Novel genetic aberrations in breast phyllodes tumours: comparison between prognostically distinct groups.

Phyllodes tumours of the breast are uncommon fibroepithelial neoplasms which pose management challenges due to difficulties in accurate prediction of clinical behaviour, as histological assessment has its limitations. Molecular studies have improved the understanding of these rare tumours but such findings are scant. We aimed to investigate genetic aberrations in phyllodes tumours stratified according to clinical behaviour, to identify potential genes contributing to disease progression. Twenty phyllodes tumours were separated into prognostically distinct categories depending on whether they had recurred/metastasized within the follow-up period. DNA extracted from FFPE materials was subjected to Affymetrix OncoScan™ FFPE Express molecular inversion probe microarray platform for analysis of copy number changes and mutational status. Results were cross validated with Sanger sequencing, FISH and immunohistochemistry. A higher number of chromosomal aberrations were observed in cases which recurred/metastasized, with median events of 19 compared to 3.5 in cases which did not recur/metastasize. High-level amplification and homozygous deletions were detected exclusively in the former group. Regions of high-level amplification included MDM4 (1q32.1), RAF1 (3p25), EGFR (7p12) and PDZD2 (5p13.3). EGFR amplification was confirmed on FISH and accompanied by intense EGFR immunostaining. Regions of homozygous deletion included CDKN2A (9p21) and MACROD2 (20p12.1). Homozygous deletion of 9p21 which involved CDKN2A was accompanied by loss of protein expression. No mutations were identified in all samples. These findings provide insights into identifying target genes and pathways exploited by phyllodes tumours, which would aid future development of individualised therapy.

Comparison of d-g3139 and its enantiomer L-g3139 in melanoma cells demonstrates minimal in vitro but dramatic in vivo chiral dependency.

G3139 (Genasense), an 18mer phosphorothioate antisense oligonucleotide targeted to the initiation codon region of the Bcl-2 messenger RNA (mRNA), downregulates Bcl-2 protein and mRNA expression in many cell lines. However, both the in vitro and in vivo mechanisms of action of G3139 are still uncertain. The isosequential L-deoxyribose enantiomer L-G3139, which does not downregulate Bcl-2 expression, was synthesized to study the role of the Bcl-2 protein in melanoma cells. Both D-G3139 and L-G3139 bind nonspecifically to basic fibroblast growth factor with approximately the same K(c), and cause highly effective inhibition of net formation in 518A2 melanoma cells on Matrigel. The uptakes of D-G3139 and L-G3139 in melanoma cells were also similar. However, unlike D-G3139, L-G3139 does not produce poly ADP-ribose polymerase-1 and procaspase-3 cleavage at 9.5 h after the initiation of the transfection, but can activate the intrinsic pathway of apoptosis at approximately 48 h. Furthermore, treatment of A375 melanoma human xenografts in severe combined immunodeficiency (SCID) mice demonstrates that tumor growth is not inhibited by L-G3139, whereas D-G3139 significantly inhibits the rate of tumor growth. Furthermore, the immunostimulatory properties of L-G3139 appear to be nil, which differs dramatically from those of D-G3139. In conclusion, profound differences exist between D-G3139 and L-G3139 in vivo despite their similarities in vitro.

Phosphorothioate oligonucleotides reduce mitochondrial outer membrane permeability to ADP.

G3139, an antisense Bcl-2 phosphorothioate oligodeoxyribonucleotide, induces apoptosis in melanoma and other cancer cells. This apoptosis happens before and in the absence of the downregulation of Bcl-2 and thus seems to be Bcl-2-independent. Binding of G3139 to mitochondria and its ability to close voltage-dependent anion-selective channel (VDAC) have led to the hypothesis that G3139 acts, in part, by interacting with VDAC channels in the mitochondrial outer membrane (21). In this study, we demonstrate that G3139 is able to reduce the mitochondrial outer membrane permeability to ADP by a factor of 6 or 7 with a K(i) between 0.2 and 0.5 microM. Because VDAC is responsible for this permeability, this result strengthens the aforesaid hypothesis. Other mitochondrial respiration components are not affected by [G3139] up to 1 microM. Higher levels begin to inhibit respiration rates, decrease light scattering and increase uncoupled respiration. These results agree with accumulating evidence that VDAC closure favors cytochrome c release. The speed of this effect (within 10 min) places it early in the apoptotic cascade with cytochrome c release occurring at later times. Other phosphorothioate oligonucleotides are also able to induce VDAC closure, and there is some length dependence. The phosphorothioate linkages are required to induce the reduction of outer membrane permeability. At levels below 1 microM, phosphorothioate oligonucleotides are the first specific tools to restrict mitochondrial outer membrane permeability.

