Phosphorothioate-modified Antisense Oligonucleotides Research Articles

Epidermal growth factor receptor mediates the basolateral uptake of phosphorothioate-modified antisense oligonucleotides in the kidney

Antisense oligonucleotides (ASOs) are highly biologically stable and specific therapeutic molecules that interfere with mRNA transcription and as a result effectively reduce the expression of a protein of interest. ASOs have low drug clearance and are retained in tissues. This is particularly evident in the kidneys where they accumulate in the renal proximal tubules. Receptor-mediated endocytosis (RME) plays a role in ASO uptake, however the membrane receptors facilitating the process and the full mechanism behind these transport and subsequent renal retention is still poorly understood. In the present study we employ a proximal tubule-on-a-chip (PTOC) that recapitulates the highly polarized nature of the renal epithelium and can discriminate between basal and apical uptake processes. The PTOC was used to determine the impact of cellular polarization in ASO uptake in the kidney and elucidate which receptors predominantly facilitated uptake. In the PTOC the uptake occurred predominantly from the basolateral side and was extensively inhibited using the selective epidermal growth factor receptor (EGFR) antagonist cetuximab. These results demonstrate that ASO uptake in a physiologically relevant model is predominately mediated by the EGFR and takes place from the basolateral side of the proximal tubules. In comparison to the 2D culture, PTOC could differentiate between distinct ASO uptake routes mediated by either Megalin or EGFR, which proper membrane expression is highly dependent on cellular polarity. Our results highlight the limitations of renal 2D models and demonstrate how an organ on a chip model can fill the physiological gap and is a powerful tool to study ASO, which have intricate transport mechanisms that ultimately impact both their efficacy and safety. • Continuously perfused proximal tubule-on-a-chip model recreates a highly polarized renal epithelium. • Uptake of antisense-oligonucleotides occurs from the basolateral side of the renal proximal tubules. • EGFR drives antisense-oligonucleotide uptake in our proximal tubule-on-a-chip model, an effect not observed in 2D culture.

Structures of annexin A2-PS DNA complexes show dominance of hydrophobic interactions in phosphorothioate binding.

The introduction of phosphorothioate (PS) linkages to the backbone of therapeutic nucleic acids substantially increases their stability and potency. It also affects their interactions with cellular proteins, but the molecular mechanisms that underlie this effect are poorly understood. Here, we report structural and biochemical studies of interactions between annexin A2, a protein that does not possess any known canonical DNA binding domains, and phosphorothioate-modified antisense oligonucleotides. We show that a unique mode of hydrophobic interactions between a sulfur atom of the phosphorothioate group and lysine and arginine residues account for the enhanced affinity of modified nucleic acid for the protein. Our results demonstrate that this mechanism of interaction is observed not only for nucleic acid-binding proteins but can also account for the association of PS oligonucleotides with other proteins. Using the anomalous diffraction of sulfur, we showed that preference for phosphorothioate stereoisomers is determined by the hydrophobic environment around the PS linkage that comes not only from protein but also from additional structural features within the ASO such as 5-Me groups on cytosine nucleobases.

RNA modifications can affect RNase H1-mediated PS-ASO activity

Phosphorothioate modified antisense oligonucleotides (PS-ASOs) can reduce gene expression through hybridization to target RNAs and subsequent cleavage by RNase H1. Target reduction through this mechanism is influenced by numerous features of the RNA, which modulate PS-ASO binding affinities to the RNA target, and how the PS-ASO-RNA hybrid is recognized by RNase H1 for RNA cleavage. Endogenous RNAs are frequently chemically modified, which can regulate intra- and intermolecular interactions of the RNA. The effects of PS-ASO modifications on antisense activity have been well studied; however, much less is known regarding the effects of RNA modifications on PS-ASO hybridization and RNase H1 cleavage activity. Here, we determine the effects of three different RNA modifications on PS-ASO binding and antisense activity in recombinant and cell-based systems. Some RNA modifications can reduce PS-ASO hybridization, the cleavage activity of RNase H1, or both, while other modifications had minimal effects on PS-ASO function. In addition to these direct effects, RNA modifications can also change the RNA structure, which may affect PS-ASO accessibility in a cellular context. Our results elucidate the effects of three prevalent RNA modifications on PS-ASO-mediated RNase H1 cleavage activity, and such findings will help improve PS-ASO target site selection.

