 2-fold increase in N-myc RNA. Thus, the expression of N-myc, but not the amplification state of either myc gene, appears to correlate with the tumorigenicity of the 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.     

Clonal cosegregation of tumorigenicity with overexpression of c-myc and transforming growth factor alpha genes in chemically transformed rat liver epithelial cells.

Tumorigenicity was correlated with levels of expression of the genes for transforming growth factor alpha (TGF-alpha), epidermal growth factor receptor, c-myc, c-H-ras, and c-K-ras in a series of 16 clonally derived transformed liver epithelial cell lines. The clonal lines, which varied in tumorigenicity from 0 to 97%, were established from a phenotypically heterogeneous population produced by repeated exposure of diploid WB-F344 (WB) cells to N-methyl-N'-nitro-N-nitrosoguanidine. Segregation of gene expression with tumorigenicity among clonal lines was determined by correlating rank orders of gene expression by clones relative to expression by wild-type WB cells. Only the expression of the c-myc gene correlated with tumorigenicity among all transformed clones. TGF-alpha gene expression was not correlated with tumorigenicity among all clones, but it was highly correlated with tumorigenicity among clones that expressed the c-myc gene above the median level for all clones (greater than 5-fold the level of expression by WB cells). Even high levels of expression of the TGF-alpha gene (up to 60-fold the level of expression by WB cells) were not correlated with tumorigenicity among the clones expressing the c-myc gene at levels less than 5-fold the level of expression by WB cells. Clones which simultaneously overexpressed both c-myc and TGF-alpha genes at levels above the median levels for all clones were significantly more tumorigenic than were clones which expressed either or both genes at lower than median levels. These results suggest that overexpressed c-myc and TGF-alpha genes cooperate in their association with tumorigenicity. Most of the highly tumorigenic clones that overexpressed c-myc and TGF-alpha also overexpressed the c-H-ras and/or the c-K-ras genes; clones that overexpressed neither of the c-ras genes nor the genes for c-myc and TGF-alpha were not very tumorigenic, while clones that expressed one or both c-ras genes (but not both c-myc and TGF-alpha) were variably tumorigenic over an intermediate range.     

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.     

Complementation of Myc-dependent cell proliferation by cDNA expression library screening

The targeted knockout of the c-myc gene from rat fibroblasts leads to a stable defect in cell proliferation. We used complex cDNA libraries expressed from retroviral vectors and an efficient sorting procedure to rapidly select for cDNAs that can restore the growth rate of c-myc deficient cells. All of the biologically active cDNAs contained either c-myc or N-myc, suggesting that no other cellular genes can effectively bypass the requirement for c-myc in fibroblast proliferation. This approach provides a powerful screening method for cell cycle changes in genetically defined systems.     

EXPRESSION OF c－myc GENE AND BIOSYNTHESIS OF BIOLOGICAL MACROMOLECULES IN ANTISENSE TRANSFECTANT HL_（60）~R－9

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Myc protein is differentially sensitive to quinidine in tumor versus immortalized breast epithelial cell lines

Quinidine regulates growth and differentiation in human breast tumor cells, but the immortalized mammary epithelial MCF-10A cell line is insensitive to quinidine. We found that a morphologically similar differentiation response was evoked by quinidine and c-myc antisense oligonucleotides in MCF-7 cells and this prompted us to investigate the actions of quinidine on c-myc gene expression. Myc protein levels were suppressed in human breast tumor cell lines, but not in MCF-10A cells, an observation that supports the hypothesis that suppression of c-myc gene expression is involved in the preferential growth and differentiation response of breast tumor cells to quinidine. Quinidine reduced c-myc mRNA levels in MCF-7 cells. Acute induction of c-myc mRNA by estradiol, as well as the c-myc response to sub-cultivation in fresh serum and H-ras driven elevations in c-myc mRNA were depressed by 50-60% in the presence of quinidine. Quinidine decreased c-myc promoter activity in MCF-7 cells in a transient reporter gene assay and a 168 bp region of human c-myc promoter (-100 to +68 with respect to the P1 promoter) was sufficient to confer responsiveness to quinidine. Quinidine is a potential lead compound for developing pharmacological agents to regulate Myc. In addition, the study of quinidine-regulated events is a promising approach to unravel differentiation control pathways that become disrupted in breast cancer.     

Increased expression of the proto-oncogene, c-myc, in human neuroblastoma cells by reversible inhibition of cell growth.

