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N M Dean

Publications and source records attributed to N M Dean.

At least 55 records · Page 3Linked to original sources
PubMed1998

Antitumor activity of a PKC-alpha antisense oligonucleotide in combination with standard chemotherapeutic agents against various human tumors transplanted into nude mice.

A 20-mer phosphorothioate oligodeoxynucleotide (ODN) directed against human protein kinase C-alpha (CGP 64128A = ISIS 3521) was analyzed for its antitumor activity either alone or in combination therapy. Combination studies with CGP 64128A and standard chemotherapeutic agents (cisplatin, mitomycin-C, vinblastine, estracyt and adriamycin) were performed in nude mice that had been transplanted s.c. with a variety of human tumors (breast, prostate, large cell lung and small cell lung carcinomas, and melanomas). Additive antitumor effects with CGP 64128A and the cytotoxins were found for half of the combinations studied. The combination of CGP 64128A with vinblastine or cisplatin showed superadditive antitumor activities against MCF-7 human breast carcinomas and PC3 prostate carcinomas with complete responses. CGP 64128A in combination with adriamycin resulted in superadditive antitumor effects against BT20 human breast carcinomas with complete tumor responses, and in combination with mitomycin-C superadditive antitumor effects with cures were observed against NCI-H460 human large cell carcinomas. The antitumor activity of CGP 64128A appeared to be due to a sequence-dependent mechanism of action as two 20-mer control ODNs were completely inactive as single agents against A549 and NCI-H69 human lung carcinomas. The antitumor activity of cisplatin against NCI-H69 human small cell lung carcinomas was slightly inhibited by one of the control ODNs, indicating that the superadditive antitumor activities of CGP 64128A in combination with cisplatin are the result of a sequence-dependent mechanism of action.

Animals↗
PubMed1998

Novel chemically modified oligonucleotides provide potent inhibition of P-glycoprotein expression.

One major form of multiple drug resistance (MDR) to cancer therapeutic agents is mediated by overexpression of P-glycoprotein, a membrane ATPase that serves as a drug efflux pump. In humans, this protein is the product of the MDR1 gene. We have used chemically modified antisense oligonucleotides to reduce expression of P-glycoprotein in multidrug-resistant fibroblasts and colon carcinoma cells. Although several types of oligonucleotides were tested, compounds having a phosphorothioate backbone and a methoxyethoxy (ME) group at the 2' position of the ribose ring proved to have the greatest potency. Thus, phosphorothioate 2'-ME oligonucleotides directed against either the AUG codon region or the stop codon region of the MDR1 message produced substantial (50-70%) inhibition of P-glycoprotein expression at concentrations of < or = 50 nM. In addition, such treatment resulted in augmented drug uptake as measured by flow cytometry. Unmodified phosphorothioate compounds of the same sequence were active only in the micromolar range. We also tested the ability of several potential delivery agents to enhance the pharmacological effectiveness of anti-MDR1 oligonucleotides. Both commercial Lipofectin, a well known liposomal transfection agent, and a liposomal preparation based on a novel "facial amphiphile" were effective in enhancing their pharmacological effects of antisense oligonucleotides. A Starburst dendrimer, a type of cationic polymer, was also effective in oligonucleotide delivery. This report emphasizes that significant improvements in antisense pharmacology can be made through judicious use of both chemical modifications of oligonucleotides and appropriate delivery systems.

3T3 Cells↗
PubMed1997

PKC-epsilon is required for mechano-sensitive activation of ERK1/2 in endothelial cells.

