Selective inhibition of restriction endonuclease cleavage by DNA intercalators

The preferred dye binding sites and the microenvironment of known nucleotide sequences within mitochondrial and plasmid pBR322 DNA was probed in a gross fashion with restriction endonucleases. The intercalating dyes, ethidium bromide and propidium iodide, do not inhibit a given restriction endonuclease equally at all of the restriction sites within a DNA molecule. The selective inhibition may be explained, in part, by the potential B to Z conformation transition of DNA flanking the restriction site and by preferred dye binding sites. Propidium iodide was found to be a more potent inhibitor than ethidium bromide and the inhibition is independent of the type of cut made by the enzyme.     

Enhanced transfection efficiency of a systemically delivered tumor-targeting immunolipoplex by inclusion of a pH-sensitive histidylated oligolysine peptide

Successful cancer gene therapy depends on the development of non-toxic, efficient, tumor cell- specific systemic gene delivery systems. Our laboratory has developed a systemically administered, ligand–liposome complex that can effectively and preferentially deliver its therapeutic payload to both primary and metastatic tumors. To further improve the transfection efficiency of this targeting complex, a synthetic pH-sensitive histidylated oligolysine K[K(H)KKK]₅-K(H)KKC (HoKC), designed to aid in endosomal escape and condensation of DNA, was included in the complex. The presence of HoKC increased the in vitro transfection efficiency over that of the original complex. Moreover, no increase in cytotoxicity was observed due to the presence of the HoKC peptide. In a DU145 human prostate cancer xenograft tumor model in athymic nude mice, inclusion of the HoKC peptide did not interfere with the tumor targeting specificity of the i.v. administered ligand/liposome/DNA complex. Most importantly, the level of transgene expression was significantly elevated in the tumors, but not in the normal tissue in those animals receiving the complex incorporating HoKC. The in vivo enhancement of transfection efficiency by this modified gene delivery vehicle could lead to a reduction in the number of administrations required for antitumor efficacy.     

<Emphasis Type="Italic">mab-31</Emphasis> and the TGF-β pathway act in the ray lineage to pattern <Emphasis Type="Italic">C. elegans</Emphasis>male sensory rays

Background  

C. elegans TGF-β-like Sma/Mab signaling pathway regulates both body size and sensory ray patterning. Most of the components in this pathway were initially identified by genetic screens based on the small body phenotype, and many of these mutants display sensory ray patterning defect. At the cellular level, little is known about how and where these components work although ray structural cell has been implicated as one of the targets. Based on the specific ray patterning abnormality, we aim to identify by RNAi approach additional components that function specifically in the ray lineage to elucidate the regulatory role of TGF-β signaling in ray differentiation.     

3'-End conjugates of minimally phosphorothioate-protected oligonucleotides with 1-O-hexadecylglycerol: synthesis and anti-ras activity in radiation-resistant cells

Activation of the ras oncogene has been implicated in many types of human tumors. It has been shown that downmodulation of ras expression can lead to the reversion of the transformed phenotype of these tumor cells. Antisense oligodeoxyribonucleotides (ODNs) can inhibit gene expression by hybridization to complementary mRNA sequences. To minimize toxicity associated with all-phosphorothioated ODNs and improve cellular uptake, we used partially phosphorothioate (PPS)-modified ODNs having an additional hydrophobic tail at the 3'-end (PPS-C(16)). The PPS ODNs are protected against degradation by PS internucleotide linkages at both the 3'- and 5'-ends and additionally stabilized at internal pyrimidine sites, which are the major sites of endonuclease cleavage. Here we show that anti-ras PPS-C(16) ODN retains the high sequence-specificity of PPS ODNs and provides maximal inhibition of Ras p21 synthesis with minimal toxicity even without the use of a cellular uptake enhancer. Moreover, treatment of T24, a radiation-resistant human tumor cell line that carries a mutant ras gene, with anti-ras PPS-C(16) ODN resulted in a reduction in the radiation resistance of the cells in vitro. We also demonstrate that the growth of RS504 (a human c-Ha-ras transformed NIH/3T3 cell line) mouse tumors was significantly inhibited by the combination of intratumoral injection of anti-ras PPS-C(16) ODN and radiation treatment. These findings indicate the potential of this combination of antisense and conventional radiation therapy as a highly effective cancer treatment modality.     

