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Biomedical subjects

David R Corey

Publications and source records attributed to David R Corey.

At least 37 records · Page 2Linked to original sources
PubMed2004

Telomerase reverse transcriptase (hTERT) mRNA and telomerase RNA (hTR) as targets for downregulation of telomerase activity.

Telomerase is expressed in cancer cells but not in most normal cells, leading to the hypothesis that telomerase inhibitors may be a powerful approach to cancer therapy. It is possible that telomerase plays roles in the cell other than telomere elongation and that blocking telomerase expression may have consequences that differ from simply blocking the active site through competitive inhibition. Here, we test this hypothesis by comparing the effects of antisense oligonucleotides and small interfering RNAs (siRNAs) that target the telomerase reverse transcriptase (hTERT) mRNA with the effects of oligonucleotides that target the telomerase RNA component (hTR). We find that the use of anti-hTR oligomers is more effective in blocking telomerase expression than strategies that target hTERT mRNA. Anti-hTR compounds are active on addition to cells in the absence of lipid, whereas antisense oligonucleotides are not. The modest inhibition of hTERT expression caused by antisense oligonucleotides or siRNAs does not persist, suggesting development of resistance. These data suggest that strategies for telomerase inhibition that require downregulation of hTERT mRNA may be less straightforward than those that target hTR. In addition, we have not seen evidence for a role for hTERT other than in telomere maintenance.

Breast Neoplasms↗
PubMed2004

Validating bioluminescence imaging as a high-throughput, quantitative modality for assessing tumor burden.

Bioluminescence imaging (BLI) is a highly sensitive tool for visualizing tumors, neoplastic development, metastatic spread, and response to therapy. Although BLI has engendered much excitement due to its apparent simplicity and ease of implementation, few rigorous studies have been presented to validate the measurements. Here, we characterize the nature of bioluminescence output from mice bearing subcutaneous luciferase-expressing tumors over a 4-week period. Following intraperitoneal or direct intratumoral administration of luciferin substrate, there was a highly dynamic kinetic profile of light emission. Although bioluminescence was subject to variability, strong correlations (r >.8, p <.001) between caliper measured tumor volumes and peak light signal, area under light signal curve and light emission at specific time points were determined. Moreover, the profile of tumor growth, as monitored with bioluminescence, closely resembled that for caliper measurements. The study shows that despite the dynamic and variable nature of bioluminescence, where appropriate experimental precautions are taken, single time point BLI may be useful for noninvasive, high-throughput, quantitative assessment of tumor burden.

Animals↗
PubMed2004

Synthesis of oligonucleotide-peptide and oligonucleotide-protein conjugates.

The conjugation of macromolecules offers a rapid and versatile route to improved function. Here, the methods for obtaining disulfide-linked oligonucleotide-peptide and oligonucleotide-protein conjugates are described. These hybrid molecules can be used deliver chemical functionality to specific sequences within deoxyribonucleic acid and ribonucleic acid. The peptides and proteins can also be used to enhance the hybridization properties of the attached oligonucleotides.

Cross-Linking Reagents↗
PubMed2004

Synthesis of peptide nucleic acid-peptide conjugates.

Synthetic oligonucleotides are versatile tools for recognizing ribonucleic acid and deoxyribonucleic acid. This chapter describes methods for enhancing recognition by derivatizing oligonucleotides with either proteins or peptides.

Cross-Linking Reagents↗
PubMed2003

Extending recognition by peptide nucleic acids (PNAs): binding to duplex DNA and inhibition of transcription by tail-clamp PNA-peptide conjugates.

Peptide nucleic acids (PNAs) are a powerful tool for recognition of double-stranded DNA. Strand invasion is most efficient when pyrimidine PNAs are linked to form a bisPNA in which one strand binds by Watson-Crick base pairing while the other binds by Hoogsteen base pairing to the newly formed PNA-DNA duplex. Within many genes, however, polypyrimidine target sequences may not be located in optimal positions relative to transcription factor binding sites, and this deficiency may complicate attempts to identify potent antigene PNAs. To increase the versatility of strand invasion by PNAs, we have synthesized bisPNAs and bisPNA-peptide conjugates containing a mixed base extension of the Watson-Crick polypyrimidine strand. We find that these tail-clamp PNAs (TC-PNAs) bind duplex DNA and inhibit transcription. DNA recognition occurs with single-stranded or TC-bisPNAs and requires attachment of positively charged amino acids. Association rate constants, k(a), for binding to DNA by TC-PNAs are as high as 35000 M(-1) s(-1) and are usually only a fewfold lower than for analogous PNAs that lack mixed base extensions. The ability to bind duplex DNA is not always necessary for inhibition of transcription, possibly because PNAs can bind to accessible DNA within the transcription bubble created by RNA polymerase. These results, together with similar findings independently obtained by Nielsen and colleagues [Bentin, T., Larsen, H. J., and Nielsen, P. E. (2003) Biochemistry 42, 13987-13995], expand the range of sequences within duplex DNA that are accessible to PNAs and suggest that TC-PNA-peptide conjugates are good candidates for further testing as antigene agents.

