Reduced DNase I sensitivity of the rearranged c-myc gene in somatic cell hybrids between murine plasmacytoma cells and fibroblasts

In mouse plasmacytoma (MPC) S194, the rearranged c-myc gene was much more sensitive to DNase I digestion than the nonrearranged gene. The sensitivity of the rearranged c-myc was markedly reduced to the same extent as that of the nonrearranged one in hybrids between the MPC cells and the fibroblasts, but not in a hybrid between the MPC and the spleen cells. These results suggest that trans-acting factors in fibroblasts alter the DNase I-sensitive structure of the rearranged c-myc gene.     

Selection of cells with different chromosomal localizations of the amplified c-myc gene during in vivo and in vitro growth of the breast carcinoma cell line SW 613-S

The c-myc gene is amplified in the human breast carcinoma cell line SW 613-S. At early in vitro passages, the extra copies of the gene were mainly localized in double minute chromosomes (DMs), as shown by in situ hybridization with a biotinylated c-myc probe. However, cells without DMs were also present in which the c-myc genes were found integrated into any of several distinct chromosomes (mainly 7q+, 4 and 4q+, and 1). When this cell line was propagated in vitro, the level of c-myc amplification decreased because cells with DMs and a high amplification level were lost and replaced by cells without DMs and having a low amplification level. On the contrary, when early passage SW 613-S cells were grown in vivo, as subcutaneous tumours in nude mice, cells with numerous DMs and a high level of c-myc amplification were selected for. In one cell line (SW 613-Tu1) established from such a tumour, the DM-containing cells were substituted at late passages for cells with a high number of c-myc copies integrated within an abnormally banded region, at band 17q24 of a 17q+ chromosome. When only cells with integrated genes were present, this cell line was still highly tumorigenic indicating that the localization of the c-myc genes in DMs was not required for these cells to be tumorigenic in nude mice. Furthermore, cells of the secondary tumours induced by SW 613-Tu1 did not contain any DMs showing that in vivo growth did not promote the release of integrated c-myc copies into DMs.     

The growth-inhibitory Ndrg1 gene is a Myc negative target in human neuroblastomas and other cell types with overexpressed N- or c-myc

A major prognostic marker for neuroblastoma (Nb) is N-myc gene amplification, which predicts a poor clinical outcome. We sought genes differentially expressed on a consistent basis between multiple human Nb cell lines bearing normal versus amplified N-myc, in hopes of finding target genes that might clarify how N-myc overexpression translates into poor clinical prognosis. Using differential display, we find the previously described growth-inhibitory gene Ndrg1 is strongly repressed in all tested Nb cell lines bearing N-myc amplification, as well as in a neuroepithelioma line with amplified c-myc. Overexpression of N-myc in non-amplified Nb cells leads to repression of Ndrg1, as does activation of an inducible c-myc transgene in fibroblasts. Conversely, N-myc downregulation in N-myc-amplified Nb cells results in re-expression of the Ndrg1, and stimuli known to induce Ndrg1 do so in Nb cells while simultaneously down-regulating N-myc. Relevant to these results, we demonstrate an in vitro interaction of Myc protein with the Ndrg1 core promoter. We also find that Ndrg1 levels increase dramatically during in vitro differentiation of two cell lines modeling neural and glial development, while c- and N-myc levels decline. Our results combined with previous information on the Ndrg1 gene product suggest that downregulation of this gene is an important component of N-Myc effects in neuroblastomas with poor clinical outcome. In support of this notion, we find that re-expression of Ndrg1 in high-Myc Nb cells results in smaller cells with reduced colony size in soft-agar assays, further underscoring the functional significance of this gene in human neuroblastoma cells.     

Reduced induction of c-fos but not of c-myc expressions in a nontumorigenic revertant R1 of EJ-ras-transformed NIH/3T3 cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA)

It has been reported that both c-fos and c-myc mRNAs are induced in NIH/3T3 cells after 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. We have studied the effect of TPA on the expression of c-fos and c-myc in EJ-ras-transformed NIH/3T3 and its nontumorigenic flat revertant R1 cells. Although TPA treatment induces c-myc mRNA, as in the case of NIH/3T3 cells, the induced level of c-fos mRNA is greatly reduced not only in slow-growing EJ-ras-transformed NIH/3T3 but also in quiescent R1 cells. In addition, serum-induced c-fos expression is also reduced in EJ-ras-transformed NIH/3T3 and R1 cells. These observations suggest that the pathway from TPA to c-fos gene is different from that to c-myc gene and that the former pathway is down-regulated in association not with the transformed phenotype, but with EJ-ras expression, and it is possible that this reduced induction of c-fos is not specific to TPA.     

