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Biomedical subjects

R L Juliano

Publications and source records attributed to R L Juliano.

At least 19 recordsLinked to original sources
PubMed2006

Intracellular delivery of oligonucleotide conjugates and dendrimer complexes.

Enhancing the delivery of antisense and siRNA molecules to cells and tissues is a key issue for oligonucleotide therapeutics. Cell-penetrating peptides (CPPs) have the ability to convey linked "cargo" molecules into the cytosol; thus we have explored the use of CPPs as delivery agents for oligonucleotides. We have extensively evaluated CPP-oligonucleotide conjugates, and have recently begun to explore the use of CPP-dendrimer-oligonucleotide complexes. We have found that CPP-antisense oligonucleotide conjugates can be taken up by cells and can effectively modify gene expression in cell culture and in tissues. Although not as potent in cell culture as cationic lipid delivery agents, CPP-oligonucleotide conjugates offer the advantage of being molecules rather than particles, and may have substantial advantages over particle-based delivery in the in vivo setting.

Cell Line, Tumor↗
PubMed2005

Delivery of MDR1 small interfering RNA by self-complementary recombinant adeno-associated virus vector.

Small interfering RNAs (siRNAs) are potentially powerful tools for therapeutic gene regulation. DNA cassettes encoding RNA polymerase III promoter-driven hairpin siRNAs allow long-term expression of siRNA in targeted cells. A variety of viral vectors have been used to deliver such cassettes to cells. Here we report on the development and use of a self-complementary recombinant adeno-associated virus (scAAV) vector for siRNA delivery into mammalian cells. We demonstrate that this modified vector efficiently delivers siRNA into multidrug-resistant human breast and oral cancer cells and suppresses MDR1 gene expression. This results in rapid, profound, and durable reduction in the expression of the P-glycoprotein multidrug transporter and a substantial reversion of the drug-resistant phenotype. This research suggests that scAAV-based vectors can be very effective agents for efficient delivery of therapeutic siRNA.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Epigenetic modulation of gene expression in mammalian cells.

The precisely controlled regulation of gene expression plays an important role in normal cellular function, whereas abnormalities of gene expression can contribute to cancer and other diseases. Thus, the ability to selectively regulate gene expression is of tremendous importance, both as a basic research tool and as a potential therapeutic approach. Over the last two decades, several different strategies have been used for controlling expression of specific genes at the transcriptional and post-transcriptional levels. The best-known approach involves antisense oligonucleotides that downregulate gene expression through various mechanisms, most importantly by triggering the degradation of mRNA. The use of designed transcription factors has also developed into an interesting technology since it allows either the enhancement or the reduction of expression of a targeted gene. Recently RNA interference has emerged as a powerful tool for selective inhibition of gene expression. This review examines the basic aspects, advantages, and liabilities of these three approaches to gene modulation.

Animals↗
PubMed2004

Selective transcription of p53 target genes by zinc finger-p53 DNA binding domain chimeras.

Active p53 stimulates the transcription of a number of key genes, including the pro-apoptotic gene bax, as well as p21, a cell cycle regulator. In this study we constructed novel chimeric zinc finger-p53 DNA binding domain (DBD) transcription factors designed to bind to the promoters of specific p53 regulated genes. In order to selectively increase the expression of Bax, we coupled a pre-selected three-zinc finger (Zif) peptide targeted to a sequence in the bax promoter to a minimal p53 DBD. This chimeric protein could increase reporter gene transcription from a minimal bax promoter (up to 10-fold) but not from a minimal p21 promoter in p53-deficient Saos-2 cells. However, fusion proteins carrying longer p53 DBDs displayed entirely different selectivity and potency. Thus, Zif-p53 DBD chimeras containing N- and C-terminal extensions of the minimal DBD could increase transcription driven by a minimal p21 promoter up to 800-fold. These chimeras preferred the minimal p21 promoter up to 500-fold over the minimal bax promoter. Additionally, endogenous p21 message and protein levels were increased in cells expressing the p21 selective Zif-p53 DBD chimera and expression of the chimeric proteins resulted in partial cell cycle arrest. Cell fractionation experiments indicated that the Zifs enhanced nuclear localization of the Zif-p53 DBD chimera. These studies suggest that it is possible to create chimeric transcription factors able to strongly and selectively activate genes downstream of p53.

Animals↗
PubMed2004

Integrin regulation of cell signalling and motility.