Binding Sites↗
PubMed2003

Imaging gene expression using oligonucleotides and peptide nucleic acids.

The development of methods for non-invasive, real-time imaging of gene expression would provide powerful tools for biomedical research and medical diagnostics. A broadly applicable strategy for achieving this goal is the use of complementary oligonucleotide probes for recognition of mRNA. The major challenge for molecular imaging is the development of specific and efficient transducers for signaling probe-target interaction. This review summarizes the strengths and limitations of reported molecular approaches for imaging of mRNA expression and discusses the challenges to development of in vivo methods.

Animals↗
PubMed2003

Consequences of telomerase inhibition and combination treatments for the proliferation of cancer cells.

Telomerase is expressed in most types of tumor cells but not in most somatic cells, suggesting that telomerase inhibitors may be a powerful new approach to cancer chemotherapy. Here we explore this hypothesis by treating cultured human tumor cells with a 2'-O-methoxyethyl oligonucleotide that binds the telomerase RNA template and acts as a potent inhibitor. Treatment of DU145 (Rb(-), p53(-)) and LNCaP (Rb(+), p53(+)) cells causes telomeres to shorten and cell proliferation to stop. Decreased cell proliferation in culture is not observed immediately but occurs after several weeks and is accompanied by telomere shortening. Antiproliferative effects are more profound for cells growing in soft agar or in colony formation assays, with 90% reduction in the colony-forming ability of LNCaP cells after less than 2 weeks of exposure to the inhibitor. Decreased growth of DU145 and LNCaP tumors and large reductions in prostate-specific antigen levels are also observed in vivo in xenograft models. Short-term treatment of cells with telomerase inhibitors does not increase the effects of standard antiproliferative agents paclitaxel, doxorubicin, etoposide, cisplatin, or carboplatin. Long-term inhibition and telomere shortening sensitize DU145 cells, but not LNCaP cells, to cisplatin or carboplatin. These results demonstrate that methoxyethyl oligomers directed against the template region of telomerase are potent agents and that significant antiproliferative effects can be observed after 2-3 weeks of treatment. Reduced cell proliferation and tumor growth support the hypothesis that telomerase inhibition can make a useful contribution to chemotherapy and should encourage broad testing of telomerase inhibitors.

Animals↗
PubMed2003

RNA interference in mammalian cells by chemically-modified RNA.

RNA interference (RNAi) is proving to be a robust and versatile technique for controlling gene expression in mammalian cells. To fully realize its potential in vivo, however, it may be necessary to introduce chemical modifications to optimize potency, stability, and pharmacokinetic properties. Here, we test the effects of chemical modifications on RNA stability and inhibition of gene expression. We find that RNA duplexes containing either phosphodiester or varying numbers of phosphorothioate linkages are remarkably stable during prolonged incubations in serum. Treatment of cells with RNA duplexes containing phosphorothioate linkages leads to selective inhibition of gene expression. RNAi also tolerates the introduction of 2'-deoxy-2'-fluorouridine or locked nucleic acid (LNA) nucleotides. Introduction of LNA nucleotides also substantially increases the thermal stability of modified RNA duplexes without compromising the efficiency of RNAi. These results suggest that inhibition of gene expression by RNAi is compatible with a broad spectrum of chemical modifications to the duplex, affording a wide range of useful options for probing the mechanism of RNAi and for improving RNA interference in vivo.

Blotting, Western↗
PubMed2003

Binding of nonphysiological protein and peptide substrates to proteases: differences between urokinase-type plasminogen activator and trypsin and contributions to the evolution of regulated proteolysis.