Chemically modified short interfering hybrids (siHYBRIDS): nanoimmunoliposome delivery in vitro and in vivo for RNAi of HER-2

A blunt-ended 19-mer short interfering hybrid (siHybrid) (H) comprised of sense-DNA/antisense-RNA targeting HER-2 mRNA was encapsulated in a liposomal nanoplex with anti-transferrin receptor single-chain antibody fragment (TfRscFv) as the targeting moiety for clinically relevant tumor-specific delivery. In vitro delivery to a human pancreatic cell line (PANC-1) was shown to exhibit sequence-specific inhibition of 48-h cell growth with an IC50 value of 37 nM. The inhibitory potency of this siHybrid was increased (IC50 value of 7.8 nM) using a homologous chemically modified siHybrid (mH) in which the 19-mer sense strand had the following pattern of 2 '-deoxyinosine (dI) and 2 '-O-methylribonucleotide (2 '-OMe) residues: 5'-d(TITIT)-2'OMe(GCGGUGGUU)-d(GICIT). These modifications were intended to favor antisense strand-mediated RNAi while mitigating possible sense strand-mediated off-target effects and RNase H-mediated cleavage of the antisense RNA strand. The presently reported immunoliposomal delivery system was successfully used in vivo to inhibit HER-2 expression, and thus induce apoptosis in human breast carcinoma tumors (MDA-MB-435) in mice upon repeated i.v. treatment at a dose of 3 mg/kg of H or mH. The in vivo potency of modified siHybrid mH appeared to be qualitatively greater than that of H, as was the case in vitro.     

Tp53 gene therapy: a key to modulating resistance to anticancer therapies?

Abnormalities in the p53 tumor suppressor have been identified in over 60% of human cancers. The status of p53 within tumor cells has been proposed to be one of the major determinants of the response to anticancer therapies. In this review we examine the relationship between functional p53 and sensitivity, or resistance, to chemotherapy and radiotherapy. We also discuss the potential of current gene-therapy approaches to restore functional p53 to tumors as a means of modulating the effects of radiation and chemotherapy.     

A tumor-targeted nanodelivery system to improve early MRI detection of cancer

The development of improvements in magnetic resonance imaging (MRI) that would enhance sensitivity, leading to earlier detection of cancer and visualization of metastatic disease, is an area of intense exploration. We have devised a tumor-targeting, liposomal nanodelivery platform for use in gene medicine. This systemically administered nanocomplex has been shown to specifically and efficiently deliver both genes and oligonucleotides to primary and metastatic tumor cells, resulting in significant tumor growth inhibition and even tumor regression. Here we examine the effect on MRI of incorporating conventional MRI contrast agent Magnevist into our anti-transferrin receptor single-chain antibody (TfRscFv) liposomal complex. Both in vitro and in an in vivo orthotopic mouse model of pancreatic cancer, we show increased resolution and image intensity with the complexed Magnevist. Using advanced microscopy techniques (scanning electron microscopy and scanning probe microscopy), we also established that the Magnevist is in fact encapsulated by the liposome in the complex and that the complex still retains its nanodimensional size. These results demonstrate that this TfRscFv-liposome-Magnevist nanocomplex has the potential to become a useful tool in early cancer detection.     

High throughput screening to investigate the interaction of stem cells with their extracellular microenvironment

Stem cells in vivo are housed within a functional microenvironment termed the “stem cell niche.” As the niche components can modulate stem cell behaviors like proliferation, migration and differentiation, evaluating these components would be important to determine the most optimal platform for their maintenance or differentiation. In this review, we have discussed methods and technologies that have aided in the development of high throughput screening assays for stem cell research, including enabling technologies such as the well-established multiwell/microwell plates and robotic spotting, and emerging technologies like microfluidics, micro-contact printing and lithography. We also discuss the studies that utilized high throughput screening platform to investigate stem cell response to extracellular matrix, topography, biomaterials and stiffness gradients in the stem cell niche. The combination of the aforementioned techniques could lay the foundation for new perspectives in further development of high throughput technology and stem cell research.     

