Characteristics of Oligonucleotide Uptake in Human Keratinocyte Cultures

Oligodeoxyribonucleotides have the potential to interfere selectivity with cellular protein synthesis by sequence-specific hybridization to DNA or RNA molecules. We have, investigated the properties of uptake and intracellular localization of fluorescently labeled oligonucleotides in cultured human keratinocytes using confocal laser scanning microscopy. Unlike many other cell types studied, keratinocytes can internalize oligonucleotides without apparent sequestration in endosomes or cell surface accumulation. Uptake is primarily nuclear and unaltered by sodium azide, monensin, or chloroquin pretreatment. We have verified our results with two different fluorophores, fluorescein and Bodipy, and found similar uptake and distribution patterns in both live and fixed cell populations. Surprisingly, we have found uptake to be heterogeneous within a population, with 15-30% of cells internalizing the oligonucleotides. This percentage is drastically increased to roughly 80% at cell population margins, and after release from M phase arrest. These results on uptake and intracellular localization suggest that keratinocytes may have increased sensitivity as target cells for oligonucleotide based gene regulation strategies

Impact of Enzalutamide on Skeletal Related Events (Sres), Pain and Quality of Life (Qol) in the Prevail Trial

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Insoluble γ-Tubulin–Containing Structures Are Anchored to the Apical Network of Intermediate Filaments in Polarized Caco-2 Epithelial Cells

We have previously shown that a thin (∼1 μm) layer of intermediate filaments located beneath the apical membrane of a variety of simple epithelial cells participates in the organization of apical microfilaments and microtubules. Here, I confirmed the apical distribution of γ-tubulin–containing structures (potential microtubule-organizing centers) in CACO-2 cells and demonstrated perfect colocalization of centrosomes and nearly 50% of noncentrosomal γ-tubulin with apical intermediate filaments, but not with apical F-actin. Furthermore, the antisense-oligonucleotide–mediated downregulation of cytokeratin 19, using two different antisense sequences, was more efficient than anticytoskeletal agents to delocalize centrosomes. Electron microscopy colocalization suggests that binding occurs at the outer boundary of the pericentriolar material. Type I cytokeratins 18 and 19 present in these cells specifically coimmunoprecipitated in multi-protein fragments of the cytoskeleton with γ-tubulin. The size and shape of the fragments, visualized at the EM level, indicate that physical trapping is an unlikely explanation for this result. Drastic changes in the extraction protocol did not affect coimmunoprecipitation. These results from three independent techniques, indicate that insoluble γ-tubulin–containing structures are attached to apical intermediate filaments

Tumor inhibition by genomically integrated inducible RNAi-cassettes

RNA interference (RNAi) has emerged as a powerful tool to induce loss-of-function phenotypes by post-transcriptional silencing of gene expression. In this study we wondered whether inducible RNAi-cassettes integrated into cellular DNA possess the power to trigger neoplastic growth. For this purpose inducible RNAi vectors containing tetracycline (Tet)-responsive derivatives of the H1 promoter for the conditional expression of short hairpin RNA (shRNA) were used to target human polo-like kinase 1 (Plk1), which is overexpressed in a broad spectrum of human tumors. In the absence of doxycycline (Dox) HeLa clones expressing TetR, that carry the RNAi-cassette stably integrated, exhibited no significant alteration in Plk1 expression levels. In contrast, exposure to Dox led to marked downregulation of Plk1 mRNA to 3% and Plk1 protein to 14% in cell culture compared to mismatch shRNA/Plk1-expressing cells. As a result of Plk1 depletion cell proliferation decreased to 17%. Furthermore, for harnessing RNAi for silencing disease-related genes in vivo we transplanted inducible RNAi-HeLa cells onto nude mice. After administration of Dox knockdown of Plk1 expression was observed correlating to a significant inhibition of tumor growth. Taken together, our data revealed that genomically integrated RNAi-elements are suitable to hamper tumor growth by conditional expression of shRNA

In vitro selection of oligonucleotides that bind double-stranded DNA in the presence of triplex-stabilizing agents

A SELEX approach has been developed in order to select oligonucleotides that bind double-stranded DNA in the presence of a triplex-stabilizing agent, and was applied to a target sequence containing an oligopurine–oligopyrimidine stretch. After only seven rounds of selection, the process led to the identification of oligonucleotides that were able to form triple helices within the antiparallel motif. Inspection of the selected sequences revealed that, contrary to GC base pair which were always recognized by guanines, recognition of AT base pair could be achieved by either adenine or thymine, depending on the sequence context. While thymines are strongly preferred for several positions, some others can accommodate the presence of adenines. These results contribute to set the rules for designing oligonucleotides that form stable triple helices in the presence of triplex-stabilizing agents at physiological pH. They set the basis for further experiments regarding extension of potential target sequences for triple-helix formation or recognition of ligand–DNA complexes

Effect of Visceral Disease Site on Outcomes in Patients With Metastatic Castration-resistant Prostate Cancer Treated With Enzalutamide in the PREVAIL Trial.

