Transrepression of the N-myc expression by c-myc protein

In the human neuroblastoma cell line IMR32 the N-myc gene happens to be amplified and actively expressed, whereas no stable c-myc RNA can be detected in the same cells. In this report, we show that in IMR32 cells the expression of the N-myc gene is repressed by introduction of a c-myc expression vector (c-myc cDNA conjugated with an SR promoter). Moreover, dose response experiments showed that the amount of endogenous c-myc protein present in HeLa cells (which express c-myc but not N-myc) is enough to repress the expression of N-myc in IMR32 cells.     

Truncation of exon 1 from the c-myc gene results in prolonged c-myc mRNa stability.

The human c-myc gene consists of three exons transcribed from two distinct promoters and the function of the first, noncoding exon is unknown. In COLO 320 cells, there co-exist normal and truncated (i.e., lacking exon 1) c-myc genes, both of which are transcribed. Studies on the turnover of c-myc mRNA show that the normal mRNA has an in vivo half-life of approximately 30 min which is approximately similar to the turnover time of the mRNA in lymphoblastoid cells. However, the truncated mRNA was found to be substantially more stable. This observation was also made with a Burkitt's lymphoma cell line which has a translocated, truncated c-myc gene. Therefore truncation of the c-myc gene can cause the mRNA to be more stable than the full size product suggesting that this can be a crucial factor in the activation of the c-myc oncogene, by exon 1 loss, in chromosomal translocation. The results also suggest a role for exon 1 in the c-myc mRNA degradative mechanism.     

Expression and characterization of the human c-myc DNA-binding protein.

In an effort to study in detail the nature of the protein product of the human protooncogene c-myc, we have expressed the gene at high levels in Escherichia coli. The c-myc coding region was taken from a full-length cDNA clone and inserted into a vector designed to express foreign gene products efficiently in E. coli. Pulse-labeling experiments indicated that the rate of expression of c-myc in this thermoinducible expression system is very efficient. The product was relatively stable and accumulated to approximately 10% of total cellular protein. A purification protocol was devised which allowed the c-myc protein to be readily purified in quantities sufficient for detailed biochemical and physical analyses. A high-titer polyclonal antiserum was raised against the pure protein and shown to immunoprecipitate the p110gag-myc fusion protein of MC-29-infected quail cells. This antiserum also selectively detects a protein with an apparent molecular weight of 64,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis from a Burkitt lymphoma cell line. We conclude that this 64-kilodalton protein is the human c-myc gene product since the E. coli-made protein exhibits an equivalent molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, even though its calculated molecular weight is 49,000. Furthermore, we demonstrate that the bacterially made human c-myc protein is a DNA-binding protein and that it exhibits a high nonspecific affinity for double-stranded DNA.     

c-myc down-regulates class I HLA expression in human melanomas

Expression of class I HLA antigen has been shown to be reduced in a number of human tumours. Here we show that in a panel of 11 melanoma cell lines with variable class I HLA expression an inverse correlation exists between the mRNA levels of c-myc and class I HLA. This suggests that high expression of the c-myc oncogene might inhibit the class I HLA expression. To test this hypothesis a melanoma cell line with a low c-myc and high class I HLA mRNA expression was transfected with a c-myc expression vector. All clones expressing the transfected c-myc gene show reduced class I HLA mRNA and beta 2-microglobulin mRNA expression. Reduced class I HLA mRNA levels result in a lowered class I protein expression on the cell surface. Treatment with gamma-interferon fully restores the class I HLA and beta 2-microglobulin expression in these cells. This effect is preceded by a transient decrease of the c-myc mRNA level. These results show that the class I HLA expression is modulated by the level of c-myc expression, thus opening up the possibility that high expression of this oncogene influences the interaction of melanoma cells with the immune system.     

Preparation and characterization of specific antisera directed against different polypeptide domains encoded by the c-myc oncogene for studying the expression of this gene introduced into quail or rat cells

By using bacterial expression vectors, we have prepared antisera directed against two polypeptidic domains encoded by exons 2 and 3 of the human c-myc oncogene. These antisera which detect specifically the human c-myc proteins allow us to analyse the expression of human c-myc gene activated by retroviral sequences and introduced in quail embryo cells (QEC) or in established rat embryo fibroblastic cell line (208 F). Although human myc mRNA are expressed in the two cell types, the p64/p67 human c-myc proteins are only detected in the QEC.     

Growth inhibition of human keratinocytes by antisense c-myc oligomer is not coupled to induction of differentiation

To study the relationship between cell growth and differentiation in human keratinocytes, we examined the effect of the antisense oligomer of c-myc mRNA. This oligomer is stable in culture medium. A 24 h incubation of cells with 5 microM antisense c-myc oligomer resulted in a 48.2% decrease in c-myc protein and inhibited cell growth by 80.7% compared to the sense c-myc oligomer. In contrast, antisense c-myc oligomer had no effect on differentiation when the population of involucrin-positive cells and cornified envelope formation were used as differentiation markers. These results show that antisense c-myc oligomer inhibits cell growth but does not induce differentiation in normal human keratinocytes. Therefore, cell growth and differentiation are not necessarily coupled in these cells.     

Activation of c-Ki-ras coexists with c-myc amplification in cells from a nude mouse tumor induced by the human breast carcinoma cell line SW 613-S

In vitro transfection experiments have shown that cooperation between two different oncogenes can confer a fully malignant phenotype to primary rodent cells. We have previously reported that SW 613-Tul cells, derived from a tumor induced in a nude mouse by the human breast carcinoma cell line SW 613-S, showed a 30-fold amplification of the c-myc gene. In the present work, we show that these cells also harbor an activated c-Ki-ras gene capable of inducing the formation of foci upon transfection of NIH 3T3 cells with SW 613-Tul genomic DNA. Our results suggest that both the c-myc and c-Ki-ras oncogenes, activated by two different mechanisms, may cooperate in the full expression of the tumorigenic phenotype of SW 613-Tul cells.     

Defective RNA-mediated c-myc gene silencing pathway in Burkitt's lymphoma

Keeping in view the fact that molecular basis of Burkitt's lymphoma (BL) is poorly understood, we attempted to explore the small interfering RNA (siRNA) mediated c-myc gene regulation using BL-derived EB-3 cell line as archetype cellular model. Such a study revealed that EB-3 cells possess 4-fold higher expression of Dicer gene coupled with 2-fold higher activity of RNA polymerase III than that observed in normal human lymphocytes. siRNAs derived from EB-3 cells had the inherent capacity to suppress c-myc gene expression in normal cells but not in native cells. Based on these findings we have proposed a novel RNA-mediated c-myc gene regulation pathway that may be responsible for BL.