Facilitation of the cellular uptake of a triplex-forming oligonucleotide by novel polyamine analogues: structure-activity relationships.

The inefficient uptake of oligodeoxynucleotides, including that of TFO, through the cell membrane is a limiting factor in developing gene therapy approaches for cancer and other diseases. To develop a new strategy for oligonucleotide delivery into the nucleus, we synthesized a series of novel polyamine analogues and examined their effects on the uptake of a 37-mer [32P]-labeled TFO, targeted to the promoter region of c-myc oncogene. We used MCF-7 breast cancer cells to investigate the efficacy of polyamines on the internalization of the TFO. The uptake of TFO was enhanced by complexing it with several unsubstituted polyamine analogues at 0. 1-5 microM concentrations, with up to 6-fold increase in TFO uptake in the presence of a hexamine, 1,21-diamino-4,9,13, 18-tetraazahenicosane (H2N(CH2)(3)NH(CH2)(4)NH(CH2)(3)NH(CH2)(4)NH(CH2)(3)NH(2) or 3-4-3-4-3). TFO uptake increased with the cationicity of the polyamines; however, bis(ethyl) substitution and structural features of the methylene bridging region had significant effects on TFO uptake. The majority of labeled TFO was recovered from the nuclear fraction containing genomic DNA. Electrophoretic mobility shift assay revealed enhanced binding of TFO to a target duplex containing promoter region sequence of c-myc oncogene. Treatment of MCF-7 cells with the TFO complexed with 0.5 microM 3-4-3-4-3 suppressed c-myc mRNA level by 65%, as determined by Northern blot analysis. These data indicate a novel approach to deliver oligodeoxynucleotides to the cell nucleus, and suppress the expression of target genes, and provide new insights into the mechanism of oligonucleotide transport in living cells.     

Differential effects of cyclopolyamines on the stability and conformation of triplex DNA

Linear polyamines are excellent promoters of triplex DNA formation. The effects of structural rigidization of polyamines on triplex DNA stability are not known at present. We wished to develop a series of polyamine analogs as secondary ligands for triplex DNA stabilization for antigene applications. To accomplish this goal, we synthesized cyclopolyamines by interconnecting the two amino or imino groups of linear polyamines with a --(CH2)n-bridge (n=3,4,5). Melting temperature (Tm) data showed that [4,3]-spermine and [4,4]-spermine stabilized poly(dA) x 2poly(dT) triplex at >25 microM concentrations (Tm = 71 degrees C at 100 microM). The dTm/dlog [polyamine] values for these compounds were 26 and 40, respectively. [4,3]-Spermine and [4,4]-spermine also stabilized triplex DNA formed by a purine-motif triplex-forming oligonucleotide, TG3TG4TG4TG3T with its target duplex, as determined by Tm, circular dichroism (CD) spectroscopy, and electrophoretic mobility shift assay (EMSA). In contrast, [4,4]-putrescine and [4,5]-putrescine as well as [4,5]-spermine had no triplex DNA stabilizing effect. CD spectra also showed triplex DNA aggregation and psi-DNA formation at >100 microM [4,3]-spermine. These data demonstrate that structural rigidization of linear polyamines has a profound effect on their ability to stabilize triplex DNA and provoke conformational transitions.     

Inhibition of cell adhesion to plastic substratum by phosphorothioate oligonucleotide.

Antisense oligonucleotides have been widely used to achieve specific inhibition of targeted gene expression. However, the mechanism of action is not well understood and in many systems sequence-independent effects occur. We have recently shown that chronic administration of an antisense c-myc phosphorothioate oligonucleotide can specifically inhibit expression of the c-myc protein and growth in human breast cancer cells. We now identify an additional effect of the same oligonucleotide on cell adhesion. Transient delivery through electroporation of 2.5 microM antisense-myc oligonucleotide to MCF-7 cells results in 85% inhibition of adhesion to plastic substratum within 24 h. Both the onset of this effect and the subsequent recovery occur without a change in cell viability, growth, or alteration of adhesion to Matrigel, collagen IV, laminin, or fibronectin. However, no parallel changes in c-myc mRNA or protein expression are detectable, suggesting that in this instance inhibition of adhesion caused by antisense-myc oligonucleotide may involve a mechanism independent of the target sequence.     