Mechano-sensitive regulation of endothelial cells (EC) function by shear stress is critical for flow-induced vasodilation and gene expression. Previous studies by our laboratory demonstrated that shear stress activates the 44- and 42-kDa extracellular signal-regulated kinases (ERK1/2) in EC in a time- and force-dependent manner. ERK1/2 activation was inhibited by protein kinase C (PKC) down-regulation with phorbol 12,13-dibutyrate (1 microM for 24 h) but not by calcium chelation with BAPTA-AM (acetoxymethyl ester of BAPTA) (75 microM for 30 min), suggesting that a novel PKC isoform (delta, epsilon, eta, theta) mediates shear stress-induced ERK1/2 activation. Western blotting with PKC isoform-specific antibodies demonstrated expression of PKC-alpha, -epsilon, and -zeta isoforms in EC. PKC-epsilon was specifically inhibited by transfection with antisense PKC-epsilon phosphorothioate oligonucleotides (1,000 nM for 6 h). Antisense treatment decreased PKC-epsilon protein levels by 80 +/- 13% after 72 h and completely inhibited shear stress-stimulated ERK1/2 activation. Scrambled PKC-epsilon oligonucleotides and antisense PKC-alpha and PKC-zeta oligonucleotides had no effect on ERK1/2 activity. PKC-epsilon appeared specific for mechano-sensitive ERK1/2 activation, as antisense PKC-epsilon oligonucleotides did not inhibit ERK1/2 activation by EGF or bradykinin but did inhibit ERK1/2 activation upon EC adhesion to fibronectin. These results define a pathway for shear stress-mediated ERK1/2 activation and establish a new function for PKC-epsilon as part of a mechano-sensitive signal transduction pathway in EC.

Animals↗
PubMed1997

Antisense oligonucleotides targeting human protein kinase C-alpha inhibit phorbol ester-induced reduction of bradykinin-evoked calcium mobilization in A549 cells.

Regulation of the bradykinin-evoked increase in intracellular Ca2+ concentration by protein kinase C (PKC)-alpha was investigated in A549 human lung carcinoma cells. Bradykinin, a potent and selective kinin B2 receptor agonist, induces calcium mobilization in a concentration-dependent fashion in this cell line. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a potent activator of PKC, is known to reduce the amplitude of agonist-induced calcium mobilization in various cell lines. Because PKC-alpha is a major PKC isozyme in A549 cells, we investigated whether this isozyme plays a role in this process. A 20-mer phosphorothioate oligonucleotide targeting the 3'-untranslated region of the human PKC-alpha mRNA, which contains 2'-methoxyethyl modifications incorporated into the 5' and 3' segments of the oligonucleotide, was used to assess the putative role of PKC-alpha in the receptor regulation. ISIS 9606 reduced PKC-alpha mRNA for > or = 72 hr after the initial treatment and the reduction was concentration dependent, whereas the mismatch control, ISIS 13009, had no effect. Concentrations of ISIS 9606 of 150 nM specifically reduced the level of immunoreactive PKC-alpha protein by 66.3 +/- 2.5% at 72 hr after treatment, without an effect on immunoreactive PKC-delta protein. This reduction in PKC-alpha was sufficient to inhibit the reduction of bradykinin-induced calcium mobilization by TPA. This finding is corroborated by the use of staurosporine, a nonselective PKC inhibitor, that prevented the effect of TPA. These results suggest that PKC-alpha is involved in kinin B2 receptor regulation by phorbol esters in A549 cells.

Bradykinin↗
PubMed1997

Definition by specific antisense oligonucleotides of a role for protein kinase C alpha in expression of differentiation markers in normal and neoplastic mouse epidermal keratinocytes.

