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Saghir Akhtar

Publications and source records attributed to Saghir Akhtar.

At least 19 recordsLinked to original sources
PubMed2005

Signal transduction involving Ras-GTPase contributes to development of hypertension and end-organ damage in spontaneously hypertensive rats-treated with L-NAME.

The purpose of this study was to examine the effect of inhibition of Ras-GTPase mediated signalling on the development of hypertension and end-organ damage in spontaneously hypertensive rats chronically treated with nitric oxide synthesis inhibitor L-NAME (SHR-L-NAME). Administration of L-NAME in drinking water (80 mg/L) for 3 weeks significantly elevated mean arterial blood pressure (MABP) (223+/-4 mmHg) as compared to that of SHR controls (165+/-3 mmHg). The administration of Ras-GTPase inhibitor FPTIII (232 ng/min) to SHR-L-NAME during the last 6 days significantly attenuated high blood pressure (192+/-4 mmHg). Morphological studies of the kidneys and hearts showed that treatment with FPTIII minimized the extensive arterial fibrinoid necrosis, arterial thrombosis, narrowing of arterial lumen with marked arterial hyperplastic arterial changes that were observed in vehicle treated SHR-L-NAME. L-NAME-induced increase in urine volume and protein was also significantly lower in FPTIII-treated animals. The impaired vascular responsiveness to isoprenaline in the perfused mesenteric vascular bed of SHR-L-NAME-treated animals was significantly attenuated by FPTIII treatment. In isolated perfused hearts, recovery of left ventricular function from a 40 min of global ischemia was significantly better in FPTIII-treated SHR-L-NAME. Treatment with FPTIII also significantly reduced expression of cardiac sodium-hydrogen exchanger-1 (NHE-1) which was elevated in SHR-L-NAME. These data indicate that inhibition of Ras-GTPase-mediated signalling can attenuate end-organ damage during severe hypertension and endothelial dysfunction.

Animals↗
PubMed2005

Diabetes-induced renal vascular dysfunction is normalized by inhibition of epidermal growth factor receptor tyrosine kinase.

Contribution of receptor tyrosine kinase activation to development of diabetes-induced renal artery dysfunction is not known. We investigated the ability of a chronic administration of genistein, a broad-spectrum inhibitor of tyrosine kinases (TKs), and AG1478, a specific inhibitor of epidermal growth factor receptor (EGFR) TK activity, to modulate the altered vasoreactivity of isolated renal artery ring segments to common vasoconstrictors in streptozotocin-induced diabetes. In diabetic renal artery, the vasoconstrictor responses induced by norepinephrine, endothelin-1 and angiotensin II were significantly increased. Inhibition of TKs or the EGFR pathway did not affect the agonist-induced vasoconstrictor responses in the non-diabetic control animals. However, inhibition of TKs by genistein or EGFR TK by AG1478 treatment produced a significant normalization of the altered agonist-induced vasoconstrictor responses without affecting blood glucose levels. Treatment with diadzein, an inactive analogue of genistein, did not affect the vasoconstrictor responses in the diabetic animals. Western blotting showed that phosphorylated EGFR protein levels were increased in vehicle-treated diabetic animals. In renal arteries from AG1478-treated diabetic animals, EGFR protein levels were similar to non-diabetic control animals. These data suggest that activation of TK-mediated pathways, including the EGFR TK signalling pathway, are involved in the development of diabetic vascular dysfunction in the renal artery.

Angiotensin II↗
PubMed2005

Signal transduction mechanisms involved in cardiac preconditioning: role of Ras-GTPase, Ca2+/calmodulin-dependent protein kinase II and epidermal growth factor receptor.