The effects of hydrocortisone on c-fos, c-myc and c-ras oncogene expression in IMR-90 fibroblasts

The effects of hydrocortisone on oncogene expression in human IMR-90 fibroblasts was analyzed by Northern blotting of total RNA. In synchronized fibroblasts stimulated with serum alone, there were two time periods of increased c-fos expression during the G1 phase of the cell cycle. There was no significant difference between cells treated with serum plus hydrocortisone, and cells treated with serum alone with respect to c-fos expression. Quiescent cells showed no change in c-fos expression during the G1 phase of the cell cycle. Three peaks of c-fos expression occur when cells are treated with hydrocortisone alone, but hydrocortisone in the absence of serum is insufficient to initiate DNA synthesis. Hydrocortisone has no effect on c-myc or c-Ha-ras expression in the presence or absence of serum in synchronized fibroblasts. Therefore, the control of mRNA production of the nuclear oncogenes c-fos and c-myc, and the cytoplasmic oncogene c-ras are independent and hydrocortisone may enhance DNA synthesis by increasing c-fos expression.     

The role of polyamines, Na(+) and K(+) in the formation of triple helices between purine oligonucleotides and the promoter region of the human c-src proto-oncogene

Binding constants for triplex formation between purine-rich oligonucleotides and a pyrimidine·purine tract of the human c-src proto-oncogene were measured by fluorescence polarization in the presence of polyamines, Na⁺ and K⁺. In both the hexamine and tetramine series, the longer polyamines had the larger binding constants for triplex formation at low concentrations of polyamine. At higher concentrations all values tended to plateau in the 10⁹/M range. In contrast to previous reports, K⁺ did not inhibit triplex formation and at 150 mM the binding constants were again in the 10⁹/M range for both an 11mer and 22mer oligonucleotide. At 150 mM K⁺ the addition of polyamines did not lead to any significant increase in the binding constants. It was determined that the lack of inhibition by K⁺ was due to the low concentration (1 nM) of purine oligonucleotide required for the fluorescence polarization technique. At higher concentrations (1 µM) self-association of the oligonucleotide was observed. These results suggest that in vivo, at least for the c-src promoter, the inhibition of triplex formation by K⁺ may not be detrimental. However, it may be difficult to achieve binding constants above ~10⁹/M even in the presence of polycations.     

Molecular dynamics simulation study of oriented polyamine- and Na-DNA: sequence specific interactions and effects on DNA structure

Molecular dynamics (MD) computer simulations have been carried out on four systems that correspond to an infinite array of parallel ordered B-DNA, mimicking the state in oriented DNA fibers and also being relevant for crystals of B-DNA oligonucleotides. The systems were all comprised of a periodical hexagonal cell with three identical DNA decamers, 15 water molecules per nucleotide, and counterions balancing the DNA charges. The sequence of the double helical DNA decamer was d(5'-ATGCAGTCAG)xd(5'-TGACTGCATC). The counterions were the two natural polyamines spermidine(3+) (Spd(3+)) and putrescine(2+) (Put(2+)), the synthetic polyamine diaminopropane(2+) (DAP(2+)), and the simple monovalent cation Na(+). This work compares the specific structures of the polyamine- and Na-DNA systems and how they are affected by counterion interactions. It also describes sequence-specific hydration and interaction of the cations with DNA. The local DNA structure is dependent on the nature of the counterion. Even the very similar polyamines, Put(2+) and DAP(2+), show clear differences in binding to DNA and in effect on hydration and local structure. Generally, the polyamines disorder the hydration of the DNA around their binding sites whereas Na(+) being bound to DNA attracts and organizes water in its vicinity. Cation binding at the selected sites in the minor and in the major groove is compared for the different polyamines and Na(+). We conclude that the synthetic polyamine (DAP(2+)) binds specifically to several structural and sequence-specific motifs on B-DNA, unlike the natural polyamines, Spd(3+) and Put(2+). This specificity of DAP(2+) compared to the more dynamic behavior of Spd(3+) and Put(2+) may explain why the latter polyamines are naturally occurring in cells.     