A pharmacologic target of G3139 in melanoma cells may be the mitochondrial VDAC.

G3139, an 18-mer phosphorothioate antisense oligonucleotide targeted to the initiation codon region of the Bcl-2 mRNA, can induce caspase-dependent apoptosis via the intrinsic mitochondrial pathway in 518A2 and other melanoma cells. G3139-mediated apoptosis appears to be independent of its ability to down-regulate the expression of Bcl-2 protein, because the release of mitochondrial cytochrome c precedes in time the down-regulation of Bcl-2 protein expression. In this study, we demonstrate the ability of G3139 and other phosphorothioate oligonucleotides to bind directly to mitochondria isolated from 518A2 cells. Furthermore, we show that this interaction leads to the release of cytochrome c in the absence of a mitochondrial membrane permeability transition. Our data further demonstrate that there is an interaction between G3139 and VDAC, a protein that can facilitate the physiologic exchange of ATP and ADP across the outer mitochondrial membrane. Evidence from the electrophysiologic evaluation of VDAC channels reconstituted into phospholipid membranes demonstrates that G3139 is capable of producing greatly diminished channel conductance, indicating a closed state of the VDAC. This effect is oligomer length-dependent, and the ability of phosphorothioate homopolymers of thymidine of variable lengths to cause the release of cytochrome c from isolated mitochondria of 518A2 melanoma cells can be correlated with their ability to interact with VDAC. Because it has been suggested that the closure of VDAC leads to the opening of another outer mitochondrial membrane channel through which cytochrome c can transit, thus initiating apoptosis, it appears that VDAC may be an important pharmacologic target of G3139.

Phosphorothioate oligodeoxynucleotides and G3139 induce apoptosis in 518A2 melanoma cells.

In a previous study, we showed that G3139, an antisense phosphorothioate oligonucleotide that down-regulates the expression of Bcl-2 protein, did not cause chemosensitization of 518A2 melanoma cells. In this work, we show that G3139, and the 2-base mismatch, G4126, can initiate apoptosis in this and other melanoma cell lines as shown by increased cell surface Annexin V expression, typical nuclear phenotypic changes as assessed by 4',6-diamidino-2-phenylindole staining, activation of caspase-3 (but not caspase-8) and Bid, appearance of DEVDase (but not IETDase) activity, and cleavage of poly(ADP-ribose)-polymerase 1. Depolarization of the mitochondrial membrane occurs as a relatively late event. All of these processes seem to be substantially, but perhaps not totally, Bcl-2 independent as shown by experiments employing an anti-Bcl-2 small interfering RNA, which as shown previously down-regulated Bcl-2 protein expression but did not produce apoptosis or chemosensitization in melanoma cells. In fact, these G3139-induced molecular events were not dramatically altered in cells that forcibly overexpressed high levels of Bcl-2 protein. Addition of irreversible caspase inhibitors (e.g., the pan-caspase inhibitor zVAD-fmk) to G3139-treated cells almost completely blocked cytotoxicity. Examination of the time course of the appearance of caspase-3 and cleaved poly(ADP-ribose)-polymerase 1 showed that this could be correlated with the release of cytochrome c from the mitochondria, an event that begins only approximately 4 hours after the end of the oligonucleotide/LipofectAMINE 2000 5-hour transfection period. Thus, both G3139 and cytotoxic chemotherapy activate the intrinsic pathway of apoptosis in these cells, although Bcl-2 expression does not seem to contribute strongly to chemoresistance. These findings suggest that the attainment of G3139-induced chemosensitization in these cells will be difficult.

Induction of apoptosis by G3139 in melanoma cells.

G3139 is an 18mer phosphorothioate oligonucleotide targeted to the initiation codon region of the Bcl-2 mRNA. Because of the ability of this antisense construct to downregulate the expression of Bcl-2 mRNA and protein, it has entered phase III clinical trials in a number of human cancers, including advanced melanoma. However, the actual mechanism of this agent is far from certain. In this work, we demonstrate that G3139 induces the relatively rapid release of cytochrome c into the cytoplasm of treated 518A2 melanoma cells. This release activates the intrinsic pathway of apoptosis, eventually leading to a mitochondrial permeability transition and cell death. By employing an siRNA strategy, we also show that this entire process appears to be Bcl-2 independent, as downregulation of Bcl-2 protein expression does not alter the induction of apoptosis by G3139. Furthermore, forced overexpression of Bcl-2 protein contributes relatively little to chemoresistance in this cell line. While these results may or may not be reflective of the in vivo situation, the value of Bcl-2 as a target in advanced melanoma must at least be questioned.