Hsc70 Facilitates Mannose-6-Phosphate Receptor-Mediated Intracellular Trafficking and Enhances Endosomal Release of Phosphorothioate-Modified Antisense Oligonucleotides.

Phosphorothioate-modified antisense oligonucleotide (PS-ASO) drugs are commonly used to modulate gene expression through RNase H1-mediated cleavage of target RNAs. Upon internalization through endocytic pathways into cells, PS-ASOs must be released from membraned endosomal organelles to act on target RNAs, a limiting step of PS-ASO activity. Here we report that Hsc70 protein mediates productive release of PS-ASOs from endosomes. Hsc70 protein was enriched in endosome fractions shortly after PS-ASO incubation with cells. Reduction of Hsc70 significantly decreased the activities of PS-ASOs in reducing target RNAs. PS-ASO uptake and transport from early endosomes to late endosomes (LEs) were not affected upon Hsc70 reduction; however, endosomal release of PS-ASOs was impaired. Reduction of Hsc70 led to more scattered mannose-6-phosphate receptor (M6PR) localization at LEs in the cytoplasm, in contrast to the perinuclear localization at trans-Golgi network (TGN) in control cells, suggesting that retrograde transport of M6PR from LEs to TGN was affected. Consistently, reduction of Hsc70 increased colocalization of M6PR and PS-ASOs at LEs, and also delayed M6PR antibody transport from LE to TGN. Together, these results suggest that Hsc70 protein is involved in M6PR vesicle escape from LEs and may thus enhance PS-ASO release from LEs.

Phosphorothioate Antisense Oligonucleotides Bind P-Body Proteins and Mediate P-Body Assembly

Antisense oligonucleotides (ASOs) regulate gene expression by binding to complementary target RNA, and ASOs can be designed to take advantage of a growing array of post RNA binding molecular mechanisms. Intracellular trafficking of ASOs influences their efficacy. We have identified a number of membrane-less structures in the nucleus, nucleolus, and cytoplasm where phosphorothioate-modified ASOs (PS-ASOs) accumulate and have shown that PS-ASOs can induce the formation of new nuclear structures such as PS-bodies and paraspeckle-like structures. In this study, we report that PS-ASOs can localize to cytoplasmic processing bodies (P-bodies) and increase the number of P-bodies in cells. The antisense activity of PS-ASOs was not affected by the absence of essential P-body assembly proteins DDX6 and LSm14A. Moreover, the effects of PS-ASOs on P-body assembly were independent of their antisense activities. The phosphorothioate modification stabilizes the association between ASOs and cellular proteins and is essential for the P-body localization of ASOs. Since PS-ASOs bind to major P-body components, PS-ASOs may serve as scaffolds for P-body formation. Taken together, these results indicate that interactions of PS-ASO with proteins, rather than antisense activities, are essential for the dynamic interplay between PS-ASOs and P-bodies.

Site-specific replacement of phosphorothioate with alkyl phosphonate linkages enhances the therapeutic profile of gapmer ASOs by modulating interactions with cellular proteins.

Phosphorothioate-modified antisense oligonucleotides (PS-ASOs) interact with a host of plasma, cell-surface and intracellular proteins which govern their therapeutic properties. Given the importance of PS backbone for interaction with proteins, we systematically replaced anionic PS-linkages in toxic ASOs with charge-neutral alkylphosphonate linkages. Site-specific incorporation of alkyl phosphonates altered the RNaseH1 cleavage patterns but overall rates of cleavage and activity versus the on-target gene in cells and in mice were only minimally affected. However, replacing even one PS-linkage at position 2 or 3 from the 5′-side of the DNA-gap with alkylphosphonates reduced or eliminated toxicity of several hepatotoxic gapmer ASOs. The reduction in toxicity was accompanied by the absence of nucleolar mislocalization of paraspeckle protein P54nrb, ablation of P21 mRNA elevation and caspase activation in cells, and hepatotoxicity in mice. The generality of these observations was further demonstrated for several ASOs versus multiple gene targets. Our results add to the types of structural modifications that can be used in the gap-region to enhance ASO safety and provide insights into understanding the biochemistry of PS ASO protein interactions.

COPII vesicles can affect the activity of antisense oligonucleotides by facilitating the release of oligonucleotides from endocytic pathways.