Anti-c-myc DNA increases differentiation and decreases colony formation by HL-60 cells

Summary The proto-oncogene c-myc, whose gene product has a role in replication, is overexpressed in the human promyelocytic leukemia HL-60 cell line. Treatment of HL-60 cells with an antisense oligodeoxyribonucleotide complementary to the start codon and the next four codons of c-myc mRNA has previously been observed to inhibit c-myc protein expression and cell proliferation in a sequence-specific, dose-dependent manner. Comparable effects are seen upon treatment of HL-60 cells with dimethylsulfoxide (Me₂SO), which is also know to induce granulocytic differentiation of HL-60 cells. Hence, the effects of antisense oligomers on cellular differentiation were examine and compared with Me₂SO. Differentiation of HL-60 cells into forms with granulocytic characteristics was found to be enhanced in a sequence-specific manner by the anti-c-myc oligomer. No synergism was observed between the anti-c-myc oligomer and Me₂SO in stimulating cellular differentiation. In contrast, synergism did appear in the inhibition of cell proliferation. Finally, the anti-c-myc oligomer uniformly inhibited colony formation in semisolid medium. It is possible that further reduction in the level of c-myc expression by antisense oligomer inhibition may be sufficient to allow terminal granulocytic differentiation and reverse transformation. This work was supported by grants to E. W. from the National Institutes of Health, Bethesda, MD (CA 42960), and the Leukemia Society of America.     

Influence of myc overexpression on the phenotypic properties of Chinese hamster lung cells resistant to antitumor agents

In the Chinese hamster lung fibroblast cell line DC-3F, the development of resistance to different drugs, through several mechanisms like MDR expression or alteration of the DNA topoisomerase II activity, has been shown to be associated with a decreased tumorigenicity. Multiple studies have shown that the myc oncogene, in cooperation with ras, plays a major role in the oncogenic transformation of fibroblasts. As an approach to a better understanding of the relationship between the different phenotypic traits, we analyzed the expression of myc and ras oncogenes in the drug-sensitive DC-3F cells and in variants resistant to 9-hydroxy-ellipticine (9-OH-E) (DNA topoisomerase II alteration) or to actinomycin D (AD) (multidrug (MDR) expression). Southern and Northern blot analyses revealed about a 10-fold amplification and a 20-fold overexpression of the c-myc gene in the DC-3F cells as compared to the normal lung fibroblasts. Both amplification and overexpression are markedly decreased in the two resistant variants. ras gene copy number and expression were found to be identical in all cell types. In order to analyze the contribution of the decreased myc expression on the different phenotypic traits, the DC-3F/9-OH-E cells were transfected with the plasmid pSV-c-myc, and six clones expressing high amounts of the transfected myc were isolated and characterized. Morphological and caryological alterations, as well as an increased cloning efficiency in soft agar, indicated that the myc gene product was made in these cells. However, the tumorigenicity of the sensitive parental cells was not restored, thus showing that the decreased myc expression alone does not account for the loss of tumorigenicity in the resistant cells. 9-OH-E resistance was not modified in the transfected cells, while the cross-resistance of these cells to MDR-sensitive drugs, such as vincristine, actinomycin D, and taxol, was reversed roughly in proportion of the expression of the transfected myc.     

c-fos antisense RNA blocks expression of c-fos gene in F9 embryonal carcinoma cells

To study the function of proto-oncogene c-fos, we prepared an antisense plasmid that expresses in mammalian cells c-fos antisense RNA which is complementary to the endogenous c-fos mRNA. Upon transfection into undifferentiated F9 EC cells, the antisense plasmid directed constitutive expression of a large amount of c-fos antisense RNA. These cells were very low in the basal level of c-fos message and were unable to induce c-fos message when stimulated with interferon or phorbol ester. The failure to induce c-fos message led to the blockade of c-fos protein expression in these cells. Thus, these cells represented a c-fos defective phenotype. The blockade of c-fos gene expression seen in antisense-cells could be caused by rapid degradation of the c-fos message, since c-fos mRNA expression was rescued in these cells when treated with protein synthesis inhibitor, cycloheximide. We found that expression of c-myc gene was down-regulated in c-fos antisense-cells: Although control undifferentiated F9 cells constitutively expressed a high level of c-myc message, the antisense cells had a much lower amount of c-myc mRNA. Since p53 and heat shock gene 70 were expressed at comparable levels in control and antisense cells, c-myc gene expression appears to be regulated by c-fos gene in F9 EC cells. Lastly, these antisense cells grew as rapidly as control F9 cells and underwent differentiation after retinoic acid treatment, indicating that c-fos expression is not a prerequisite for differentiation of F9 cells.     

Normal and transforming Ras are differently regulated for posttranslational modifications

Point mutation of the c-H-ras gene significantly increases cellular transforming activities of Ras. Since posttranslational modification and subsequent membrane localization are essential for the biological activities of Ras, we examined whether or not the mutation also affects these two factors. The normal (Gly¹²) or the transforming (Val¹²) c-H-ras gene was expressed in NIH3T3 cells using a metallothionein promoter. Expression of either type of Ras was efficiently induced by the cadmium treatment of these cells, and immunoprecipitation of metabolically labeled cell extracts revealed that both normal and transforming Ras were expressed as four differently migrating forms on SDS-polyacrylamide gels, two of which were slower migrating cytosolic precursors and the other two were faster migrating membrane-bound forms. There was no significant difference in half lives between normal and transforming Ras; however, posttranslational modification was quite different between the two types of Ras. Transforming Ras was processed and became membrane-bound forms much more efficiently than normal Ras. Interestingly, posttranslational modification and membrane localization of Ras was significantly inhibited when the c-myc oncogene was co-expressed with Ras. In contrast to the c-myc oncogene, expression of either wild type or mutant p53 did not affect the posttranslational modification of Ras, suggesting that the c-myc oncogene specifically impairs the posttranslational modification of Ras. © 1996 Wiley-Liss, Inc.