It is well established that brief episodes of ischemia/reperfusion (I/R) [preconditioning (PC)] protect the myocardium from the damage induced by subsequent more prolonged I/R. However, the signaling pathways activated during PC or I/R are not well characterized. In this study, the role of Ras-GTPase, tyrosine kinases (TKs), epidermal growth factor receptor (EGFR) and Ca2+/calmodulin-dependent protein kinase II (CaMK II) in mediating PC in a perfused rat heart model was investigated. A 40-min episode of global ischemia in perfused rat hearts produced significantly impaired cardiac function, measured as left ventricular developed pressure (Pmax) and left ventricular end-diastolic pressure (LVEDP), and impaired coronary hemodynamics, measured as coronary flow (CF) and coronary vascular resistance (CVR). PC significantly enhanced cardiac recovery after IR. Combination of PC and FPT III (Ras-GTPase inhibitor FPT III; 232 ng/min for 6 days) treatment did not produce any additive benefits as compared to PC alone. In contrast, PC-induced improvements in cardiac function after I/R were significantly attenuated by pretreatment with genistein (1mg/kg/day for 6 days), a broad-spectrum inhibitor of TKs, or AG1478 (1mg/kg/day for 6 days), a specific inhibitor of EGFR tyrosine kinase or KN-93 (578 ng/min for 6 days), a CaMK II inhibitor, before PC. These observations suggest that PC and FPT III pretreatment may produce cardioprotection via similar mechanisms. Present results also indicate that activation of TKs and specifically activation of EGFR-mediated TKs and CaMK II-mediated regulation of calcium homeostasis are part of the PC mechanisms that improve recovery after IR.

Animals↗
PubMed2005

Inhibition of Ca2+/calmodulin-dependent protein kinase II, RAS-GTPase and 20-hydroxyeicosatetraenoic acid attenuates the development of diabetes-induced vascular dysfunction in the rat carotid artery.

Diabetes causes accelerated vascular dysfunction through mechanisms that are poorly understood. This study examined the role of Ca2+/calmodulin-dependent protein kinase II (CaMKII), Ras-GTPase and 20-hydroxyeicosatetraenoic acid (20-HETE) in the development of abnormal reactivity to vasoactive agents in the carotid artery of diabetic rats. The vasoconstrictor response induced by endothelin-1 (ET-1) was significantly increased, whereas vasodilator response to carbachol was significantly reduced in the carotid artery segments of the STZ-diabetic rats. In contrast, the vasoconstrictor response to depolarization of the carotid arterial rings with 50mM KCl was similar in control and diabetic animals. Chronic intraperitoneal administration of KN-93 (5 mg/kg/alt diem), an inhibitor of CaMKII, FPTIII (1.5 mg/kg/alt diem), an inhibitor of Ras-GTPase, and inhibitors of 20-HETE formation 1-aminobenzotriazole (ABT, 50 mg/kg/alt diem) and N-hydroxy-N'-(4-butyl-2-methylphenyl)formamidine (HET0016, 2.5mg/kg/day), produced significant normalization of the altered agonist-induced vasoconstrictor and vasodilator responses without affecting blood glucose levels. All the inhibitors were administered for 4 weeks starting from the day 1 of diabetes induction. Inhibition of CaMKII, Ras-GTPase or 20-HETE formation did not affect the agonist-induced vasoconstrictor and vasodilator responses in the non-diabetic control animals. These data indicate that chronic blockade of CaMKII, Ras-GTPase or the production of 20-HETE normalizes the altered vascular reactivity to ET-1 and carbachol in the carotid artery of STZ-induced diabetic rats.

Amidines↗
PubMed2005

Epidermal growth factor receptor tyrosine kinase-mediated signalling contributes to diabetes-induced vascular dysfunction in the mesenteric bed.