Relative Bcl-2 independence of drug-induced cytotoxicity and resistance in 518A2 melanoma cells.

PURPOSE: Inhibition of the function of Bcl-2 protein has been postulated to sensitize cells to cytotoxic chemotherapy. G3139 (Genasense) is a phosphorothioate anti-Bcl-2 antisense oligonucleotide, but its mechanism of action is uncertain. The aim of the present work is to investigate inhibition of Bcl-2 expression in 518A2 melanoma cells, the cell line on which recent phase II and phase III clinical trials employing this agent were based. EXPERIMENTAL DESIGN: We down-regulated the expression of Bcl-2 protein by two different strategies in these cells: one employing G3139 and controls, and the other using a small interfering RNA approach. Cell viability after treatment with oligonucleotides or small interfering RNA and cytotoxic agents including gemcitibine, DDP, docetaxel, and thapsigargin was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. A 518A2 melanoma cell line stably overexpressing Bcl-2 protein was constructed and treated with either these cytotoxic agents or G3139. RESULTS: The cytotoxic effects of either G3139 or small interfering RNA treatment of 518A2 melanoma cells are Bcl-2 independent. In addition, in the Bcl-2-overexpressing cells, only a modest increment in chemoresistance was observed, and treatment with G3139 not only did not down-regulate Bcl-2 expression but produced essentially identical toxicity as was observed in the wild-type or mock-transfected cells. CONCLUSIONS: Our results suggest that the mechanism whereby G3139 produces drug-induced cytotoxicity in the 518A2 melanoma line is not dependent on levels of Bcl-2. These findings emphasize the nonsequence specific effects of this phosphorothioate oligonucleotide and call into question the validity of Bcl-2 as a target in this cell line.

Changes in gene expression induced by phosphorothioate oligodeoxynucleotides (including G3139) in PC3 prostate carcinoma cells are recapitulated at least in part by treatment with interferon-beta and -gamma.

PURPOSE: G3139 is an antisense bcl-2 phosphorothioate oligodeoxyribonucleotide that is currently being evaluated in Phase III clinical trials in several human cancers. The aim of the present work was to further identify the apparent non-bcl-2-dependent mechanism of this action of this compound in PC3 prostate cancer cells. EXPERIMENTAL DESIGN: We performed Affymetrix U95A oligonucleotide microarray studies on mRNA isolated from cells treated with G3139 and related oligonucleotides. RESULTS: Hierarchical clustering revealed the presence of a set of genes of which the expression was elevated on both 1 and 3 days after oligonucleotide treatment. Significantly, the persistence of expression of the up-regulation of these genes, many of which are members of the IFN cascade, was greater for G3139 than for any other oligomer evaluated. Furthermore, many of the genes with the greatest up-regulation of expression are also those of which the expression is up-regulated after treatment of cells with IFNs. Treatment of PC3 cells with either IFN-beta or -gamma recapitulated some of the aspects of the molecular and phenotypic changes observed after treatment with a G3139/Lipofectin complex. These include down-regulation of bcl-2 protein expression itself, down-regulation of protein kinase C alpha protein expression (but not that of other protein kinase C isoforms), alteration in p21/Waf1/Cip1 protein expression, up-regulation of MHC-I cell surface expression, and profound suppression of cell growth in the absence of a notable increase in cellular apoptosis. However, G3139 (when complexed with Lipofectin) did not induce the up-regulation of expression of either type I or type II IFNs, nor could IFNs be found in conditioned media from treated cells. CONCLUSIONS: Oligonucleotide microarray experiments demonstrated that G3139 could induce elements of the IFN cascade in PC3 cells in vitro. In addition, the cellular phenotype obtained after treatment with exogenous IFN could, at least in part, recapitulate that obtained after G3139 treatment. Nevertheless, the oligonucleotide microarray experiments we performed also demonstrated that there are extremely large qualitative and quantitative differences between the two treatments.

