Antisense inhibition of c-myc expression reveals common and distinct mechanisms of growth inhibition by TGF beta and TNF alpha

Downregulation of the c-myc gene in HL-60 cells is associated with growth inhibition and induction of differentiation. Previous studies have reported that the growth inhibitors TGF beta and TNF alpha downregulate c-myc mRNA levels, suggesting the possibility that these agents may exert some of their phenotypic effects via c-myc downregulation. Our study demonstrates that although both growth inhibitors produce a similar decrease in c-myc protein synthesis, TNF alpha produces a greater growth inhibition and differentiation induction in HL-60 cells. Combined addition of anti-myc oligomer with either growth inhibitor produces no additive effect. In fact, 4 microM anti-myc oligomer produces the same growth and differentiation effects as does 10 ng/ml TGF beta 1. We conclude that downregulation of c-myc expression represents a common mechanism of growth inhibition by TGF beta and TNF alpha, but that TNF alpha possesses an additional effect that is independent of c-myc expression.     

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.     

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.     

Modulation of c-myc expression in the HL-60 cell line

A decrease in the expression of the myc proto-oncogene of HL-60 cells has been reported as an accompaniment of myeloid differentiation induced by either dimethylsulfoxide or retinoic acid. We report herein that several inhibitors of poly(ADP-ribose)-polymerase induced myeloid differentiation in HL-60 cultures. Studies on the expression of the c-myc gene in total cell RNA populations indicate that expression of this gene is inversely correlated with the state of differentiation, either myeloid or monocytic, of the cultured cells independent of the inducer and the rate of cell proliferation.     

Relationships between the cell cycle and the expression of c-myc and transferrin receptor genes during induced myeloid differentiation

We examined the relationship of cellular oncogene c-myc and transferrin receptor (TfR) gene expression to cell proliferation and cell cycle progression during myeloid differentiation in the HL-60 myeloid leukemia cell line. In order to determine levels of mRNA for these genes in HL-60 cells induced to differentiate along the myeloid pathway, RNA was isolated from HL-60 cells incubated with retinoic acid for 24 h and Northern blots were probed with labeled cDNAs for c-myc and TfR. c-myc mRNA decreased within 3 h of retinoic acid addition, and TfR mRNA decreased after 9 h; both mRNAs continued to decrease over 24 h. RNA was also isolated from HL-60 cells separated by centrifugal elutriation into cell cycle phases. TfR and c-myc cDNA probes hybridized equally to RNA from uninduced cells in all phases of the cell cycle. However, after 24 h incubation with the differentiation inducer retinoic acid, TfR mRNA was expressed substantially less in the G1 stage, whereas c-myc mRNA was still expressed equally in all cell cycle phases. These data indicate that, although TfR and c-myc expression are both associated with cell proliferation in the HL-60 line, TfR is down-regulated specifically in G1 upon induction of terminal differentiation whereas c-myc expression is disassociated from cell cycle control in these cells.     

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.     

Inhibition of murine monocyte proliferation by a colony-stimulating factor-1 antisense oligodeoxynucleotide. Evidence for autocrine regulation

Using a combination of v-myc and v-ras oncogenes, we have established a growth factor-independent monocyte cell line from murine fetal liver (FL-ras/myc). Biologic and molecular characterization demonstrated that the gene for the macrophage growth factor CSF-1 and the c-fms proto-oncogene (CSF-1 receptor) are expressed in this cell line, thus suggesting autocrine regulation as a possible mechanism for the unregulated growth of these cells. To study this possibility, we used 1) mAb, to neutralize the CSF-1 protein produced by the cell line, and 2) antisense oligomers, to inhibit CSF-1 gene products by specific base-pairing of complementary nucleic acids. We report here that both approaches inhibited in vitro cell growth by 60 to 70%, whereas the combination of oligomer and mAb inhibited proliferation by 95%. However, control antisense oligomers (50% bp mismatch with CSF-1 mRNA) did not inhibit FL-ras/myc cell growth. Furthermore, the inhibitory effects of mAb and oligomers were reversible when they were removed from the media. Detection of cell-associated CSF-1 protein by immunofluorescence showed that cells treated with the antisense oligomer expressed significantly less CSF-1 protein. These results indicate that the FL-ras/myc cell line requires CSF-1 for autonomous growth and that oligomers can efficiently block production of autocrine growth factors.     