Expression of the proto-oncogenes N-myc and c-myc in the human neuroblastoma cell line, IMR32, was examined after reversible inhibition of DNA synthesis by treatment with deferoxamine. After 48 h treatment with 10(-5) M deferoxamine, the number of c-myc expressing cells doubled, while N-myc expressing cells did not change. The expression level of c-myc in each cell also increased. These results suggested that the synthesis of cellular factor(s) involved in the degradation of c-myc RNA or its product was suppressed by reversible inhibition of cell growth. The regulatory mechanisms of expression of N-myc and c-myc may be distinct.     

Growth inhibition of murine neuroblastoma cells by c-myc with cell cycle arrest in G2/M

c-myc and N-myc belong to the myc family of proteins that plays an important role in cell proliferation, differentiation and apoptosis. The N-myc gene is amplified in aggressive neuroblastoma and c-myc is overexpressed in many lymphomas and cancers. However, c-myc has not been implied in tumorigenesis or progression of neuroblastoma. We therefore investigated the so far unknown effects of c-myc overexpression on the aggressiveness of neuroblastoma cells with single copy N-myc. c-myc overexpression in serum-deprived murine NXS2 neuroblastoma cells led to cell cycle progression and massive apoptosis, causing a net decrease of viable cells. In serum-replete medium c-myc caused NXS2 cells to arrest in G2/M. Furthermore, c-myc decreased clonogenic growth of neuroblastoma cells. Taken together, these data suggest that c-myc attenuates the malignant phenotype of NXS2 neuroblastoma cells. Thus, although c-myc increased NXS2 tumor mass in vivo, c-myc appears to have decreased malignant potency in neuroblastoma cells compared to N-myc. This may be one reason why c-myc does not play a role in neuroblastomagenesis.     

N-myc proto-oncogene expression can induce DNA replication in Balb/c 3T3 fibroblasts

N-myc and c-myc are structurally and functionally related proto-oncogenes. C-myc expression plays an essential role towards the induction of DNA replication following mitogen stimulation of quiescent cells. We have examined whether N-myc expression can stimulate cellular DNA synthesis. When growth factor-deprived quiescent Balb/c 3T3 fibroblasts harboring a glucocorticoid-inducible N-myc gene were stimulated with dexamethasone, the resultant N-myc expression induced S-phase DNA synthesis. Hence, functional homology between N-myc and c-myc extends to their joint ability to stimulate DNA replication.     

重组反义c-myc腺病毒对HL-60细胞PCNA表达及其生长影响的研究

目的:研究重组反义c-myc腺病毒(Ad-AS-c-myc)对人急性早幼粒HL-60细胞系的c-myc、增殖细胞核抗原(PCNA)表达及细胞生长抑制。方法:采用重组腺病毒Lac-Z(Ad-LacZ)及Ad-AS-c-myc联合多聚阳离子转染HL-60细胞,X-gal染色判断转染效率。采用MTT、RT-PCR及免疫细胞化学等方法进行研究。结果:多聚阳离子可提高腺病毒对HL-60细胞的转染,Ad-LacZ加入鱼精蛋白硫酸盐转染率达79.8%。Ad-AS-c-myc能抑制HL-60细胞c-myc基因在转录及蛋白水平的表达。Ad-AS-c-myc抑制HL-60细胞生长及活力(抑制率51%),细胞生长停滞后PCNA蛋白表达降低。结论:Ad-AS-c-myc对HL-60细胞具有使生长减缓、增殖能力降低作用,其生物学作用与HL-60细胞c-myc和PCNA的表达受抑有关。Ad-AS-c-myc在急性早幼粒白血病基因治疗中具有潜在的临床价值,其生物学活性与HL-60细胞c-myc和PCNA表达受抑有关。     

Loss of one HuD allele on chromosome #1p selects for amplification of the N-myc proto-oncogene in human neuroblastoma cells

In human neuroblastoma tumors, amplification of the N-myc proto-oncogene and loss of all or part of the short arm of chromosome #1 are both associated with a poor prognosis. Accruing evidence indicates that it is the absence of one allele of the HuD (ELAVL4) gene, encoding the neuronal-specific RNA-binding protein HuD and localized to 1p34, that is linked to amplification. In 12 human neuroblastoma cell lines, N-myc amplification correlates with loss of one HuD allele and decreased HuD expression. Transfection experiments demonstrate that modulating HuD expression affects N-myc gene copy number as well as expression. Introduction of a sense HuD construct into two N-myc amplified cell lines considerably increases N-myc expression whereas gene copy number decreases. Conversely, expression of antisense HuD in N-myc nonamplified SH-SY5Y cells reduces HuD and N-myc mRNA levels even as cells show amplification of the N-myc gene. Thus, N-myc gene copy number is modulated by alteration of HuD expression. We propose that haploinsufficiency of HuD due to chromosome #1p deletion in neuroblastoma selects for cells that amplify N-myc genes. Application of these findings could lead to more effective therapies in the treatment of those patients with the worst prognosis.     