In order to characterize the roles of tyrosine kinases (TKs) and epidermal growth factor receptor (EGFR) in diabetes-induced vascular dysfunction, we investigated the ability of a chronic administration of genistein, a broad-spectrum inhibitor of TKs and AG1478, a specific inhibitor of EGFR TK activity to modulate the altered vasoreactivity of the perfused mesenteric bed to common vasoconstrictors and vasodilators in streptozotocin (STZ)-induced diabetes in rats. The vasoconstrictor responses induced by norepinephrine (NE), endothelin-1 (ET-1) and angiotensin II (Ang II), were significantly increased, whereas vasodilator responses to carbachol and histamine were significantly reduced in the perfused mesenteric bed of STZ-induced diabetic rats in comparison with healthy rats. Treatment of diabetic animals with genistein or AG1478 produced a significant normalization of the altered agonist-induced vasoconstrictor and vasodilator responses without affecting blood glucose levels. In contrast, neither inhibitor had any effect on the vascular responsiveness of control (nondiabetic) animals. Treatment of diabetic animals with diadzein, an inactive analogue of genistein, did not affect the vasoconstrictor and vasodilator responses in control or diabetic animals. Phosphorylated EGFR levels were markedly raised in the mesenteric bed from diabetic animals and were normalized upon treatment with AG1478 or genistein. These data suggest that activation of TK-mediated pathways, including EGFR TK signalling are involved in the development of diabetic vascular dysfunction.

Angiotensin II↗
PubMed2005

Structural rearrangements in RNA on the binding of an antisense oligonucleotide: implications for the study of intra-molecular RNA interactions and the design of cooperatively acting antisense reagents with enhanced efficacy.

We show that binding of an antisense oligonucleotide can lead to considerable changes in the target mRNA structure. The approaches described here are not only useful in the study of intra-molecular interactions in RNAs but can also be used to design oligonucleotides that facilitate binding of other antisense reagents. Such "cooperatively acting" antisense reagents have the potential to overcome several problems faced in their use, for example, low efficacy and non-specificity. To provide proof-of-principle, radiolabelled cyclin B5 transcript, a model mRNA, was hybridised with an antisense oligonucleotide array. An oligonucleotide sequence was selected from the array hybridisation data and was used in an RNase H/oligonucleotide library (dN12) assay to assess its ability to enhance cleavage of target RNA. This oligonucleotide ("facilitator") greatly enhanced cleavage of B5 RNA at a neighbouring site. The precise position and sequence of this "new" site was determined by further hybridisation of RNA-facilitator mixture to the B5 antisense array. Antisense oligonucleotides designed from the new region were used in combination with the facilitator in a cell-free system. The presence of the facilitator considerably enhanced cleavage of B5 RNA with these oligonucleotides. These approaches may be useful in designing antisense reagents against sequences of specific interest, such as, gene fusion sites, splice variants, mutant alleles and tightly structured RNA sites.

Antisense Elements (Genetics)↗
PubMed2004

Increased 5-HT2A receptor expression and function following central glucocorticoid receptor knockdown in vivo.

Central glucocorticoid receptor function may be reduced in depression. In vivo modelling of glucocorticoid receptor underfunctionality would assist in understanding its role in depressive illness. The role of glucocorticoid receptors in modulating 5-HT(2A) receptor expression and function in the central nervous system (CNS) is presently unclear, but 5-HT(2A) receptor function also appears altered in depression. With the aid of RNAse H accessibility mapping, we have developed a 21-mer antisense oligodeoxynucleotide (5'-TAAAAACAGGCTTCTGATCCT-3', termed GRAS-5) that showed 56% reduction in glucocorticoid receptor mRNA and 80% down-regulation in glucocorticoid receptor protein in rat C6 glioma cells. Sustained delivery to rat cerebral ventricles in slow release biodegradable polymer microspheres produced a marked decrease in glucocorticoid receptor mRNA and protein in hypothalamus (by 39% and 80%, respectively) and frontal cortex (by 26% and 67%, respectively) 5 days after a single injection, with parallel significant up-regulation of 5-HT(2A) receptor mRNA expression (13%) and binding (21%) in frontal cortex. 5-HT(2A) receptor function, determined by DOI-head-shakes, showed a 55% increase. These findings suggest that central 5-HT(2A) receptors are, directly or indirectly, under tonic inhibitory control by glucocorticoid receptor.

Animals↗
PubMed2004

A novel anionic dendrimer for improved cellular delivery of antisense oligonucleotides.