Cooperative effect of 8-Cl-cAMP and rhGM-CSF on the differentiation of HL-60 human leukemia cells

In HL-60 leukemia cells the site-selective cAMP analog, 8-Cl-cAMP, at a dose of 5 microM produced growth inhibition with no signs of toxicity, whereas granulocyte-macrophage colony stimulating factor (GM-CSF) exerted an early transient increase of cell proliferation which was followed by differentiation toward monocytes. 8-Cl-cAMP in combination with GM-CSF blocked the growth stimulation due to GM-CSF and demonstrated a synergistic effect on the differentiation of HL-60 cells. The early proliferative effect of GM-CSF was correlated with an increased expression of type I regulatory subunit of cAMP-dependent protein kinase (RI alpha). Treatment with an RI alpha antisense oligodeoxynucleotide suppressed the GM-CSF-inducible cell proliferation and differentiation. Conversely, an RII beta antisense oligodeoxynucleotide, which suppresses the RII beta and causes a compensatory increase in RI alpha level, greatly enhanced the early proliferative input and the differentiation induced by GM-CSF. These results provide an insight into the mechanism of action of GM-CSF and the rationale for a combination differentiation therapy with 8-Cl-cAMP and GM-CSF.     

Changes in the gene expression of C-myc and CD38 in HL-60 cells during differentiation induced by nicotinic acid-related compounds

Changes in gene expression levels of c-myc and CD38 were examined during the differentiation of HL-60 cells to granulocytes due to three nicotinic acid-related compounds. CD38 expression was increased by isonicotinic acid and all-trans-retinoic acid (ATRA). Nicotinamide and nicotinamide N-oxide drastically decreased c-myc expression, but isonicotinic acid had no effect, suggesting that these compounds differentiate HL-60 to granulocytes through different pathways. These results should provide useful information as to the mechanisms of cell differentiation.     

Oligonucleotide N3'-->P5' phosphoramidates as antisense agents.

Uniformly modified oligonucleotide N3'-->P5' phosphoramidates, where every 3'-oxygen is replaced by a 3'-amino group, were synthesized. These compounds have very high affinity to single-stranded RNAs and thus have potential utility as antisense agents. As was shown in this study, the oligonucleotide phosphoramidates are resistant to digestion with snake venom phosphodiesterase, to nuclease activity in a HeLa cell nuclear extract, or to nuclease activity in 50% human plasma, where no significant hydrolysis was observed after 8 h. These compounds were used in various in vitro cellular systems as antisense compounds addressed to different targeted regions of c-myb, c-myc and bcr-abl mRNAs. C-myb antisense phosphoramidates at 5 microM caused sequence and dose-dependent inhibition of HL-60 cell proliferation and a 75% reduction in c-myb protein and RNA levels, as determined by Western blot and RT-PCR analysis. Analogous results were observed for anti-c-myc phosphoramidates, where a complete cytostatic effect for HL-60 cells was observed at 1 microM concentration for fully complementary, but not for mismatched compounds, which were indistinguishable from untreated controls. This was correlated with a 93% reduction in c-myc protein level. Moreover, colony formation by the primary CML cells was also inhibited 75-95% and up to 99% by anti-c-myc and anti-bcr-abl phosphoramidate oligonucleotides, respectively, in a sequence- and dose-dependent manner within a 0.5 nM-5 microM dose range. At these concentrations the colony-forming ability of normal bone marrow cells was not affected. The presented in vitro data indicate that oligonucleotide N3'-->P5' phosphoramidates could be used as specific and efficient antisense agents.     

Differential effects of c-myb and c-fes antisense oligodeoxynucleotides on granulocytic differentiation of human myeloid leukemia HL60 cells

To gain some insight into the role of c-myb and c-fes in myeloid differentiation, the authors have analyzed the ability of HL60 cells to differentiate in response to several different inducers after inhibition of c-myb and c-fes function. This function has been inhibited almost completely by using deoxynucleotides complementary to two 18-nucleotide sequences of c-myb and c-fes encoding mRNA. After 5 days in culture, in several separate experiments with different oligomer preparations, more than 90% growth inhibition was observed in c-myb antisense-treated HL60 cells. At this time, independent of the differentiation inducer used, c-myb antisense-treated HL60 cells differentiate only along the monocytic pathway, whereas in sense oligomer-treated cultures, retinoic acid and dimethyl sulfoxide induced granulocytic differentiation. No perturbation of the HL60 cell growth was observed after 5 days of treatment with antisense c-fes oligomer. However, induction to granulocytic differentiation by retinoic acid and dimethyl sulfoxide resulted in progressive cell death, whereas monocytic differentiation by other differentiation inducers was only marginally affected. These results suggest that granulocytic, unlike monocytic, differentiation requires c-myb-conditioned proliferation and the activity of the protein encoded by c-fes.