RNase H1-dependent, phosphorothioate-modified antisense oligonucleotides (PS-ASOs) can enter cells through endocytic pathways and need to be released from the membrane-enclosed organelles, a limiting step for antisense activity. Accumulating evidence has suggested that productive PS-ASO release mainly occurs from late endosomes (LEs). However, how PS-ASOs escape from LEs is not well understood. Here, we report that upon PS-ASO incubation, COPII vesicles, normally involved in ER–Golgi transport, can re-locate to PS-ASO-containing LEs. Reduction of COPII coat proteins significantly decreased PS-ASO activity, without affecting the levels of PS-ASO uptake and early-to-late endosome transport, but caused slower PS-ASO release from LEs. COPII co-localization with PS-ASOs at LEs does not require de novo assembly of COPII at ER. Interestingly, reduction of STX5 and P115, proteins involved in tethering and fusion of COPII vesicles with Golgi membranes, impaired COPII re-localization to LEs and decreased PS-ASO activity. STX5 can re-locate to LEs upon PS-ASO incubation, can bind PS-ASOs, and the binding appears to be required for this pathway. Our study reveals a novel release pathway in which PS-ASO incubation causes LE re-localization of STX5, which mediates the recruitment of COPII vesicles to LEs to facilitate endosomal PS-ASO release, and identifies another key PS-ASO binding protein.

Cellular uptake mediated by epidermal growth factor receptor facilitates the intracellular activity of phosphorothioate-modified antisense oligonucleotides.

Chemically modified antisense oligonucleotides (ASOs) with phosphorothioate (PS) linkages have been extensively studied as research and therapeutic agents. PS-ASOs can enter the cell and trigger cleavage of complementary RNA by RNase H1 even in the absence of transfection reagent. A number of cell surface proteins have been identified that bind PS-ASOs and mediate their cellular uptake; however, the mechanisms that lead to productive internalization of PS-ASOs are not well understood. Here, we characterized the interaction between PS-ASOs and epidermal growth factor receptor (EGFR). We found that PS-ASOs trafficked together with EGF and EGFR into clathrin-coated pit structures. Their co-localization was also observed at early endosomes and inside enlarged late endosomes. Reduction of EGFR decreased PS-ASO activity without affecting EGF-mediated signaling pathways and overexpression of EGFR increased PS-ASO activity in cells. Furthermore, reduction of EGFR delays PS-ASO trafficking from early to late endosomes. Thus, EGFR binds to PS-ASOs at the cell surface and mediates essential steps for active (productive) cellular uptake of PS-ASOs through its cargo-dependent trafficking processes which migrate PS-ASOs from early to late endosomes. This EGFR-mediated process can also serve as an additional model to better understand the mechanism of intracellular uptake and endosomal release of PS-ASOs.

Intra-endosomal trafficking mediated by lysobisphosphatidic acid contributes to intracellular release of phosphorothioate-modified antisense oligonucleotides

Antisense oligonucleotides (ASOs) with phosphorothioate (PS) linkages are broadly used as research tools and therapeutic agents. Chemically modified PS-ASOs can mediate efficient target reduction by site-specific cleavage of RNA through RNase H1. PS-ASOs are known to be internalized via a number of endocytotic pathways and are released from membrane-enclosed endocytotic organelles, mainly late endosomes (LEs). This study was focused on the details of PS-ASO trafficking through endocytic pathways. It was found that lysobisphosphatidic acid (LBPA) is required for release of PS-ASOs from LEs. PS-ASOs exited early endosomes (EEs) rapidly after internalization and became co-localized with LBPA by 2 hours in LEs. Inside LEs, PS-ASOs and LBPA were co-localized in punctate, dot-like structures, likely intraluminal vesicles (ILVs). Deactivation of LBPA using anti-LBPA antibody significantly decreased PS-ASO activities without affecting total PS-ASO uptake. Reduction of Alix also substantially decreased PS-ASO activities without affecting total PS-ASO uptake. Furthermore, Alix reduction decreased LBPA levels and limited co-localization of LBPA with PS-ASOs at ILVs inside LEs. Thus, the fusion properties of ILVs, which are supported by LBPA, contribute to PS-ASO intracellular release from LEs.