Amplification of the c-myc gene in human medulloblastoma cell lines and xenografts

Cultured cell lines and xenografts derived from 7 human medulloblastomas were evaluated for amplification of the c-myc, N-myc, epidermal growth factor receptor, and gli genes by Southern blot analysis. Karyotypes of the original biopsies and early passaged cells demonstrated double minute chromosomes in 4 of the 7 cases. All 7 samples (3 cell lines and 4 xenografts) from the 4 tumors with double minute chromosomes contained amplification of the c-myc gene. Cell lines and xenografts derived from the 3 biopsies without double minute chromosomes failed to demonstrate amplification of the 4 genes which were tested, but a rearrangement of the c-myc gene occurred in 1 of the 3 tumors. These observations demonstrate that the c-myc gene is often amplified and/or rearranged in human medulloblastomas and suggest that amplification of this gene provides a growth advantage for medulloblastoma cells in vitro and in vivo.     

Oncogene expression in small-cell lung cancer cell lines and a testicular germ-cell tumor: activation of the N-myc gene and decreased RB mRNA

N-myc and L-myc proto-oncogenes are expressed in many developing embryonic tissues. In contrast to expression of the closely related c-myc gene, N-myc and L-myc expression is very restricted in adult tissues. We show that small amounts of the L-myc RNA can be detected in normal adult testicular tissue. A high level of N-myc expression from a single-copy N-myc gene was found in a malignant tumor of the testis, histopathologically defined as a seminoma. This tumor also showed a decrease of mRNA from the retinoblastoma gene (RB), which is ubiquitously expressed in all normal tissues. Strikingly similar results on elevated expression of the L-myc and N-myc genes and a lack of RB mRNA have been reported in small-cell lung cancer cell lines, and are confirmed in our study.     

The human breast carcinoma cell line SW 613-S: an experimental system to study tumor heterogeneity in relation to c-myc amplification, growth factor production and other markers (review)

Amplification of the c-myc gene has been frequently reported in breast carcinomas. However the precise function of the c-myc protein is still unknown and the nature of the selective advantage offered to a cell by an overexpression of such a protein is unclear. We are addressing this question using the SW 613-S human breast carcinoma cell line as a model system. This cell line harbours an amplified c-myc gene and a mutated c-Ki-ras gene. By various criteria the amplified c-myc gene of SW613-S cells appears undistinguishable from a normal human c-myc gene. The SW613-S cell line is heterogeneous: it contains cells with a high level of amplification and carrying the extra copies of the c-myc gene in double minute chromosomes (DMs) and cells with few c-myc genes integrated into chromosomes. DM-containing cells are progressively lost upon in vitro cultivation but are selected for during in vivo growth, as tumors in nude mice, or by cultivating the cells in a chemically defined, serum-free medium or under conditions preventing anchorage. Clones with different levels of amplification and different chromosomal localization of the c-myc copies were isolated from the SW 613-S cell population. Those with a high level of amplification and expression of the c-myc gene are tumorigenic in nude mice, whereas those with a low level are not. Introduction of c-myc gene copies by transfection confers tumorigenicity to the nontumorigenic clones, indicating that a high level of amplification of the c-myc gene contributes to the tumorigenic phenotype of SW 613-S cells. Tumorigenic clones grow unattached, are able to proliferate in a chemically defined medium, and produce high levels of several growth factors (e.g. TGF-alpha, IGF2). Nontumorigenic clones are more dependent upon anchorage for growth, show a restricted growth in defined medium, and produce low or undetectable level of the growth factors tested. We have identified several genes, besides c-myc, the expression level of which is markedly different in the two types of clones. TGF-alpha, IGF2, PDGF-A, int-2, cytokeratins K8 and K18 and ferritin H chain are overexpressed in tumorigenic clones. In contrast, c-erbB1 (EGF receptor), c-jun, vimentin and p53 are expressed at a higher level in the nontumorigenic clones. Finally the major histocompatibility class I antigens, ferritin L chain, TGF-beta and c-Ki-ras, are examples of genes expressed at the same level in both types of clones.(ABSTRACT TRUNCATED AT 400 WORDS)