The optimal design of hybridisation-competent antisense oligonucleotides (ODNs) coupled with an efficient delivery system appear to be important prerequisites for the successful use of antisense reagents for gene silencing. We selected an antisense ODN complementary to an accessible region of the epidermal growth factor receptor (EGFR) mRNA with the aid of an antisense oligonucleotide scanning array. The scanning array comprised 2684 antisense ODN sequences targeting the first 120 nts in the coding region of EGFR mRNA. The array-designed antisense ODN was covalently conjugated to a novel anionic dendrimer using a pentaerythritol-based phosphoroamidite synthon via automated DNA synthesis and the ability of this conjugate to effectively deliver and down-regulate EGFR expression in cancer cells was evaluated. Each dendrimeric structure had nine ODN molecules covalently linked to a common centre at their 3' termini. This dendrimer conjugate was markedly more stable to serum nucleases compared to the free ODNs and the cellular uptake of ODN-dendrimer conjugates was up to 100-fold greater as compared to mannitol, a marker for fluid phase endocytosis, and up to 4-fold greater than naked ODN in cancer cells. ODN-dendrimer uptake was energy-dependent and mediated, at least in part, via binding to cell surface proteins; a process that was inhibited by self-competition and by competition with free ODN, salmon sperm DNA, heparin and dextran sulphate. Fluorescent microscopy studies showed a combination of punctate and more diffuse cytosolic distribution pattern for fluorescently labelled ODN-dendrimer conjugate in A431 cells implying internalization by endocytosis followed by release and sequestration of the conjugate into the cytosol. Little or no conjugate appeared to be present in the nuclei of A431 cells. In vitro RNase H-mediated cleavage assays confirmed that covalently conjugated antisense ODNs in the dendrimer conjugate were able to hybridize and cleave the array-defined hybridisation target site within the EGFR mRNA without the need for ODN dissociation from the conjugate. In cell culture, ODN-dendrimer conjugates were effective in inhibiting cancer cell growth that correlated with a marked knockdown in EGFR protein expression. These data highlight a novel anionic dendrimer delivery system for gene silencing oligonucleotides that improved their biological stability, cellular delivery and antisense activity in cultured cancer cells.

Animals↗
PubMed2004

Inhibition of Ras-GTPase, but not tyrosine kinases or Ca2+/calmodulin-dependent protein kinase II, improves recovery of cardiac function in the globally ischemic heart.

The signaling pathways involved in ischemic heart disease are not well characterized. In this study, the roles of Ras-GTPase, tyrosine kinases (TKs) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) in global ischemia and reperfusion (I/R) in a perfused rat heart model were investigated and compared to beneficial effects produced by preconditioning (PC). A 40 min episode of global ischemia followed by a 30 min reperfusion in perfused rat hearts produced significantly impaired cardiac function, measured as left ventricular developed pressure (Pmax) and left ventricular end-diastolic pressure (LVEDP), and impaired coronary hemodynamics, measured as coronary flow (CF) and coronary vascular resistance (CVR). Hearts from male Wistar rats pre-treated with the tyrosine kinase inhibitor, genistein (1 mg/kg/day for 6 days), or the CaMKII inhibitor, KN-93 (578 ng/min for 6 days), produced detrimental effects on recovery of cardiac function and coronary hemodynamics. In contrast, pre-treatment with Ras-GTPase inhibitor FPT III (232 ng/min for 6 days) significantly enhanced cardiac recovery in terms of left ventricular contractility and coronary vascular hemodynamics. Treatment with FPT III also significantly reduced expression of the sodium-hydrogen exchanger-1 (NHE-1) which was elevated during I/R as detected by Western blotting. These data suggest that TKs and CaMKII are involved in signaling pathways leading to recovery from cardiac ischemia, whereas activation of Ras-GTPase signaling pathways are critical in the development of cardiac dysfunction due to I/R.

Alkyl and Aryl Transferases↗
PubMed2004

Evaluation of generation 2 and 3 poly(propylenimine) dendrimers for the potential cellular delivery of antisense oligonucleotides targeting the epidermal growth factor receptor.