Antisense RNA down-regulation of bcl-2 expression in DU145 prostate cancer cells does not diminish the cytostatic effects of G3139 (Oblimersen).

PURPOSE: Inhibition of the function of the bcl-2 protein has been postulated to sensitize cells to cytotoxic chemotherapy, and thus provides an attractive target for investigative therapies. G3139, a phosphorothioate antisense oligonucleotide targeted to the initiation codon region of the bcl-2 mRNA, is currently being evaluated in several Phase II and Phase III clinical trials. However, the mechanism of action of this molecule appears to depend on a combination of antisense plus nonantisense events. Indeed, the very idea that bcl-2 is a critical target is, at least in part, an extrapolation from experiments in which intracellular bcl-2 protein concentrations have been dramatically increased, yielding chemoresistant cells. EXPERIMENTAL DESIGN: In this work, we down-regulated the expression of bcl-2 protein by 80-90% by two different antisense RNA strategies (antisense RNA and small interfering RNA) in DU145 prostate cancer cells. RESULTS: Even after down-regulation of bcl-2 protein expression by either one of these strategies, the cellular phenotype induced by subsequent G3139 treatment (inhibition of cellular growth and the generation of reactive oxygen species) was essentially identical to that induced in mock-infected or wild-type DU145 cells in which bcl-2 protein expression had not been down-regulated previously. CONCLUSIONS: These results strongly suggest that bcl-2 expression in DU145 cells is not strongly associated with the prolife phenotype and that the mechanism by which G3139 produces its cytostatic effects in this cell line is bcl-2 independent.

Single-cell measurements of polyamidoamine dendrimer binding.

The binding of fluorescein-labeled polyamidoamine dendritic polymers (dendrimers) to the surface of individual fibroblast cells has been quantitatively measured using confocal fluorescence microscopy. Because the binding of these polymers is not strong, significant fluorescence from polymers in solution complicates the measurement, even using confocal optics. To overcome this difficulty, we have developed a new method employing a cell-entrapped marker dye to determine the extracellular volume in each voxel. This allows for a correct subtraction of fluorescence due to polymers in solution. We have found that, as expected, the larger dendrimers bound more tightly than the smaller generations, which may partially account for their increased efficacy in DNA transfection applications. The binding varied significantly from cell to cell, and an analysis of the cell surface area showed that this variability was not caused simply by variations in cell size.

Protamine-fragment peptides fused to an SV40 nuclear localization signal deliver oligonucleotides that produce antisense effects in prostate and bladder carcinoma cells.

The development of antisense technology has focused on improving methods for oligonucleotide delivery into cells. In the present work, we describe a novel strategy for oligonucleotide delivery based on a bifunctional peptide composed of a C-terminal protamine-fragment that contains a DNA-binding domain and an N-terminal nuclear localization signal sequence derived from the SV40 large-T antigen (The sequences of two of the peptides are R6WGR6-PKKKRKV [s-protamine-NLS] and R4SR6FGR6VWR4-PKKKRKV [l-protamine-NLS]). We demonstrated, by intrinsic fluorescence quenching, that peptides of this class form complexes with oligodeoxynucleotides. To evaluate delivery, we used a 20-mer phosphorothioate oligomer (Isis 3521) targeted to the 3'-untranslated region of the PKC-alpha mRNA and G3139, an 18-mer phosphorothioate targeted to the first six codons of the human bcl-2 open reading frame, and complexed them with either of two peptides (s- or l-protamine-NLS). These peptides bind to and deliver antisense oligonucleotides to the nucleus of T24 bladder and PC3 prostate cancer cells, as demonstrated by confocal microscopy. Furthermore, as shown by Western and Northern blotting, the peptide-oligonucleotide complexes produced excellent downregulation of the expression of the complementary mRNAs, which in turn resulted in downregulation of protein expression. However, under certain circumstances (predominantly in PC3 cells), incubation of the cells with chloroquine was required to produce antisense activity. Using this strategy, PKC-alpha protein and mRNA expression in T24 and PC3 cells and bcl-2 expression in PC3 cells was reduced by approximately 75 +/- 10% at a minimum concentration of oligomer of 0.25 microM, in combination with 12-15 microM peptide. On the basis of our results, we conclude that arginine-rich peptides of this class may be potentially useful delivery vehicles for the cellular delivery of antisense oligonucleotides. This new strategy may have several advantages over other methods of oligonucleotide delivery and may complement already existing lipid-based technologies.