Epidermal keratinocyte differentiation is a tightly regulated, stepwise process that requires protein kinase C (PKC) activation. Studies using cultured mouse keratinocytes induced to differentiate with Ca2+ have indirectly implicated the alpha isoform of PKC in upregulation of "late" (granular cell) epidermal differentiation markers. Activation of this isoform is also implicated in the suppression of "early" differentiation markers keratin (K) 1 and 10 that characterizes the neoplastic phenotype produced by the v-Ha-ras oncogene. We used antisense oligonucleotides (AS) to directly address the role of PKC alpha in regulating expression of these markers in normal and v-Ha-ras-transduced primary keratinocytes and a keratinocyte cell line (SP-1) containing an activating mutation of the c-Ha-ras gene. Transfection of PKC alpha AS reduced the PKC alpha protein level in a dose-dependent manner, with a maximum effect at doses of 100 nM or higher. Immunoblot analysis with antibodies against PKC alpha, PKC delta, PKC epsilon, and PKC eta confirmed that PKC alpha AS selectively reduced the level of PKC alpha but not the other isoforms. In vitro kinase assays also revealed suppression of Ca(2+)-dependent PKC activity, which is the PKC alpha activity in this cell type, after transfection of PKC alpha AS. When PKC alpha AS-treated normal keratinocytes were stimulated to terminally differentiate with Ca2+, induction of the late differentiation markers loricrin, filaggrin, and SPR-1, as well as transglutaminase K mRNA, was suppressed when compared with their induction in scrambled AS-treated controls. In neoplastic v-Ha-ras-transduced keratinocytes and SP-1 cells, transfection of PKC alpha AS, but not the scrambled AS control, selectively downregulated PKC alpha and restored differentiation-specific expression of K1. These findings directly confirm that PKC alpha is an important component of the signaling pathway regulating terminal differentiation of normal keratinocytes and that activation of PKC alpha contributes to the altered differentiation program of neoplastic murine keratinocytes.

Animals↗
PubMed1996

Reduction of expression of the multidrug resistance protein (MRP) in human tumor cells by antisense phosphorothioate oligonucleotides.

Multidrug resistance protein (MRP) is a member of the ATP-binding cassette superfamily of transport proteins which has been demonstrated to cause multidrug resistance when transfected into previously sensitive cells. Sixteen eicosomeric oligonucleotides complementary to different regions along the entire length of the MRP mRNA reduced MRP mRNA and protein levels in drug-resistant small cell lung cancer cells that highly overexpress this protein. In MRP-transfected HeLa cells that express intermediate levels of MRP, one oligonucleotide, ISIS 7597, targeted to the coding region of the MRP mRNA, decreased the levels of MRP mRNA to < 10% of control levels in a concentration-dependent manner. This effect was rapid but transient with a return to control levels of MRP mRNA 72 hr after treatment. A double treatment with ISIS 7597 produced a sustained inhibition, resulting in a greater than 90% reduction in MRP mRNA for 72 hr and a comparable decrease in protein levels. Increased sensitivity to doxorubicin was observed under these conditions. Northern blotting analyses using two DNA probes corresponding to sequences 5' and 3' of the ISIS 7597 target sequence, respectively, revealed the presence of low levels of two smaller sized RNA fragments as expected from an RNase H-mediated mechanism of action of the antisense oligonucleotide. These studies indicate that a specific reduction in MRP expression can be achieved with antisense oligonucleotides, a finding that has potential implications for the treatment of drug-resistant tumors.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Enhanced activity of an antisense oligonucleotide targeting murine protein kinase C-alpha by the incorporation of 2'-O-propyl modifications.

We have previously described the characterization of a 20mer phosphorothioate oligodeoxynucleotide (ISIS 4189) which inhibits murine protein kinase C-alpha (PKC-alpha) gene expression, both in vitro and in vivo. In an effort to increase the antisense activity of this oligonucleotide, 2'-O-propyl modifications have been incorporated into the 5'- and 3'-ends of the oligonucleotide, with the eight central bases left as phosphorothioate oligodeoxynucleotides. Hybridization analysis demonstrated that these modifications increased affinity by approximately 8 and 6 degrees C per oligonucleotide for the phosphodiester (ISIS 7815) and phosphorothioate (ISIS 7817) respectively when hybridized to an RNA complement. In addition, 2'-O-propyl incorporation greatly enhanced the nuclease resistance of the oligonucleotides to snake venom phosphodiesterase or intracellular nucleases in vivo. The increase in affinity and nuclease stability of ISIS 7817 resulted in a 5-fold increase in the ability of the oligonucleotide to inhibit PKC-alpha gene expression in murine C127 cells, as compared with the parent phosphorothioate oligodeoxynucleotide. Thus an RNase H-dependent phosphorothioate oligodeoxynucleotide can be modified as a 2'-O-propyl 'chimeric' oligonucleotide to provide a significant increase in antisense activity in cell culture.