Stabilin-1 and Stabilin-2 are specific receptors for the cellular internalization of phosphorothioate-modified antisense oligonucleotides (ASOs) in the liver.

Phosphorothioate (PS)-modified antisense oligonucleotides (ASOs) have been extensively investigated over the past three decades as pharmacological and therapeutic agents. One second generation ASO, Kynamro™, was recently approved by the FDA for the treatment of homozygous familial hypercholesterolemia and over 35 second generation PS ASOs are at various stages of clinical development. In this report, we show that the Stabilin class of scavenger receptors, which were not previously thought to bind DNA, do bind and internalize PS ASOs. With the use of primary cells from mouse and rat livers and recombinant cell lines each expressing Stabilin-1 and each isoform of Stabilin-2 (315-HARE and 190-HARE), we have determined that PS ASOs bind with high affinity and these receptors are responsible for bulk, clathrin-mediated endocytosis within the cell. Binding is primarily dependent on salt-bridge formation and correct folding of the intact protein receptor. Increased internalization rates also enhanced ASO potency for reducing expression of the non-coding RNA Malat-1, in Stabilin-expressing cell lines. A more thorough understanding of mechanisms by which ASOs are internalized in cells and their intracellular trafficking pathways will aid in the design of next generation antisense agents with improved therapeutic properties.

Phosphorothioate oligonucleotides can displaceNEAT1RNA and form nuclear paraspeckle-like structures

Nuclear paraspeckles are built co-transcriptionally around a long non-coding RNA, NEAT1. Here we report that transfected 20-mer phosphorothioate-modified (PS) antisense oligonucleotides (ASOs) can recruit paraspeckle proteins to form morphologically normal and apparently functional paraspeckle-like structures containing no NEAT1 RNA. PS-ASOs can associate with paraspeckle proteins, including P54nrb, PSF, PSPC1 and hnRNPK. NEAT1 RNA can be displaced by transfected PS-ASO from paraspeckles and rapidly degraded. Co-localization of PS-ASOs with P54nrb was observed in canonical NEAT1-containing paraspeckles, in perinucleolar caps upon transcriptional inhibition, and importantly, in paraspeckle-like or filament structures lacking NEAT1 RNA. The induced formation of paraspeckle-like and filament structures occurred in mouse embryonic stem cells expressing little or no NEAT1 RNA, suggesting that PS-ASOs can serve as seeding molecules to assemble paraspeckle-like foci in the absence of NEAT1 RNA. Moreover, CTN, an RNA reported to be functionally retained in paraspeckles, was also observed to localize to paraspeckle-like structures, implying that paraspeckle-like structures assembled on PS-ASOs are functional. Together, our results indicate that functional paraspeckles can form with short nucleic acids other than NEAT1 RNA.

WS1.5 Restoration of the CFTR function by antisense oligonucleotide splicing modulation

10−15% of mutations in the CFTR gene affect its correct splicing. The disease severity in patients carrying these mutations is highly variable, correlated with the level of aberrantly spliced transcripts. We aim to develop a splicing mutationspecific therapy using antisense oligonucleotides (AOs), to modulate the splicing pattern in patients carrying common splicing mutations. We focused on a common splicing mutation, the 3849+10kb C-to-T, which leads to inclusion of an 84 bp cryptic exon between exons 22−23 in the mature mRNA. This cryptic exon contains an in-frame stop codon that leads to degradation of a significant fraction of the mRNA by the NMD pathway as well as to the production of prematurely truncated nonfunctional proteins. We designed 4 2′-O-methyl phosphorothioatemodified AOs, targeted to prevent the recognition of enhancer splice motifs in the cryptic exon or to mask the junctions between this exon and its flanking sequences. Epithelial cell line established from a nasal polyp of a CF patient carrying the 3849+10kb C-to-T mutation was transfected with 2 of the designed AOs. AOstreated cells showed a highly significant decrease in the level of aberrantly spliced CFTR mRNA, along with a significant increase in normal spliced CFTR mRNA levels, reflecting transcripts that under normal conditions are degraded by the NMD pathway. These results indicate that AOs targeted to mask splicing motifs around the cryptic exon generated due to the 3849+10kb C-to-T splicing mutation, can modulate increase the correct splicing. Further studies are required to investigate whether these AOS can restore the CFTR function and improve patients’ clinical state. WS1.6 OligoG normalizes the CF mucus phenotype