Integrins clearly play a key role in regulating both mitogenic signalling and cell migration. Thus integrins modulate the efficiency of the Erk (extracellular-signal-regulated kinase)/MAP kinase (mitogen-activated protein kinase) pathway, acting at several distinct levels. We have shown that both cAMP-dependent protein kinase and PAKs (p21-activated kinases) play a role in integrin regulation of the Erk pathway, acting primarily at the level of Raf-1. Integrins and PAKs also play a role in the control of cell migration. Thus we have discovered a novel protein that links the alpha5beta1 integrin to migration controlled by Rho-family GTPases. This protein, termed Nischarin, is a large cytosolic macromolecule that is not related to well-known protein families. The N-terminus of Nischarin interacts with a short segment of the cytoplasmic domain of the alpha5 integrin subunit. Overexpression of Nischarin alters actin organization and inhibits Rac-driven cell migration and tumour cell invasion. Use of effector domain mutants of Rac suggest that Nischarin acts downstream of Rac, probably at the level of PAK-family kinases. These studies emphasize the intricate connection between integrins and Rho-family GTPases and their effectors in controlling both mitogenesis and migration.

Animals↗
PubMed2004

Strategies for inhibition of MDR1 gene expression.

Several distinct strategies have been used to modulate the expression of cancer-associated genes, including antisense oligonucleotides, small interfering RNAs (siRNAs), and artificial transcriptional factors. One major cause for chemotherapeutic treatment failure in cancer is the overexpression of P-glycoprotein, the product of the multidrug resistance gene MDR1. In this study, we tested the ability of siRNAs to inhibit MDR1 gene expression. We evaluated the efficiency of chemically synthesized dsRNAs as well as vector-based hairpin siRNAs and investigated the behavior of clones of multidrug-resistant NCI/ADR-RES breast carcinoma cells stably transfected with hairpin siRNA vectors. The effects of siRNA on the MDR phenotype were compared with those elicited by antisense oligonucleotides or by designed transcription factors targeting the MDR1 promoter. These studies suggest that there are several comparably effective strategies for inhibiting MDR1 expression.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2003

Design of artificial transcription factors to selectively regulate the pro-apoptotic bax gene.

The tumor suppressor p53 is the most commonly mutated gene in human cancers. Active p53 is able to stimulate the transcription of a variety of genes including the pro-apoptotic gene bax, as well as p21, a cell cycle regulator. In this study we produced novel zinc finger transcription factors that would selectively increase the expression of bax, but not of other p53 targets. Reporter gene assays in p53-negative Saos-2 cells showed that the novel zinc finger proteins stimulated transcription driven by a minimal bax promoter, but not that driven by a minimal p21 promoter. Moreover, electromobility shift assays demonstrated that the novel transcription factors could bind the bax promoter sequence with high affinity and selectivity. Expression of a five zinc finger protein (5ZFAV) in COS-7 cells resulted in an increase in Bax protein levels, indicating that this novel transcription factor could act on endogenous gene expression. Expression of 5ZFAV also drastically reduced Saos-2 cell survival; this effect could be reversed by the general caspase inhibitor B-D-FMK. These data suggest that 5ZFAV is able to induce apoptosis through increased Bax expression. Further, while expression of 5ZFAV in p53-deficient Saos-2 cells reduced cell survival, there was little effect on U-2 OS cells which have wild-type p53. Thus the selective induction of the pro-apoptotic bax gene may be a valuable adjunct to cancer chemotherapy by diminishing survival of p53-deficient tumor cells.

Animals↗
PubMed2003

P53-dependent cell-killing by selective repression of thymidine kinase and reduced prodrug activation.

Selective killing of tumor cells is an important goal for cancer therapeutics. The tumor suppressor transcription factor p53 is absent or mutated in more than 50% of human tumors. Thus, determining approaches that use p53 status to regulate therapy may be an important strategy for attaining cancer selectivity. We have shown previously that a designed transcriptional repressor, K2-5F, strongly and selectively reduces the expression of its target gene MDR1. In this study, we exploited p53 status and the strong repressor activity of K2-5F to establish a system for preferential killing of p53-negative cells. In this system, the expression of K2-5F is induced by p53 in normal cells, and the K2-5F repressor then inhibits the expression of herpes simplex virus thymidine kinase (HSV-TK) driven by an MDR1 minipromoter. In p53-deficient cells, little K2-5F is expressed, and thus HSV-TK is expressed, allowing the cells to be killed by ganciclovir (GCV). K2-5F induced by exogenous p53 dramatically reduced the expression of HSV-TK in human embryonic kidney 293 cells, and it subsequently increased cell survival in response to GCV. To further evaluate this approach in a uniform genetic background, we developed Saos-2 cells stably expressing physiological levels of p53 and paired them with wild-type p53-negative Saos-2 cells. Stable expression of moderate levels of p53 in Saos-2 cells was able to induce the expression of K2-5F and reduce HSV-TK expression and resulted in a modest but distinct protection from GCV toxicity. Thus, this system may be suitable for further development as an approach to selective cancer therapy.