Alkylation↗
PubMed1996

Inhibition of interleukin-1 type I receptor expression in human cell-lines by an antisense phosphorothioate oligodeoxynucleotide.

A 20 mer oligodeoxynucleotide designed to hybridize to specific 3'-untranslated sequences in the type I receptor IL-1r mRNA was identified which inhibited the expression of both IL-1r mRNA and protein in human A549 lung carcinoma cells and human dermal fibroblasts. The oligodeoxynucleotide exhibited an IC50 value of approximately 100 nM in both cell lines and reduced IL-1r mRNA expression for up to 48 h. Multiple scrambled control oligonucleotides were without effect on IL-1r mRNA expression. Treatment of A549 cells with this oligodeoxynucleotide (but not scrambled controls) inhibited the IL-1 stimulated expression of the cell adhesion molecule ICAM-1 but was without effect on the TNF-alpha induction of this molecule. This study demonstrates that phosphorothioate oligodeoxynucleotides can selectively inhibit IL-1r expression leading to a reduction in IL-1 dependent gene expression.

Carcinoma↗
PubMed1996

Treatment of glioblastoma U-87 by systemic administration of an antisense protein kinase C-alpha phosphorothioate oligodeoxynucleotide.

Glioblastoma multiforme is the most common form of malignant brain cancer in adults and, unfortunately, is not amenable to treatment with current therapeutic modalities. Human glioblastoma U-87 has many of the distinguishing phenotypic features of primary glioblastoma, including an autocrine form of proliferation, high levels of protein kinase C alpha (PKC alpha), and infiltration via white matter tracts. We show that treatment of mice bearing U-87 xenografts with an antisense phosphorothioate oligodeoxynucleotide (S-oligodeoxynucleotide) against the 3'-untranslated region of PKC alpha mRNA results in suppression of tumor growth. Growth was inhibited in both subcutaneous and intracranial tumors, and in the latter instance, treatment with the antisense PKC alpha S-oligodeoxynucleotide resulted in a doubling in median survival time ( > 80 days), with 40% long term survivors. The antisense S-oligodeoxynucleotide did not produce systemic toxicity in mice with subcutaneous or intracranial tumors after daily intraperitoneal injection for 21 or 80 days, respectively, and a scrambled S-oligodeoxynucleotide with the same nucleotide composition as the antisense S-oligodeoxynucleotide did not produce an antitumor effect. The intratumoral levels of both antisense and scrambled S-oligodeoxynucleotide in subcutaneous tumors were 2 microM after 21 daily doses of 20 mg/kg S-oligodeoxynucleotide. The antisense S-oligodeoxynucleotide selectively reduced the levels of PKC alpha in subcutaneous tumors but not those of protein kinase C epsilon or protein kinase C zeta. This is the first demonstration that the growth of glioblastoma multiforme can be suppressed by an antisense PKC alpha S-oligodeoxynucleotide and suggests that this may represent an effective therapy for this type of malignancy.

Animals↗
PubMed1996

Inhibition of expression of the multidrug resistance-associated P-glycoprotein of by phosphorothioate and 5' cholesterol-conjugated phosphorothioate antisense oligonucleotides.