Background The Multinational Phase 3, Randomized, Double-Blind, Placebo-Controlled Efficacy and Safety Study of Oral MDV3100 in Chemotherapy-Naive Patients With Progressive Metastatic Prostate Cancer Who Have Failed Androgen Deprivation Therapy (PREVAIL) trial was unique as it included patients with visceral disease. This analysis was designed to describe outcomes for the subgroup of men from PREVAIL with specific sites of visceral disease to help clinicians understand how these patients responded to enzalutamide prior to chemotherapy.Patients and methods Prespecified analyses examined the coprimary endpoints of radiographic progression-free survival (rPFS) and overall survival (OS) only. All other efficacy analyses were post hoc. The visceral subgroup was divided into liver or lung subsets. Patients with both liver and lung metastases were included in the liver subset.Results Of the 1717 patients in PREVAIL, 204 (12%) had visceral metastases at screening (liver only or liver/lung metastases, n = 74; lung only metastases, n = 130). In patients with liver metastases, enzalutamide was associated with an improvement in rPFS (hazard ratio [HR], 0.44; 95% confidence interval [CI], 0.22-0.90) but not OS (HR, 1.04; 95% CI, 0.57-1.87). In patients with lung metastases only, the HR for rPFS (0.14; 95% CI, 0.06-0.36) and the HR for OS (0.59; 95% CI, 0.33-1.06) favored enzalutamide over placebo. Patients with liver metastases had worse outcomes than those with lung metastases, regardless of treatment. Enzalutamide was well tolerated in patients with visceral disease.Conclusions Enzalutamide is an active first-line treatment option for men with asymptomatic or mildly symptomatic chemotherapy-naive metastatic castration-resistant prostate cancer and visceral disease. Patients with lung-only disease fared better than patients with liver disease, regardless of treatment

Hyperstable U1snRNA complementary to the K-ras transcripts induces cell death in pancreatic cancer cells

One of the critical steps that governs the inhibitory effect of antisense RNA on target gene expression is the association of the antisense RNA with the target RNA molecules. However, until now, no systematic method has been available to select the suitable parts of a gene as antisense targets. In this study, we utilised U1 small nuclear RNA (snRNA) that binds physiologically to the 5′ splice site (5′ss) of pre-mRNA, to develop a novel vector system that permits imposed binding of antisense RNA to its target. The 5′ free end of U1snRNA was replaced with the antisense sequence against the K-ras gene to generate a hyperstable U1snRNA, whose binding stability to 5′ss of the K-ras transcript is ten-fold higher than that of wild-type U1snRNA. The efficacy of such hyperstable U1snRNA was examined by transducing the expression plasmids into human pancreatic cancer cell lines. This revealed that two of the hyperstable U1snRNAs induced cell death after gene transduction, and significantly reduced the number of G418-resistant colonies to less than 10% of the controls. Furthermore, hyperstable U1snRNA suppressed intraperitoneal dissemination of pancreatic cancer cells in vivo. Hyperstable U1snRNA might be a novel approach to express effective antisense RNA in target cells

Telomeric DNA induces apoptosis and senescence of human breast carcinoma cells

INTRODUCTION: Cancer is a leading cause of death in Americans. We have identified an inducible cancer avoidance mechanism in cells that reduces mutation rate, reduces and delays carcinogenesis after carcinogen exposure, and induces apoptosis and/or senescence of already transformed cells by simultaneously activating multiple overlapping and redundant DNA damage response pathways. METHODS: The human breast carcinoma cell line MCF-7, the adriamycin-resistant MCF-7 (Adr/MCF-7) cell line, as well as normal human mammary epithelial (NME) cells were treated with DNA oligonucleotides homologous to the telomere 3' overhang (T-oligos). SCID mice received intravenous injections of MCF-7 cells followed by intravenous administration of T-oligos. RESULTS: Acting through ataxia telangiectasia mutated (ATM) and its downstream effectors, T-oligos induced apoptosis and senescence of MCF-7 cells but not NME cells, in which these signaling pathways were induced to a far lesser extent. In MCF-7 cells, experimental telomere loop disruption caused identical responses, consistent with the hypothesis that T-oligos act by mimicking telomere overhang exposure. In vivo, T-oligos greatly prolonged survival of SCID mice following intravenous injection of human breast carcinoma cells. CONCLUSION: By inducing DNA damage-like responses in MCF-7 cells, T-oligos provide insight into innate cancer avoidance mechanisms and may offer a novel approach to treatment of breast cancer and other malignancies