Understanding the regulation of physiological processes requires detailed knowledge of the recognition of substrates by enzymes. One of the most productive model systems for the study of enzyme-substrate interactions is the serine protease family; however, most studies of protease action have used small substrates that contain an activated, non-natural scissile bond. Because few kinetic or structural studies have used protein substrates, the physiologically relevant target of most proteases, it seems likely that important mechanisms of substrate recognition and processing by proteases have not yet been fully elucidated. Consistent with this hypothesis, we have observed that K(m) values for protein substrates are reduced as much as 200-15000-fold relative to those of analogous peptide substrates. Here we examine the thermodynamic consequences of interactions between proteases and their substrates using staphylococcal nuclease (SNase) and SNase variants as model protein substrates. We have obtained values for enthalpy, entropy, and K(d) for binding of proteins and peptides by the nonspecific protease trypsin and the highly specific protease urokinase-type plasminogen activator (u-PA). To avoid cleavage of substrates during these measurements, we used inactive variants of trypsin and u-PA whose catalytic serine S195 had been replaced by alanine. Differences in the K(d) values for binding of protein and peptide substrates closely approximate the large differences observed in the corresponding K(m) values. Improved binding of protein substrates is due to decreased enthalpy, and this effect is pronounced for the selective protease u-PA. Fundamental differences in recognition of analogous protein and peptide substrates may have influenced the evolution of protease specificity.

Catalytic Domain↗
PubMed2003

Inhibition of telomerase by BIBR 1532 and related analogues.

BIBR 1532 has been reported to be a potent, small molecule inhibitor of human telomerase, suggesting it as a lead for the development of anti-telomerase therapy. We confirm the ability of BIBR 1532 to inhibit telomerase and report the discovery of an equally potent analogue. Importantly, IC(50) values in cell extract are considerably higher than those previously reported using assays for purified enzyme, indicating that substantial improvement may be necessary.

Aminobenzoates↗
PubMed2003

The synaptic complex of RecA protein participates in hybridization and inverse strand exchange reactions.

RecA protein catalyzes strand exchange between homologous single-stranded and double-stranded DNAs. In the presence of ATPgammaS, the post-strand exchange synaptic complex is a stable end product that can be studied. Here we ask whether such complexes can hybridize to or exchange with DNA, 2'-OMe RNA, PNA, or LNA oligonucleotides. Using a gel mobility shift assay, we show that the displaced strand of a 45 bp synaptic complex can hybridize to complementary oligonucleotides with different backbones to form a four-stranded (double D-loop) joint that survives removal of the RecA protein. This hybridization reaction, which confirms the single-stranded character of the displaced strand in a synaptic complex, might initiate recombination-dependent DNA replication if it occurs in vivo. We also show that either strand of the heteroduplex in a 30 bp synaptic complex can be replaced with a homologous DNA oligonucleotide in a strand exchange reaction that is mediated by the RecA filament. Consistent with the important role that deoxyribose plays in strand exchange, oligonucleotides with non-DNA backbones did not participate in this reaction. The hybridization and strand exchange reactions reported here demonstrate that short synaptic complexes are dynamic structures even in the presence of ATPgammaS.

Adenosine Triphosphate↗
PubMed2003

Telomerase inhibitors: a new option for chemotherapy.

Telomerase is expressed in most types of tumors but not in most somatic cells. This observation has led to two hypotheses: (i) telomerase activity is necessary for the proliferation of cancer cells and (ii) telomerase inhibitors are a powerful strategy for cancer chemotherapy. The properties and function of human telomerase are reviewed briefly. The effects of telomerase inhibitors on telomere length and tumor cell proliferation are then described. Much evidence, both pro and con, has accumulated about the value of telomerase as a target for therapy. Resolving the controversy will require a thorough understanding of telomere biology and clinical trials designed around potent inhibitors.

Antineoplastic Agents↗
PubMed2002

Telomerase inhibition, telomere shortening, and decreased cell proliferation by cell permeable 2'-O-methoxyethyl oligonucleotides.

Telomerase is an attractive target for chemotherapy. Testing this hypothesis will require potent inhibitors with favorable pharmacokinetic properties. We report that 2'-methoxyethyl oligonucleotides complementary to the telomerase RNA component diffuse across cell membranes without the need for cationic carrier lipid, inhibit telomerase, and cause telomeres to shorten. The ability of antitelomerase oligomers to enter cells without the need to add lipid will simplify preclinical studies and may suggest advantages for clinical use.

Cell Division↗
PubMed2002

Antisense inhibition of gene expression in cells by oligonucleotides incorporating locked nucleic acids: effect of mRNA target sequence and chimera design.

Use of antisense oligonucleotides is a versatile strategy for achieving control of gene expression. Unfortunately, the interpretation of antisense-induced phenotypes is sometimes difficult, and chemical modifications that improve the potency and specificity of antisense action would be useful. The introduction of locked nucleic acid (LNA) bases into oligonucleotides confers exceptional improvement in binding affinity, up to 10 degrees C per substitution, making LNAs an exciting option for the optimization of antisense efficacy. Here we examine the rules governing antisense gene inhibition within cells by oligonucleotides that contain LNA bases. LNA- containing oligomers were transfected into cells using cationic lipid and accumulated in the nucleus. We tested antisense gene inhibition by LNAs and LNA-DNA chimeras complementary to the 5'-untranslated region, the region surrounding the start codon and the coding region of mRNA, and identified effective antisense agents targeted to each of these locations. Our data suggest that LNA bases can be used to develop antisense oligonucleotides and that their use is a versatile approach for efficiently inhibiting gene expression inside cells.