Multiple drug resistance (MDR) as a result of overexpression of the P-glycoprotein drug transporter, a product of the MDR1 gene, is a significant problem in cancer therapeutics. We demonstrate that phosphorothioate antisense oligonucleotides can reduce levels of MDR1 message, inhibit expression of P-glyco protein, and affect drug uptake in MDR mouse 3T3 fibroblasts. An obligonucleotide (5995) directed against a sequence overlapping the AUG start codon was effective in reduction MDR1 transcript and protein levels when used at submicromolar concentrations in conjunction with cationic liposomes, whereas a scrambled control oligonucleotide (10221) was ineffective. Substantial and specific antisense effects could also be attained with a 5' cholesterol conjugate of the 5995 sequence. In this case, use of cationic liposomes was unnecessary. The 5' cholesterol 5995, but the not 5' cholesterol 10221, reduced MDR1 message and P-glycoprotein levels by 50-60% when used at low micromolar concentrations. In parallel, treatment with 5' cholesterol 5995 also enhanced cellular accumulation of rhodamine 123, a well-known substrate of the P-glycoprotein transporter. The effectiveness of the cholesterol-conjugated 5995 may be due to its rapid and extensive cell uptake, as indicated in flow cytometry and confocal microscopy studies. These observations suggest that cholesterol-conjugated anti-sense oligonucleotides may offer a novel approach to inhibition of P-glycoprotein-mediated MDR and to the modulation of other tumor cell genes whose overexpression contributes to the neoplastic state or to resistance to therapy.

3T3 Cells↗
PubMed1995

EGF-induced increase in diacylglycerol, choline release, and DNA synthesis is extracellular calcium dependent.

Previous studies have demonstrated a strict extracellular Ca2+ dependence for the G0 to G1 and G1 to S transition in growth factor-treated T51B rat liver cells that is associated with increased levels of protein kinase C activity. Consequently, we have examined these cells for changes in phospholipid-derived second messengers in response to epidermal growth factor (EGF) and thrombin in order to determine which signals are generated during the initiation of the G0 to G1 transition. Thrombin is coupled to a phosphoinositide hydrolyzing phospholipase C, as we have found a rapid Ca(2+)-independent increase in the levels of inositol 1,4,5-trisphosphate (Ins[1,4,5]P3), inositol 1,4-bisphosphate (Ins[1,4]P2), and inositol 4-monophosphate (Ins[4]P), as well as a concomitant, transient elevation in diacylglycerol. No changes in either intracellular or extracellular choline metabolites, or an increase in DNA synthesis, were found in response to thrombin. By contrast, treatment of T51B cells with EGF results in a slower, more prolonged extracellular Ca(2+)-dependent increase in both [3H]-glycerol radiolabeled diacyl-glycerol, and diacylglycerol mass, an increase in choline release into the extracellular medium, and eventually a substantial DNA synthesis. We were, however, unable to detect any changes in phosphatidylinositol (PtdIns) turnover, either by accumulation of inositol phosphates or by changes in phospholipids in response to EGF. These results indicate that DNA synthesis can readily occur in the absence of stimulated PtdIns turnover, and that PtdIns turnover is not sufficient in itself or necessary to induce DNA synthesis and is not necessary for a Ca(2+)-dependent increase in diacylglycerol. Moreover, we have demonstrated that the extracellular Ca(2+)-dependent increase in diacylglycerol levels in response to EGF is associated with an increase in extracellular choline release, which is indicative of an activation of a phosphatidylcholine-linked phospholipase D. These results suggest that diacylglycerol sources other than PtdIns's may be important in the extracellular Ca(2+)-dependent regulation of EGF-mediated cell replication.

Animals↗
PubMed1994

Inhibition of protein kinase C-alpha expression in mice after systemic administration of phosphorothioate antisense oligodeoxynucleotides.