WS1.6 OligoG normalizes the CF mucus phenotype

10−15% of mutations in the CFTR gene affect its correct splicing. The disease severity in patients carrying these mutations is highly variable, correlated with the level of aberrantly spliced transcripts. We aim to develop a splicing mutationspecific therapy using antisense oligonucleotides (AOs), to modulate the splicing pattern in patients carrying common splicing mutations. We focused on a common splicing mutation, the 3849+10kb C-to-T, which leads to inclusion of an 84 bp cryptic exon between exons 22−23 in the mature mRNA. This cryptic exon contains an in-frame stop codon that leads to degradation of a significant fraction of the mRNA by the NMD pathway as well as to the production of prematurely truncated nonfunctional proteins. We designed 4 2′-O-methyl phosphorothioatemodified AOs, targeted to prevent the recognition of enhancer splice motifs in the cryptic exon or to mask the junctions between this exon and its flanking sequences. Epithelial cell line established from a nasal polyp of a CF patient carrying the 3849+10kb C-to-T mutation was transfected with 2 of the designed AOs. AOstreated cells showed a highly significant decrease in the level of aberrantly spliced CFTR mRNA, along with a significant increase in normal spliced CFTR mRNA levels, reflecting transcripts that under normal conditions are degraded by the NMD pathway. These results indicate that AOs targeted to mask splicing motifs around the cryptic exon generated due to the 3849+10kb C-to-T splicing mutation, can modulate increase the correct splicing. Further studies are required to investigate whether these AOS can restore the CFTR function and improve patients’ clinical state. WS1.6 OligoG normalizes the CF mucus phenotype

Antisense correction of SMN2 splicing in the CNS rescues necrosis in a type III SMA mouse model

Increasing survival of motor neuron 2, centromeric (SMN2) exon 7 inclusion to express more full-length SMN protein in motor neurons is a promising approach to treat spinal muscular atrophy (SMA), a genetic neurodegenerative disease. Previously, we identified a potent 2'-O-(2-methoxyethyl) (MOE) phosphorothioate-modified antisense oligonucleotide (ASO) that blocks an SMN2 intronic splicing silencer element and efficiently promotes exon 7 inclusion in transgenic mouse peripheral tissues after systemic administration. Here we address its efficacy in the spinal cord--a prerequisite for disease treatment--and its ability to rescue a mild SMA mouse model that develops tail and ear necrosis, resembling the distal tissue necrosis reported in some SMA infants. Using a micro-osmotic pump, we directly infused the ASO into a lateral cerebral ventricle in adult mice expressing a human SMN2 transgene; the ASO gave a robust and long-lasting increase in SMN2 exon 7 inclusion measured at both the mRNA and protein levels in spinal cord motor neurons. A single embryonic or neonatal intracerebroventricular ASO injection strikingly rescued the tail and ear necrosis in SMA mice. We conclude that this MOE ASO is a promising drug candidate for SMA therapy, and, more generally, that ASOs can be used to efficiently redirect alternative splicing of target genes in the CNS.

Activation of extracellular signal-regulated protein kinases 5 in the spinal cord contributes to the neuropathic pain behaviors induced by CCI in rats

Objective: To investigate whether activation and translocation of extracellular signal-regulated kinase 5 (ERK5) is involved in the induction and maintenance of neuropathic pain and observe the effects of activation and translocation of ERK5 on the expression of phosphorylated cAMP response element binding (pCREB) in the chronic neuropathic pain.Methods: Lumbar intrathecal catheters were chronically implanted in male Sprague–Dawley rats. The left sciatic nerve was loosely ligated proximal to the sciatica's trifurcation at ∼ 1.0 mm intervals with 4-0 silk sutures. The phosphorothioate-modified antisense oligonucleotides (AS-ODNs) were intrathecally administered every 12 hours, 1 day pre-chronic constriction injury (CCI) and 3 day post-CCI. Thermal and mechanical nociceptive thresholds were assessed with the paw withdrawal latency to a radiant heat and von Frey filaments. Expressions of phosphorylated ERK5 (pERK5), pCREB, were assessed by both Western blotting and immunohistochemical analysis.Results: Intrathecal injection of ERK5 AS-ODN significantly attenuated CCI-induced mechanical allodynia and thermal hyperalgesia. CCI significantly increased the expression of pERK5 neurons in the ipsilateral spinal dorsal horn to injury, not in the contralateral spinal dorsal horn. The time courses of pERK5 expression showed that the levels of both cytosol and nuclear pERK5 were increased at all points after CCI and reached a peak level on post-operative day 5. CCI significantly increased the expression of pERK5 neurons in the laminae I and II of ipsilateral spinal dorsal horn to injury, not in the contralateral spinal dorsal horn. Phospho-CREB-positive neurons were distributed in all laminae of the bilateral spinal cord. Intrathecal injection AS-ODN markedly suppressed the increase of CCI-induced pERK5, pCREB expression in the spinal cord.Conclusion: The activation of ERK5 pathways contributes to neuropathic pain in CCI rats, and the function of pERK5 may partly be accomplished via the CREB protein-dependent gene expression.