5' Untranslated Regions↗
PubMed2002

Enhanced strand invasion by peptide nucleic acid-peptide conjugates.

Efficient and selective recognition of DNA by proteins is due to sequence-specific interactions with a target site and nonselective electrostatic interactions that promote the target's rapid location. If synthetic molecules could mimic these functions, they would render a wide range of chromosome sequences accessible to rationally designed probes. Here we describe conjugates between bispeptide nucleic acids (bisPNAs) designed to specifically recognize duplex DNA and peptides that have been designed to promote rapid sequence recognition. Peptide design was based on the surface of staphylococcal nuclease, a cationic DNA binding protein with low sequence selectivity. We observe that attachment of the designed peptide increases rates of strand invasion by 100-fold relative to unmodified bisPNA. The peptide can contain D-amino acids, increasing the likelihood that it will be stable in cell extract and inside cells. Binding of the conjugate containing the D-amino acid peptide occurred over a broad range of experimental conditions and was sensitive to a single mismatch. Strand invasion was efficient at neutral to basic pH, a wide range of temperatures (0-65 degrees C), and in the presence of up to 7 mM Mg(2+) and 100 mM Na(+) or K(+). Our data suggest that attachment of peptides that mimic cationic protein surfaces to PNAs can afford conjugates that mimic the rapid and selective binding that characterizes native DNA binding proteins. Rapid strand invasion over a wide range of experimental conditions should further expand the utility of strand invasion by PNAs.

Amino Acid Sequence↗
PubMed2002

Implications of high-affinity hybridization by locked nucleic acid oligomers for inhibition of human telomerase.

Oligonucleotides that contain locked nucleic acid (LNA) bases have remarkably high affinity for complementary RNA and DNA sequences. This increased affinity may facilitate the recognition of nucleic acid targets inside cells and thus improve our ability to use synthetic oligonucleotides for controlling cellular processes. Here we test the hypothesis that LNAs offer advantages for inhibiting human telomerase, a ribonucleoprotein that is critical for tumor cell proliferation. We observe that LNAs complementary to the telomerase RNA template are potent and selective inhibitors of human telomerase. LNAs can be introduced into cultured tumor cells using cationic lipid, with diffuse uptake throughout the cell. Transfected LNAs effectively inhibited intracellular telomerase activity up to 40 h post-transfection. Shorter LNAs of eight bases in length are also effective inhibitors of human telomerase. The melting temperatures of these LNAs for complementary sequences are superior to those of analogous peptide nucleic acid oligomers, emphasizing the value of LNA bases for high-affinity recognition. These results demonstrate that high-affinity binding by LNAs can be exploited for superior recognition of an intracellular target.

Animals↗
PubMed2002

DNA assembly using bis-peptide nucleic acids (bisPNAs).

DNA nanostructures are ordered oligonucleotide arrangements that have applications for DNA computers, crystallography, diagnostics and material sciences. Peptide nucleic acid (PNA) is a DNA/RNA mimic that offers many advantages for hybridization, but its potential for application in the field of DNA nanotechnology has yet to be thoroughly examined. We report the synthesis and characterization of tethered PNA molecules (bisPNAs) designed to assemble two individual DNA molecules through Watson-Crick base pairing. The spacer regions linking the PNAs were varied in length and contained amino acids with different electrostatic properties. We observed that bisPNAs effectively assembled oligonucleotides that were either the exact length of the PNA or that contained overhanging regions that projected outwards. In contrast, DNA assembly was much less efficient if the oligonucleotides contained overhanging regions that projected inwards. Surprisingly, the length of the spacer region between the PNA sequences did not greatly affect the efficiency of DNA assembly. Reasons for inefficient assembly of inward projecting DNA oligonucleotides include non-sequence-specific intramolecular interactions between the overhanging region of the bisPNA and steric conflicts that complicate simultaneous binding of two inward projecting strands. These results suggest that bisPNA molecules can be used for self-assembling DNA nanostructures provided that the arrangement of the hybridizing DNA oligonucleotides does not interfere with simultaneous hybridization to the bisPNA molecule.

Chromatography, High Pressure Liquid↗