Relationship of c-myc gene copy number and gene expression: cellular effects of elevated c-myc protein

The relationship between gene copy number and expression and cellular consequences of elevated levels of c-myc protein has been investigated using recombinant Chinese hamster ovary cell lines transfected with DNA coding for the murine c-myc gene. HC-8 and LC-5 recombinant cells carry approximately 800 and 50 copies of c-myc sequences, respectively, under control of an inducible heat shock promoter. Multivariate flow cytometric analysis and clonogenic assays were used to measure the relationship among c-myc expression, rate of DNA synthesis, and cell survival. Following heat exposure, maximally induced HC-8 cells produced approximately tenfold more c-myc protein than heated LC-5 cells, suggesting a close relationship between gene copy number and level of expression. However, considerable heterogeneity in the level and time of c-myc expression was observed following heat induction, even though the amounts of genomic c-myc were relatively constant. Heterogeneity in gene expression was not attributable to variation in heat induction methodologies and/or cell cycle phase distributions. The presence of high levels of recombinant c-myc protein was associated with a decreased rate of bromodeoxyuridine incorporation into DNA. High levels of c-myc protein in HC-8 cells were inversely correlated with cell survival postheating, suggesting that high levels of c-myc protein are incompatible with cell survival.     

Constitutive c-myc expression enhances proliferation of differentiating F9 teratocarcinoma cells

The c-myc protooncogene is expressed in many tumor cells as well as during normal development. In order to study the role of c-myc in differentiation, proliferation and tumorigenicity of F9 mouse teratocarcinoma cells, the pSVmyc1 plasmid constitutively expressing an active c-myc oncogene was introduced into F9 stem cells by cotransfection with the selectable marker RSVneo. Enhanced expression of c-myc did not alter the properties of F9 stem cells. Prolonged proliferation during retinoic acid induced differentiation was observed in cell clones constitutively expressing c-myc. In contrast, as determined by morphology, by immunocytochemistry for markers specific for stem cells and differentiated derivatives, and by Northern hybridization for mRNAs specific for differentiated cells, differentiation was neither inhibited nor delayed by constitutive c-myc expression. Tumorigenicity of stem cells as well as retinoic acid-treated cells--as measured by soft agar cloning efficiency and tumor formation in syngenic mice--was not altered by SVmyc1. We conclude that in F9 teratocarcinoma cells down-regulation of c-myc is related to arrest of proliferation rather than differentiation.     

Endothelin stimulates c-fos and c-myc expression and proliferation of vascular smooth muscle cells

Recently, a potent vasoconstrictor peptide, endothelin (EDT), was isolated from vascular endothelial cells. We examined its effect on rat vascular smooth muscle cells (VSMCs). EDT induced the elevation of intracellular calcium, which was dependent on extracellular calcium and inhibited by a calcium-channel antagonist in a competitive manner. EDT caused a rapid and transient increase in the c-fos and c-myc mRNA levels and stimulated the DNA synthesis of VSMCs in a dose-dependent manner. This effect of EDT on the proliferation of VSMCs might be related to the development of atherosclerosis.     

Interleukin-6 stimulates c-myc expression and proliferation of cultured vascular smooth muscle cells

The effects of interleukin-6 (IL-6), a cytokine recently found to be secreted by monocytes and macrophages, on c-myc expression and proliferation of cultured vascular smooth muscle cells (VSMC) were investigated. Treatment with IL-6 caused rapid increase in the c-myc mRNA level of VSMC. It also stimulated DNA synthesis and proliferation of the cells significantly and dose-dependently at concentrations of more than 10 U/ml. These results suggest that IL-6 may be important in the proliferation of VSMC, which is a key event in the development of arteriosclerosis, as a factor mediating immune cell-VSMC interaction.     

Possible function of the c-myc product: promotion of cellular DNA replication.