Antitumor Mechanisms of Systemically Administered Epidermal Growth Factor Receptor Antisense Oligonucleotides in Combination with Docetaxel in Squamous Cell Carcinoma of the Head and Neck

Squamous cell carcinoma of the head and neck (SCCHN) is one of the most common malignancies worldwide, with low 5-year survival rates. Current strategies that block epidermal growth factor receptor (EGFR) have limited effects when administered as single agents. Targeting EGFR via intratumoral administration of phosphorothioate-modified antisense oligonucleotides has antitumor efficacy in xenograft models of SCCHN. Because intratumoral delivery of therapeutic agents has limited clinical application, the present study was undertaken to examine the therapeutic mechanisms of systemically delivered phosphorothioate-modified EGFR antisense oligonucleotides alone, or in combination with docetaxel, in a SCCHN xenograft model. EGFR antisense oligonucleotides were administered at 5 mg/kg i.p. daily in athymic mice bearing 1483 human SCCHN xenografts alone or in combination with docetaxel at 2.5 mg/kg i.p. once a week for 4 weeks. Administration of EGFR antisense oligonucleotides in combination with docetaxel improved antitumor efficacy and resulted in lower expression levels of EGFR, fewer proliferating cells, and more apoptotic cells in the tumors compared with controls. Systemic administration of phosphorothioated EGFR antisense oligonucleotides for 30 days increased the retention of docetaxel in the tumor by approximately 4-fold compared with tumors treated with docetaxel alone or docetaxel and EGFR sense oligonucleotides (P < 0.05). Combination of EGFR antisense oligonucleotides with low doses of docetaxel has antitumor efficacy, and it may be an effective treatment strategy for SCCHN.

MicroRNAs: A new class of regulatory genes affecting metabolism

MicroRNAs (miRNAs) are short noncoding RNAs that regulate gene expression by binding to target mRNAs, which leads to reduced protein synthesis and sometimes decreased steady-state mRNA levels. Although hundreds of miRNAs have been identified, much less is known about their biological function. Several studies have provided evidence that miRNAs affect pathways that are fundamental for metabolic control in higher organisms such as adipocyte and skeletal muscle differentiation. Furthermore, some miRNAs have been implicated in lipid, amino acid, and glucose homeostasis. These studies open the possibility that miRNAs may contribute to common metabolic diseases and point to novel therapeutic opportunities based on targeting of miRNAs.

Activation of ERK/CREB pathway in spinal cord contributes to chronic constrictive injury-induced neuropathic pain in rats1