Antiviral Agents↗
PubMed2002

Cell adhesion differentially regulates the nucleocytoplasmic distribution of active MAP kinases.

Cells decide whether to undergo processes, such as proliferation, differentiation and apoptosis, based upon the cues they receive from both circulating factors and integrin-mediated adhesion to the extracellular matrix. Integrins control the activation of the early signaling pathways. For example, growth factor activation of the ERK cascade is enhanced when cells are adherent. In addition, adhesion receptors oversee the cellular localization of critical signaling components. We have recently shown that ERK signaling to the nucleus is regulated by cell adhesion at the level of nucleocytoplasmic trafficking. Since the ERKs are only one class of MAP kinase, we extended these studies to include both JNK and p38 MAP kinases. We have rendered JNK and p38 activation in NIH 3T3 fibroblasts anchorage-independent either by treatment with anisomycin or by expression of upstream activators. Under conditions whereby JNK activation is anchorage-independent, we show that localization of JNK to the nucleus and JNK-mediated phosphorylation of c-Jun and Elk-1 is not altered by loss of adhesion. Likewise, the ability of activated p38 to accumulate in the nucleus was similar in suspended and adherent cells. Finally, we show that expression of a form of ERK, which is activated and resistant to nuclear export, reverses the adhesion-dependency of ERK phosphorylation of Elk-1. Thus, adhesion differentially regulates the nucleocytoplasmic distribution of MAP kinase members; ERK accumulation in the nucleus occurs more efficiently in adherent cells, whereas nuclear accumulation of active p38 and active JNK are unaffected by changes in adhesion.

3T3 Cells↗
PubMed2002

Regulation of vasodilator-stimulated phosphoprotein phosphorylation and interaction with Abl by protein kinase A and cell adhesion.

Members of the vasodilator-stimulated phosphoprotein (VASP) family are important regulators of actin cytoskeletal dynamics whose functions and protein-protein interactions are regulated by phosphorylation by the cAMP-dependent protein kinase (PKA). Herein, we show that phosphorylation of VASP is dynamically regulated by cellular adhesion to extracellular matrix. Detachment of cells stimulated PKA activity and induced PKA-dependent phosphorylation of VASP and the related murine-Enabled (Mena) protein. VASP and Mena were rapidly dephosphorylated immediately following reattachment but showed an intermediate level of phosphorylation during active cell spreading. This pattern correlated closely with adhesion-dependent changes in PKA activity. The in vivo interaction of VASP with the Abl tyrosine kinase, shown here for the first time, was readily apparent in adherent cells, lost following cellular detachment, and induced upon reattachment to matrix. Importantly, inhibition of PKA activity prevented phosphorylation of VASP and dissociation of VASP-Abl complexes after cellular detachment, whereas activation of PKA completely eliminated the co-immunoprecipitation of Abl activity with VASP. These data establish a new biochemical link between cell adhesion and regulation of VASP proteins and provide the first demonstration of a regulated interaction between VASP and Abl in mammalian cells.

1-Methyl-3-isobutylxanthine↗
PubMed2002

The alpha5beta1 integrin selectively enhances epidermal growth factor signaling to the phosphatidylinositol-3-kinase/Akt pathway in intestinal epithelial cells.

We have investigated EGF-driven signaling processes in rat intestinal epithelial cell lines that overexpress either the alpha5beta1 integrin or the alpha2beta1 integrin. Both cell types display efficient activation of Erk/MAP kinase, but only the alpha5beta1 expressing cells display a strong activation of Akt. A complex is formed between activated EGFR and alpha5beta1, but not with alpha2beta1; this complex also contains ErbB3 and p85. Thus alpha5beta1 can support efficient activation of both the Erk and the phosphatidylinositol-3-kinase/Akt branches of the EGFR signaling cascade, whereas alpha2beta1 can support only the Erk branch.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Biological aspects of signal transduction by cell adhesion receptors.

Cell adhesion receptors such as integrins, cadherins, selectins, and immunoglobulin family receptors profoundly modulate many signal transduction cascades. In this review we examine aspects of adhesion receptor signaling and how this impinges on key biological processes. We have chosen to focus on cell migration and on programmed cell death. We examine many of the cytoplasmic signaling molecules that interface with adhesion receptors, including focal adhesion kinase (FAK), phosphatidylinositol-3-kinase (PI3K), and elements of the Erk/MAP kinase pathway. In many cases these molecules impinge on both the regulation of cell movement and on control of apoptosis.

Animals↗
PubMed2002

Anchorage-dependent ERK signaling--mechanisms and consequences.