PURPOSE: To evaluate low generation, G2 and G3, poly(propylenimine) dendrimers for the potential cellular delivery of antisense oligonucleotides (ODNs) targeting the epidermal growth factor receptor (EGFR) in A431 epidermoid carcinoma cells. METHODS: Cell cytotoxicity of the dendrimers was evaluated using trypan blue exclusion assays. Cellular uptake studies of fluorescently labeled ODNs were performed using fluorescence-activated cell sorting analysis. Intracellular fate of dendrimer-delivered ODNs was assessed in both fixed and live cells using fluorescent microscopy. Antisense ODN activity was assessed in terms of cancer cell growth, inhibition of target EGFR protein, and reduction in mRNA levels. RESULTS: G2 dendrimer (DAB-8) was less toxic than G3 (DAB-16) dendrimer in A431 cells, with IC50 of >175 and approximately 30 microg/ml, respectively. Uptake of fluorescently labeled ODN:dendrimer complexes was increased by up to 100-fold compared to a marker of fluid-phase endocytosis and up to 9-fold over free ODN at the optimal dendrimer:ODN (w/w) ratio of 5:1. Uptake of dendrimer:ODN complexes was significantly reduced at 4 degrees C (p < 0.05). Live cell fluorescent microscopy resulted in an intracellular distribution of dendrimer:ODN complexes that was suggestive of endocytic uptake; in contrast, cell fixation resulted in an artefactual nuclear localization. Treatment of A431 cells with anti-EGFR antisense ODN:dendrimer complexes inhibited cell growth, protein, and mRNA expression to levels comparable to Oligofectamine-mediated delivery. CONCLUSIONS: G2 and G3 poly(propylenimine) dendrimers markedly improved the delivery and activity of ODNs and thus may represent general reagents for the delivery of ODNs to cells in culture.

Dendrimers↗
PubMed2004

Sustained polymeric delivery of gene silencing antisense ODNs, siRNA, DNAzymes and ribozymes: in vitro and in vivo studies.

Small interfering RNA (siRNA), antisense oligonucleotides (ODNs), ribozymes and DNAzymes have emerged as sequence-specific inhibitors of gene expression that may have therapeutic potential in the treatment of a wide range of diseases. Due to their rapid degradation in vivo, the efficacy of naked gene silencing nucleic acids is relatively short lived. The entrapment of these nucleic acids within biodegradable sustained-release delivery systems may improve their stability and reduce the doses required for efficacy. In this study, we have evaluated the potential in vitro and in vivo use of biodegradable poly (D,L-lactide-co-glycolide) copolymer (PLGA) microspheres as sustained delivery devices for ODNs, ribozyme, siRNA and DNA enzymes. In addition, we investigated the release of ODN conjugates bearing 5'-end lipophilic groups. The in vitro sustained release profiles of microsphere-entrapped nucleic acids were dependent on variables such as the type of nucleic acid used, the nature of the lipophilic group, and whether the nucleic acid used was single or double stranded. For in vivo studies, whole body autoradiography was used to monitor the bio-distribution of either free tritium-labelled ODN or that entrapped within PLGA microspheres following subcutaneous administration in Balb-c mice. The majority of the radioactivity associated with free ODN was eliminated within 24 h whereas polymer-released ODN persisted in organs and at the site of administration even after seven days post-administration. Polymer microsphere released ODN exhibited a similar tissue and cellular tropism to the free ODN. Micro-autoradiography analyses of the liver and kidneys showed similar bio-distribution for polymer-released and free ODNs with the majority of radioactivity being concentrated in the proximal convoluted tubules of the kidney and in the Kupffer cells of the liver. These findings suggest that biodegradable PLGA microspheres offer a method for improving the in vivo sustained delivery of gene silencing nucleic acids, and hence are worthy of further investigation as delivery systems for these macromolecules.