In vitro effects of bovine dialyzable leukocyte extract (bDLE) in cancer cells

BACKGROUND: Bovine dialyzable leukocyte extract (bDLE) is a dialyzate of a heterogeneous mixture of low molecular weight substances released from disintegrated blood leukocytes or lymphoid tissue obtained from homogenized bovine spleen. The purpose of this study was to determine if bDLE had cytotoxic effects and modulated apoptosis gene expression in breast cancer cells. METHODS: The MCF-7, BT-474, MDA-MB-453, A-427, Calu-1, U937 and L5178Y cancer cell lines and PBMC human cells were treated with bDLE (0-0.66 U/mL) for 72 h. The bDLE effect on cell growth proliferation was evaluated by MTT assay, and the MCF-7 was evaluated by ethidium bromide-acridine orange staining; total DNA was evaluated for DNA fragmentation, and total RNA was isolated for p53, bag-1, c-myc, bim, bax, bcl-2 and bad mRNA expression. RESULTS: The bDLE had dose-dependent cytotoxic effects and demonstrated an IC50 at a dosage of 0.06 U/mL (P<0.05). The bDLE did not affect the viability of normal human PBMC. The bDLE induced DNA fragmentation at doses of 0.06 and 0.13 U/mL in MCF-7 breast cancer cells. The bDLE induced cytotoxic effects and suppressed the p53, bag-1, c-myc, bax, bcl-2, and bad mRNA expression that influences apoptosis in MCF-7 breast cancer cells. Bim mRNA expression was not detected. DISCUSSION: This may open up interesting prospects for the treatment of human breast cancer.     

Estrogen induces c-myc gene expression via an upstream enhancer activated by the estrogen receptor and the AP-1 transcription factor

c-myc oncogene is implicated in tumorigenesis of many cancers, including breast cancer. Although c-myc is a well-known estrogen-induced gene, its promoter has no estrogen-response element, and the underlying mechanism by which estrogen induces its expression remains obscure. Recent genome-wide studies by us and others suggested that distant elements may mediate estrogen induction of gene expression. In this study, we investigated the molecular mechanism by which estrogen induces c-myc expression with a focus on these distal elements. Estrogen rapidly induced c-myc expression in estrogen receptor (ER)-positive breast cancer cells. Although estrogen had little effect on c-myc proximal promoter activity, it did stimulate the activity of a luciferase reporter containing a distal 67-kb enhancer. Estrogen induction of this luciferase reporter was dependent upon both a half-estrogen response element and an activator protein 1 (AP-1) site within this enhancer, which are conserved across 11 different mammalian species. Small interfering RNA experiments and chromatin immunoprecipitation assays demonstrated the necessity of ER and AP-1 cross talk for estrogen to induce c-myc expression. TAM67, the AP-1 dominant negative, partially inhibited estrogen induction of c-myc expression and suppressed estrogen-induced cell cycle progression. Together, these results demonstrate a novel pathway of estrogen regulation of gene expression by cooperation between ER and AP-1 at the distal enhancer element and that AP-1 is involved in estrogen induction of the c-myc oncogene. These results solve the long-standing question in the field of endocrinology of how estrogen induces c-myc expression.     

Silencing of human c-myc oncogene expression by poly-DNP-RNA

Deregulation of c-myc oncogene expression drives the progression of many different types of cancer. Recent experimental data suggest that even brief inhibition of c-myc expression may be sufficient to permanently stop tumor growth and induce regression of tumors. Previous efforts in developing an inhibitor to silence the c-myc gene were hampered by low efficacy and lack of sequence specificity. Here, we report the synthesis of an antisense RNA inhibitor based on a new 21-nt sequence on a poly- DNP-RNA platform that can specifically inhibit cancer cell growth by silencing c-myc gene expression. Both c-myc mRNA and protein levels were significantly decreased in MCF-7 cells following treatment with this antisense DNP-RNA inhibitor. The control compounds with sense or mismatched sequence were inactive. When c-myc transgenic mice were each treated with a single dose of the antisense RNA inhibitor, in vivo silencing of c-myc gene expression was observed for up to 72 hours by real-time RT-PCR. Similar treatment of c-myc transgenic mice with unmodified (native) homologous small interfering RNA (siRNA) had no effect on the mRNA concentration of the c-myc gene. Injection of this short antisense poly-DNP-RNA into mice did not induce the synthesis of DNP-binding immunoglobulins in the host. The observed in vivo gene silencing by this antisense RNA inhibitor suggests its possible use as a therapeutic agent for cancers involving the deregulation of c-myc gene expression.     