Integrin-mediated adhesion to the extracellular matrix regulates the cellular response to mitogens. Anchorage-dependent growth factor activation of the extracellular signal-regulated kinase (ERK) is an intensely studied example of this regulation. Given the central role of ERK in mediating cell migration, division, and survival, it is also an extremely important example. Recent work has demonstrated that cell adhesion can regulate ERK signaling at several checkpoints and has begun to define the mechanism and consequences associated with anchorage-dependent effects on the ERK cascade.

Animals↗
PubMed2002

Conjugates of antisense oligonucleotides with the Tat and antennapedia cell-penetrating peptides: effects on cellular uptake, binding to target sequences, and biologic actions.

PURPOSE: The attainment of effective intracellular delivery remains an important issue for pharmacologic applications of antisense oligonucleotides. Here, we describe the synthesis, binding properties, and biologic properties of peptide-oligonucleotide conjugates comprised of the Tat and Ant cell-penetrating peptides with 2'-O-methyl phosphorothioate oligonucleotides. METHODS: The biologic assay used in this study measures the ability of the antisense molecule to correct splicing of an aberrant intron inserted into the Luciferase gene; thus, this assay clearly demonstrates the delivery of functional antisense molecules to the splicing machinery within the nucleus. The binding affinities of the conjugates to their target sequences were measured by surface plasmon resonance (BIAcor) techniques. RESULTS: The peptide-oligonucleotide conjugates progressively entered cells over a period of hours and were detected in cytoplasmic vesicles and in the nucleus. Peptide-oligonucleotide conjugates targeted to the aberrant splice site, but not mismatched controls, caused an increase in Luciferase activity in a dose-responsive manner. The kinetics of Luciferase appearance were consistent with the course of the uptake process for the conjugates. The effects of peptide conjugation on the hybridization characteristics of the oligonucleotides were also examined using surface plasmon resonance. The peptide-oligonucleotide conjugates displayed binding affinities and selectivities similar to those of unconjugated oligonucleotides. CONCLUSIONS: Conjugation with cell-penetrating peptides enhances oligonucleotide delivery to the nucleus without interfering with the base-pairing function of antisense oligonucleotides.

Amino Acid Sequence↗
PubMed2002

Evaluation of strategies for the intracellular delivery of proteins.

PURPOSE: The intracellular delivery of functionally active protein represents an important emerging strategy for laboratory investigation and therapeutic applications. Although a number of promising approaches for protein delivery have been developed, thus far there has been no attempt to compare the merits of the various deliver technologies. This issue is addressed in the current study. METHODS: In this study we utilize a sensitive luciferase reporter gene assay to provide unambiguous and quantitative evaluation of several strategies for the intracellular delivery of a biologically active protein comprised of the Gal4 DNA binding domain and the VP16 transactivating domain. RESULTS: Both a cationic lipid supramolecular complex and a poly meric complex were able to effectively deliver the chimeric transcription factor to cultured cells. In addition, protein chimeras containing the Tat cell penetrating peptide, but not those containing the VP22 peptide, were somewhat effective in delivery. CONCLUSIONS: Both supramolecular protein-carrier complexes and protein chimeras with certain cell penetrating peptides can support intracellular delivery of proteins. In the cell culture setting the supramolecular complexes are more effective, but their large size may present problems for in vivo applications.

Cell Line↗
PubMed2002

Selective inhibition of P-glycoprotein expression in multidrug-resistant tumor cells by a designed transcriptional regulator.

Selective inhibition of the multidrug resistance 1 (MDR1) gene and its product, the P-glycoprotein, a membrane transporter responsible for multidrug resistance, could be an important approach for enhancing cancer therapeutics. An emerging strategy for selective gene regulation involves designed zinc finger proteins that can recognize specific sequences in the promoter regions of disease-related genes. Herein, we investigate the behavior of clones of multidrug-resistant NCI/ADR-RES breast carcinoma cells displaying ponasterone-inducible expression of a designed transcriptional repressor targeted to the MDR1 promoter. The controlled production of this novel repressor resulted in major reductions in P-glycoprotein levels in these otherwise highly drug-resistant tumor cells. The regulated reduction of MDR1 expression in NCI/ADR-RES cells was accompanied by a marked increase in the rate of uptake of the P-glycoprotein substrate rhodamine 123. In addition, the cytotoxicity profile of the antitumor drug doxorubicin was dramatically altered in the induced cells compared with controls. The expression levels of other genes were examined both by a DNA array analysis of approximately 2000 genes and by biochemical techniques. Although some changes were observed in mRNA levels of nontargeted genes, the most dramatic effect by far was on MDR1, indicating that the action of the designed transcriptional repressor was quite selective. This study suggests that designed transcriptional regulators can be used to strongly and selectively influence expression of cancer-related genes, even under circumstances of extensive amplification of the target gene.

ATP Binding Cassette Transporter, Subfamily B↗