We have recently cloned a plasmid, pARS65, containing the sequences derived from mouse liver DNA which can autonomously replicate in mouse and human cells (Ariga et al., 1987). In this report, we show that replication of pARS65 in HL-60 cells can be inhibited by co-transfection with anti-c-myc antibody. In an in-vitro replication system using HL-60 nuclear extract, pARS65 functioned as a template. This in-vitro replication was also blocked by addition of anti-c-myc antibody. Specific binding activity of the c-myc product to pARS65 was detected by an immunobinding assay, suggesting that the c-myc protein promotes DNA replication through binding to the initiation site of replication. This has been substantiated using the antibody to help isolate a human DNA segment that can autonomously replicate in the cells.     

A hemodynamic load in vivo induces cardiac expression of the cellular oncogene, c-myc

To establish whether a hemodynamic load that causes cardiac hypertrophy in the intact animal might interact with cellular pathways that are thought to transduce growth signals in model systems, we have analyzed expression of the cellular oncogene, c-myc, after a systolic pressure load. Aortic constriction increased c-myc mRNA abundance in both the atria and left ventricle of 28-day rats, but did not activate a second "competence" gene, r-fos, whose expression by cardiac cells ceases upon termination of mitotic growth. In 80-day rats, c-myc was induced in the atria alone. Induction of c-myc by aortic constriction in vivo may correlate with the respective capacity of atrial and ventricular myocytes to replicate DNA during cardiac hypertrophy. Activation of c-myc was not sufficient to account for inhibition of muscle creatine kinase (mck) mRNA, which was decreased only in 28-day rats.     

Amplification and expression of a cellular oncogene (c-myc) in human gastric adenocarcinoma cells.

Three of 16 human gastric adenocarcinoma samples, maintained as solid tumors in nude mice, were found to carry amplified c-myc genes. In two samples with a high degree of c-myc DNA amplification (15- to 30-fold), double minute chromosomes were observed in karyotype analysis. The level of c-myc RNA was markedly elevated in a rapidly growing and poorly differentiated tumor, whereas it was only slightly elevated in a slowly growing and more differentiated tumor.     

Inhibition of protein kinase C arrests proliferation of human tumors

We have shown that inhibition of protein kinase C by 1-5-isoquinolinylsulfonyl-2-methylpiperazine, H7, induces differentiation and inhibits proliferation of Neuro 2a cells. We have now tested if H7 is able to inhibit proliferation of: 1) human tumor cell lines from tissues other than brain; and 2) primary cultured cells from several human brain tumors. H7 inhibits, in a dose-dependent manner, proliferation of all human tumor cell lines tested and of primary cultured cells from human brain tumors. These results indicate that inhibition of protein kinase C inhibits proliferation of tumoral cells, therefore, H7, and likely other inhibitors of protein kinase C, could be useful in the clinical treatment of brain (and probably other) tumors.     

A combination treatment of c-myc antisense DNA with all-trans-retinoic acid inhibits cell proliferation by downregulating c-myc expression in small cell lung cancer

The dysregulation of both c-myc expression and retinoid signaling pathways commonly occurs in small cell lung cancers (SCLC), frequently accompanying tumor relapse, and contributing to the poor prognosis of patients with SCLC. In this study, we investigated whether c-myc antisense oligodeoxynucleoside phosphorothioate (OPT) covering the translational initiation site of c-myc mRNA used in combination with all-trans-retinoic acid (RA) would be more effective than either agent alone in inhibiting the growth of an SCLC cell line, NCI-H82, overexpressing c-myc with amplification of this gene, and whether this combination could be an experimental therapeutic tool against SCLC. c-myc antisense OPT decreased c-myc expression in Northern and Western blot analyses, thus inducing 40% and 20% cell growth inhibition compared with scrambled and sense OPT and with scrambled four guanosine-containing OPT (p < 0.01, and p < 0.01, respectively). All-trans-RA also inhibited cell proliferation at the rate of 40% by downregulating c-myc expression. Having obtained these results, we tested the hymothesis that c-myc antisense OPT in combination with all-trans-RA may further reduce c-myc expression and lead to improved cell growth control. This combination showed a greater inhibition of cell proliferation than either agent given alone (p < 0.01) (60% inhibition of cell growth compared with treatment of control scrambled or sense OPT alone, p < 0.01) through enhanced downregulation of c-myc expression. In conclusion, c-myc antisense DNA in combination with other modalities for c-myc downregulation may represent an attractive gene regulation-based therapy of SCLC in the future. Further efforts, however, using new oligodeoxynucleotide analogs, specific interventions for DNA delivery into cells, and more potent therapeutic agents are required to increase the potentiation of c-myc downregulation and cell growth inhibition.     