Differential regulation of c-myc by progestins and antiestrogens in T-47D human breast cancer cells.

In order to investigate further the mechanisms associated with growth inhibition of human breast cancer cells by progestins and nonsteroidal antiestrogens, their effect on c-myc gene expression in T-47D-5 and T-47D cells has been investigated. The c-myc mRNA levels were differentially regulated by the synthetic progestin, medroxyprogesterone acetate and the nonsteroidal antiestrogen, monohydroxytamoxifen, in both cell lines. Antiestrogen treatment caused a persistent decrease in c-myc mRNA levels while the progestin caused a more complex response. Initially c-myc mRNA levels increased approx. 2-fold, this was followed by a decrease and then partial recovery. The end result, however, of each of these treatments is decreased cell number.     

Monovalent cation effects on intermolecular purine-purine-pyrimidine triple-helix formation.

The binding of a 19-mer guanosine-rich oligodeoxyribonucleotide, TG3TG4TG4TG3T (ODN 1), to a complementary polypurine DNA target was investigated by DNase I footprinting and restriction endonuclease protection assays. Monovalent cations inhibited intermolecular purine-purine-pyrimidine triple-helical DNA formation, with K+ and Rb+ being most effective, followed by NH4+ and Na+. Li+ and Cs+ had little to no effect. Similar results were observed with the G/A-rich oligonucleotide AG3AG4AG4AG3AGCT. Kinetic studies indicated that monovalent cations interfered with oligonucleotide-duplex DNA association but did not significantly promote triplex dissociation. The observed order of monovalent cation inhibition of triplex formation is reminiscent of their effect on tetraplex formation with G/T-rich oligonucleotides. However, using electrophoretic mobility shift assays we found that the oligonucleotide ODN 1 did not appear to form a four-stranded species under conditions promoting tetraplex formation. Taken together, our data suggest that processes other than the self-association of oligonucleotides into tetraplexes might be involved in the inhibitory effect of monovalent cations on purine-pyrimidine-purine triplex formation.     

Oligodeoxyribonucleotide length and sequence effects on intermolecular purine-purine-pyrimidine triple-helix formation.

The binding of guanosine/thymidine-rich oligodeoxyribonucleotides containing various deletions, extensions, and point mutations to polypurine DNA targets was investigated by DNase I footprinting. Intermolecular purine-purine-pyrimidine triple-helical DNA formation was best achieved using oligonucleotides 12 nucleotides in length. Longer oligonucleotides were slightly weaker in binding affinity, whereas shorter oligonucleotides were considerably weaker. Oligonucleotide extensions had a slight effect on triplex formation, while single point mutations located near the oligonucleotide ends had a greater effect. In the cases of extensions and point mutations, changes to the 3' end of the oligonucleotide had a consistently greater effect on triplex formation than changes to the 5' end. Such differences in triplex-forming ability were not caused by an intrinsic property of these oligonucleotides, since the same point mutated oligonucleotides could bind with high affinity to duplex DNAs containing complementary sites. Taken together, our data suggest that there may be an asymmetry involved in the process of purine-motif triplex formation, with interactions between the 3' end of the oligonucleotide and complementary sequences on the target duplex DNA being dominant.     

Turning the Tables: Myc Activates Wnt in Breast Cancer

Previous molecular and genetic data implicate the c-myc gene as a critical downstream effector of the Wnt/TCF pathway in colon cancer. However, the involvement of c-myc in mammary epithelial cell transformation had not been explored. We recently showed that c-Myc induces a profound morphological transformation in human mammary epithelial cells accompanied by anchorage-independent growth. The mechanism of c-Myc transformation was revealed in part through the finding that, in contrast to colon cancer, c-Myc activates the Wnt pathway and endogenous TCF activity by suppressing the Wnt inhibitors DKK1 and SFRP1. Notably, DKK1 and SFRP1 were found to be strongly suppressed in human breast cancer cell lines and their re-expression inhibited the transformed phenotype. We demonstrated that breast cancer cells become dependent on repression of the Wnt inhibitors for cell proliferation, i.e. they have acquired an “oncogene addiction”, suggesting that the Myc-Wnt pathway is an attractive therapeutic target. We propose that a positive feedback loop of c-myc and Wnt signaling operates in breast cancer.