Animals↗
PubMed2004

The design and exogenous delivery of siRNA for post-transcriptional gene silencing.

RNA interference (RNAi) is a natural cellular process that effects post-transcriptional gene silencing in eukaryotic systems. Small interfering RNA (siRNA) molecules are the key intermediaries in this process which when exogenously administered can inhibit or "silence" the expression of any given target gene. Thus, siRNA molecules hold great promise as biological tools and as potential therapeutic agents for targeted inhibition of disease-causing genes. However, key challenges to the effective and widespread use of these polyanionic, macromolecular duplexes of RNA are their appropriate design and efficient delivery to cells in vitro and in vivo. This review highlights the current strategies used in the design of effective siRNA molecules and also summarises the main strategies being considered for the exogenous delivery of siRNA for both in vitro and in vivo applications.

Animals↗
PubMed2003

Messenger RNA expression profiling of genes involved in epidermal growth factor receptor signalling in human cancer cells treated with scanning array-designed antisense oligonucleotides.

Scanning oligodeoxynucleotide (ODN) arrays appear promising in vitro tools for the prediction of effective antisense reagents but their usefulness has not yet been reported in mammalian systems. In this study, we have evaluated the use of scanning ODN arrays to predict efficacious antisense ODNs targeting the human epidermal growth factor receptor (EGFR) mRNA in a human epidermoid cancer cell line and in primary human glioma cells. Hybridisation accessibility profile of the first 120nt in the coding region of the human EGFR mRNA was determined by hybridising a radiolabelled EGFR transcript to a scanning array of 2684 antisense sequences ranging from monomers to 27-mers. Two ODNs, AS1 and AS2, complementary to accessible sequences within the EGFR mRNA, were designed and their ability to hybridise to EGFR mRNA was further confirmed by in vitro RNase H-mediated cleavage assays. Phosphorothioate-modified 21-mer AS1 and AS2 ODNs inhibited the growth of an established human A431 cancer cell line as well as primary glioma cells from human subjects when delivered as cationic lipoplexes. In contrast, scrambled controls and AS3-an antisense ODN complementary to an inaccessible site in EGFR mRNA-were inactive. Western blots showed that AS1 ODN exhibited a dose-dependent inhibition of EGFR protein expression in A431 cells in the nanomolar range. Microarray-based gene expression profiling studies of A431 cells treated with the 21-mer phosphorothioate AS1 ODN demonstrated successful inhibition of downstream signalling molecules further confirming the effective inhibition of EGFR expression in human cancer cells by antisense ODNs designed by scanning ODN array technology.

Cell Division↗
PubMed2003

Toxicogenomics of non-viral vectors for gene therapy: a microarray study of lipofectin- and oligofectamine-induced gene expression changes in human epithelial cells.

Of the non-viral vectors, cationic lipid (CL) formulations are the most widely studied for the delivery of genes, antisense oligonucleotides and gene silencing nucleic acids such as small interfering RNAs. However, little is known about the impact of these delivery systems on global gene expression in target cells. In an attempt to study the geno-compatibility of CL formulations in target cells, we have used microarrays to examine the effect of Lipofectin and Oligofectamine on the gene expression profiles of human A431 epithelial cells. Using the manufacturer's recommended CL concentrations routinely used for gene delivery, cDNA microarray expression profiling revealed marked changes in the expression of several genes for both Lipofectin- and Oligofectamine-treated cells. Data from the 200 spot arrays housing 160 different genes indicated that Lipofectin or Oligofectamine treatment of A431 cells resulted in more than 2-fold altered expression of 10 and 27 genes, respectively. The downstream functional consequences of CL-induced gene expression alterations led to an increased tendency of cells to enter early apoptosis as assessed by annexin V-FITC flow cytometry analyses. This effect was greater for Oligofectamine than Lipofectin. Observed gene expression changes were not sufficient to induce any significant DNA damage as assessed by single cell gel electrophoresis (COMET) assay. These data highlight the fact that inadvertent gene expression changes can be induced by the delivery formulation alone and that these may, ultimately, have important safety implications for the use of these non-viral vectors in gene-based therapies. Also, the induced non-target gene changes should be taken into consideration in gene therapy or gene silencing experiments using CL formulations where they may potentially mask or interfere with the desired genotype and/or phenotype end-points.