c-myc expression affects proliferation but not terminal differentiation or survival of explanted erythroid progenitor cells

The expression of c-myc was analyzed in murine and human erythroblasts throughout their differentiation in vitro into reticulocytes. The murine cells were splenic erythroblasts from animals infected with the anemia strain of Friend virus (FVA cells). In FVA cells cultured without EPO, the c-myc mRNA and protein levels decrease sharply within 3 to 4 h, showing that continual EPO stimulation is required to maintain c-myc expression. When cultured with EPO, the c-myc mRNA level of FVA cells is raised within 30 min of exposure. The c-myc mRNA and protein reach maxima at 1 to 3 h, then decline slowly to very low levels by 18 h. In contrast, c-fos and c-jun mRNA levels are not regulated by EPO in FVA cells. The human cells analyzed were colony-forming units-erythroid, CFU-E, derived in vitro by the culture of peripheral blood burst-forming units-erythroid (BFU-E). When grown in EPO and insulin-like growth factor 1 (IGF-1) these cells differentiate into reticulocytes over 6 days rather than the 2 days required for murine cells, but the c-myc mRNA kinetics and response to EPO parallel those of mouse cells at similar stages of differentiation. Both IGF-1 and c-kit ligand (SCF) cause an additive increase in c-myc mRNA in human CFU-E in conjunction with EPO. These additive effects suggest that EPO, IGF-1, and SCF affect c-myc mRNA accumulation by distinct mechanisms. Addition of an antisense oligonucleotide to c-myc in cultures of human CFU-E specifically inhibited cell proliferation but did not affect erythroid cell differentiation or apoptosis. When human cells were grown in high SCF concentrations, an environment which enhances proliferation and retards differentiation, antisense oligonucleotide to c-myc strongly inhibited proliferation, but such inhibition did not induce differentiation. This latter result indicates that differentiation requires signals other than depression of c-myc and resultant depression of proliferation. © 1996 Wiley-Liss, Inc.     

Hypoxia regulates VEGF expression and cellular proliferation by osteoblasts in vitro.

Numerous studies have demonstrated the critical role of angiogenesis for successful osteogenesis during endochondral ossification and fracture repair. Vascular endothelial growth factor (VEGF), a potent endothelial cell-specific cytokine, has been shown to be mitogenic and chemotactic for endothelial cells in vitro and angiogenic in many in vivo models. Based on previous work that (1) VEGF is up-regulated during membranous fracture healing, (2) the fracture site contains a hypoxic gradient, (3) VEGF is up-regulated in a variety of cells in response to hypoxia, and (4) VEGF is expressed by isolated osteoblasts in vitro stimulated by other fracture cytokines, the hypothesis that hypoxia may regulate the expression of VEGF by osteoblasts was formulated. This hypothesis was tested in a series of in vitro studies in which VEGF mRNA and protein expression was assessed after exposure of osteoblast-like cells to hypoxic stimuli. In addition, the effects of a hypoxic microenvironment on osteoblast proliferation and differentiation in vitro was analyzed. These results demonstrate that hypoxia does, indeed, regulate expression of VEGF in osteoblast-like cells in a dose-dependent fashion. In addition, it is demonstrated that hypoxia results in decreased cellular proliferation, decreased expression of proliferating cell nuclear antigen, and increased alkaline phosphatase (a marker of osteoblast differentiation). Taken together, these data suggest that osteoblasts, through the expression of VEGF, may be in part responsible for angiogenesis and the resultant increased blood flow to fractured bone segments. In addition, these data provide evidence that osteoblasts have oxygen-sensing mechanisms and that decreased oxygen tension can regulate gene expression, cellular proliferation, and cellular differentiation.