To investigate whether activation and translocation of extracellular signal-regulated kinase (ERK) is involved in the induction and maintenance of neuropathic pain, and effects of activation and translocation of ERK on expression of pCREB and Fos in the chronic neuropathic pain. Lumbar intrathecal catheters were chronically implanted in male Sprague-Dawley rats. The left sciatic nerve was loosely ligated proximal to the sciaticaos trifurcation at approximately 1.0 mm intervals with 4-0 silk sutures. The mitogen-activated protein kinase kinase (MEK) inhibitor U0126 or phosphorothioate-modified antisense oligonucleotides (ODN) were intrathecally administered every 12 h, 1 d pre-chronic constriction injury (CCI) and 3 d post-CCI. Thermal and mechanical nociceptive thresholds were assessed with the paw withdrawal latency (PWL) to radiant heat and von Frey filaments. The expression of pERK, pCREB, and Fos were assessed by both Western blotting and immunohistochemical analysis. Intrathecal injection of U0126 or ERK antisense ODN significantly attenuated CCI-induced mechanical allodynia and thermal hyperalgesia. CCI significantly increased the expression of p-ERK-IR neurons in the ipsilateral spinal dorsal horn to injury, not in the contralateral spinal dorsal horn. The time courses of pERK expression showed that the levels of both cytosol and nuclear pERK, but not total ERK, were increased at all points after CCI and reached a peak level on postoperative d 5. CCI also significantly increased the expression of pCREB and Fos. Phospho-CREB-positive neurons were distributed in all laminae of the bilateral spinal cord and Fos was expressed in laminae I and II of the ipsilateral spinal dorsal horn. Intrathecal injection of U0126 or ERK antisense ODN markedly suppressed the increase of CCI-induced pERK, pCREB and c-Fos expression in the spinal cord. The activation of ERK pathways contributes to neuropathic pain in CCI rats, and the function of pERK may partly be accomplished via the cAMP response element binding protein (CREB)-dependent gene expression.

Co-activation of ERK, NF-κB, and GADD45β in Response to Ionizing Radiation

NF-kappaB has been well documented to play a critical role in signaling cell stress reactions. The extracellular signal-regulated kinase (ERK) regulates cell proliferation and survival. GADD45beta is a primary cell cycle element responsive to NF-kappaB activation in anti-apoptotic responses. The present study provides evidence demonstrating that NK-kappaB, ERK and GADD45beta are co-activated by ionizing radiation (IR) in a pattern of mutually dependence to increase cell survival. Stress conditions generated in human breast cancer MCF-7 cells by the administration of a single exposure of 5 Gy IR resulted in the activation of ERK but not p38 or JNK, along with an enhancement of the NF-kappaB transactivation and GADD45beta expression. Overexpression of dominant negative Erk (DN-Erk) or pre-exposure to ERK inhibitor PD98059 inhibited NF-kappaB. Transfection of dominant negative mutant IkappaB that blocks NF-kappaB nuclear translocation, inhibited ERK activity and GADD45beta expression and increased cell radiosensitivity. Interaction of p65 and ERK was visualized in living MCF-7 cells by bimolecular fluorescence complementation analysis. Antisense inhibition of GADD45beta strikingly blocked IR-induced NF-kappaB and ERK but not p38 and JNK. Overall, these results demonstrate a possibility that NF-kappaB, ERK, and GADD45beta are able to coordinate in a loop-like signaling network to defend cells against the cytotoxicity induced by ionizing radiation.

Ki-67-directed antisense therapy in an orthotopic renal cell carcinoma model.

The Ki-67 antigen is only present in proliferating cells. We have shown previously that phosphorothioate-modified antisense oligonucleotides (ON) against this antigen are potent antitumoral agents in bladder and prostate cancer-derived cells. Since ON are known to accumulate in vivo in the kidney, high local effectivity may be expected. Here, we evaluated and characterized antitumoral effects in an orthotopic renal cell cancer (RENCA) model. RENCA cells were incubated with antisense and control ON in the presence of a cationic lipid. Uptake studies were performed with FITC-labeled ON. Ki-67 protein analysis after ON treatment was performed by immunohistochemical staining. For animal studies, 1 x 10(5) RENCA cells were implanted under the renal capsule of Balb/c mice. Antisense and control ON were injected intraperitoneally daily for 14 days. Tumor weights and status of metastasis were documented after sacrifice. Furthermore, vessel density in tumor tissues was determined by CD31 immunolabeling. Antisense treatment of RENCA cells resulted in specific reduction of the Ki-67 protein and inhibition of cell growth. A substantial cellular uptake of labeled ON was noted in vitro and in vivo. The growth of orthotopically implantated syngeneic kidney tumors in immunocompetent mice was significantly inhibited in antisense-treated animals (p < 0.05). Furthermore, lung metastases were noted in 10% of antisense-treated animals compared to 30-40% in control groups. Immunohistochemical staining of the vessel density showed no significant difference among treatment groups. The results demonstrate that Ki-67-directed antisense oligonucleotides are potent inhibitors of target protein expression and proliferation of tumor cells in vitro, and of tumor growth and lung metastasis formation in murine renal cell carcinoma whereas tumor vascularization is not significantly affected.