Annexin A5↗
PubMed2003

Gene silencing nucleic acids designed by scanning arrays: anti-EGFR activity of siRNA, ribozyme and DNA enzymes targeting a single hybridization-accessible region using the same delivery system.

Gene silencing nucleic acids such as ribozymes, DNA enzymes (DNAzymes), antisense oligonucleotides (ODNs), and small interfering (si)RNA rely on hybridization to accessible sites within target mRNA for activity. However, the accurate prediction of hybridization accessible sites within mRNAs for design of effective gene silencing reagents has been problematic. Here we have evaluated the use of scanning arrays for the effective design of ribozymes, DNAzymes and siRNA sequences targeting the epidermal growth factor receptor (EGFR) mRNA. All three gene silencing nucleic acids designed to be complementary to the same array-defined hybridization accessible-site within EGFR mRNA were effective in inhibiting the growth of EGFR over-expressing A431 cancer cells in a dose dependent manner when delivered using the cationic lipid (Lipofectin) delivery system. Effects on cell growth were correlated in all cases with concomitant dose-dependent reduction in EGFR protein expression. The control sequences did not markedly alter cell growth or EGFR expression. The ribozyme and DNAzyme exhibited similar potency in inhibiting cell growth with IC50 values of around 750 nM. In contrast, siRNA was significantly more potent with an IC50 of about 100 nM when delivered with Lipofectin. The potency of siRNA was further enhanced when Oligofectamine was used to further improve both the cellular uptake and subcellular distribution of fluorescently labelled siRNA. Our studies show that active siRNAs can be designed using hybridization accessibility profiles on scanning arrays and that siRNAs targeting the same array-designed hybridization accessible site in EGFR mRNA and delivered using the same delivery system are more potent than ribozymes and DNAzymes in inhibiting EGFR expression in A431 cells.

Blotting, Western↗
PubMed2002

Sequence and chemistry requirements for a novel aptameric oligonucleotide inhibitor of EGF receptor tyrosine kinase activity.

We have previously identified a phosphorothioate oligonucleotide (PS-ODN) that inhibited epidermal growth factor receptor tyrosine kinase (TK) activity both in cell fractions and in intact A431 cells. Since ODN-based TK inhibitors may have anti-cancer applications and may also help understand the non-antisense mediated effects of PS-ODNs, we have further studied the sequence and chemistry requirements of the parent PS-ODN (sequence: 5'-GGA GGG TCG CAT CGC-3') as a sequence-dependent TK inhibitor. Sequence deletion and substitution studies revealed that the 5'-terminal GGA GGG hexamer sequence in the parent compound was essential for anti-TK activity in A431 cells. Site-specific substitution of any G with a T in this 5'-terminal motif within the parent compound caused a significant loss in anti-TK activity. The fully PS-modified hexameric motif alone exhibited equipotent activity as the parent 15-mer whereas phosphodiester (PO) or 2'-O-methyl-modified versions of this motif had significantly reduced anti-TK activity. Further, T substitutions within the two 5'-terminal G residues of the hexameric PS-ODN to produce a sequence, TTA GGG, representing the telomeric repeats in human chromosomes, also did not exhibit a significant anti-TK activity. Multiple repeats of the active hexameric motif in PS-ODNs resulted in more potent inhibitors of TK activity than the parent ODN. These results suggested that PS-ODNs, but not PO or 2'-O-methyl modified ODNs, containing the GGA GGG motif can exert potent anti-TK activity which may be desirable in some anti-tumor applications. Additionally, the presence of this previously unidentified motif in antisense PS-ODN constructs may contribute to their biological effects in vitro and in vivo and should be accounted for in the design of the PS-modified antisense ODNs.

Animals↗