Tumor-targeting,systemically delivered antisense HER-2 chemosensitizes human breast cancer xenografts irrespective of HER-2 levels

BACKGROUND: The failure to respond to chemotherapy is a major obstacle in the successful treatment of breast cancer. We have previously shown that anti-HER-2 antisense oligonucleotide (AS HER-2 ODN) treatment was able to sensitize breast cancer cells to various chemotherapeutic agents in vitro irrespective of their HER-2 status, indicating that the use of AS HER-2 ODN therapy for breast cancer is not limited to tumors overexpressing the protein. One of the main drawbacks to the use of antisense therapy in the clinical setting is the lack of an efficient, tumor-targeting, systemic delivery method. We have developed a tumor-specific, ligand-targeting, cationic liposome delivery system designed for systemic gene therapy of cancer. In this study we employ this ligand-liposome strategy to enhance the delivery of the AS Her-2 ODN to breast cancer cells, including those that do not overexpress HER-2, in vitro and in vivo. MATERIALS AND METHODS: A cationic liposome complex that includes folate as the targeting ligand was designed and optimized for more efficient delivery of AS HER-2 ODN to breast tumors cells in vitro, and more significantly, for systemic delivery with tumor-specific targeting in vivo. Human breast cancer cell line MDA-MB-435, which does not overexpress HER-2, was used to compare the degree of chemosensitization to the taxanes of AS HER-2 ODN delivered via the optimized folate-liposome versuscommercial Lipofectin. MDA-MB-435 xenograft tumors were also used to evaluate the anti-tumor effect of the combination of systemically delivered folate-liposome-AS HER-2 ODN and docetaxel (Taxotere). RESULTS: The optimized folate-liposome-AS HER-2 ODN complex significantly increases the response of breast tumor cell lines to conventional chemotherapeutic agents in vitro as compared to AS HER-2 delivered via an unliganded commercially available reagent, Lipofectin. In vivo, the folate-liposome-AS HER-2 ODN complex has prolonged stability in blood and increased uptake in tumors. More significantly, the combination of intravenously administered ligand-liposome-AS HER-2 ODN and docetaxel resulted in a marked inhibition of xenograft growth in an aggressive breast cancer model that does not overexpress HER-2, even after treatment ended. CONCLUSIONS: Although there are other reports of liposomal delivery of AS ODNs, this is the first report of in vivo efficacy against human cancer cells using a tumor-targeting liposome delivery system for systemic AS therapy. Moreover, the increased stability in circulation and anti-tumor efficacy observed were obtained without the need for continuous intravenous infusion. HER-2 is an integral component within a network of cell growth pathways that can affect many different types of tumors where HER-2 may be a contributing factor, such as ovarian, esophageal, and GI malignancies including colon and pancreatic cancers. Therefore, the effectiveness of this therapy with xenograft tumors that do not overexpress HER-2 has the potential to expand the clinical usefulness of this efficacious form of therapy.     

Association of interferon-gamma induced growth inhibition and modulation of epidermal growth factor receptor gene expression in squamous cell carcinoma cell lines

Interferons (IFN) possess the ability to inhibit proliferation of certain transformed cell lines. Down modulation of the abnormal expression of certain oncogenes by IFN has been associated with phenotypic reversion of src, myc, or ras transformed cells. It has already been shown that some squamous cell carcinoma (SCCa) cell lines express elevated levels of the epidermal growth factor receptor (EGFR). Previously, in A431, an SCCa cell line, it was shown that IFN-gamma-induced growth inhibition was associated with both enhanced expression of EGFR and terminal differentiation. This study examines the effect of IFN-beta or IFN-gamma on five additional cervical SCCa cell lines. One cell line was shown to have amplification of the EGFR gene. An IFN-gamma induced antiproliferative response, observed in four of the five cell lines, was associated with increased expression of EGFR mRNA and induction of the IFN-inducible genes, HLA-A3 class I antigen and 2-5 oligoadenylate synthetase. These data suggest that the increased expression of the EGFR gene in a particular SCCa may predict response to IFN-gamma.