A 20-mer phosphorothioate oligodeoxynucleotide designed to hybridize to the AUG translation initiation codon of mRNA encoding murine protein kinase C-alpha (PKC-alpha) inhibits the expression of PKC-alpha both in vitro and in vivo. In mouse C127 mammary epithelial cells, the reduction in PKC-alpha mRNA expression was both dose and time dependent. The oligodeoxynucleotide exhibited an IC50 value of 100-200 nM and reduced PKC-alpha mRNA expression for up to 48 hr. This reduction was specific for PKC-alpha versus other PKC isozymes (delta, epsilon, and zeta) and completely dependent upon oligodeoxynucleotide sequence. When administered intraperitoneally in mice, the same oligodeoxynucleotide caused a dose-dependent, oligodeoxynucleotide sequence-dependent reduction of PKC-alpha mRNA in liver, with an IC50 value of 30-50 mg/kg of body weight. Inhibition of expression was 64 +/- 11% after a single 50-mg/kg dose. The expression of PKC-delta, epsilon, and zeta mRNA was unaffected by this treatment. The oligodeoxynucleotide activity in vivo did not require the presence of cationic liposomes or any other delivery systems, although in vitro, the oligodeoxynucleotide required cationic liposomes for inhibition of PKC-alpha expression. This study demonstrates the utility of phosphorothioate oligodeoxynucleotides as specific inhibitors of gene expression in vivo after systemic administration.

3T3 Cells↗
PubMed1994

Inhibition of protein kinase C-alpha expression in human A549 cells by antisense oligonucleotides inhibits induction of intercellular adhesion molecule 1 (ICAM-1) mRNA by phorbol esters.

We have identified 20-mer phosphorothioate oligodeoxynucleotides which potently (IC50 values of 100-200 nM) and specifically inhibit protein kinase C (PKC)-alpha mRNA and protein expression in human lung carcinoma (A549) cells. These oligonucleotides target multiple, diverse sites on PKC-alpha mRNA including the AUG translation codon and 3'-untranslated sequences. 2'-O-Methyl phosphorothioate analogs of these oligonucleotides were without effect on PKC-alpha mRNA levels, suggesting that the reduction in targeted PKC-alpha mRNA is through RNase H-mediated cleavage. One oligonucleotide, however, was effective at inhibiting PKC-alpha protein levels as a 2'-O-methyl phosphorothioate at concentrations 2-3-fold greater than its phosphorothioate/deoxy homolog. These results suggest that the ability to serve as an RNase H substrate, although not required for all oligonucleotides, certainly increases their potency. These oligonucleotides have been used to examine the role played by PKC-alpha in mediating the phorbol ester-induced changes in mRNA levels of the cell adhesion molecule ICAM-1. In A549 cells, ICAM-1 mRNA is increased 10-20-fold by treatment of cells with the phorbol ester phorbol 12-myristate 13-acetate. When PKC-alpha protein levels are depleted by oligonucleotide treatment of A549 cells, the increase in ICAM-1 expression in response to phorbol 12-myristate 13-acetate is greatly reduced, demonstrating that PKC-alpha plays a major role in this process.

Base Sequence↗
PubMed1991

Okadaic acid inhibits PDGF-induced proliferation and decreases PDGF receptor number in C3H/10T1/2 mouse fibroblasts.

Okadaic acid is both a potent inhibitor of protein serine/threonine phosphatases and a tumor promoter in the mouse skin model. We have previously shown that at non-toxic nanomolar concentrations okadaic acid reversibly inhibits induction (promotion) by PDGF of transformed cells by the 'complete' and 'two-stage' protocols in the C3H/10T1/2 mouse fibroblast transformation assay. In the present study we have demonstrated that treatment of confluent and proliferatively quiescent C3H/10T1/2 mouse fibroblasts with low doses of okadaic acid inhibits the platelet-derived growth factor (PDGF)-induced mitogenic response. This inhibition is accompanied by a loss of PDGF binding sites, a decreased PDGF-induced phosphatidylinositol turnover and a decrease in the PDGF-induced intracellular calcium signal. The decrease in the PDGF-generated intracellular signalling processes represents a mechanism by which okadaic acid inhibits PDGF-induced proliferation and the promotion of in vitro neoplastic transformation by PDGF.

Animals↗