| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Department of Physiology and Biophysics and the Institute for Molecular Cardiology at SUNY at Stony Brook, NY, USA
2 The Laboratory for Chemical Biology and the Department of Pharmacological Science at SUNY at Stony Brook, NY, USA
3 Department of Pharmacology and the Center for Molecular Therapeutics, Columbia University, New York, NY, USA
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
(Received 19 May 2005;
accepted after revision 20 July 2005;
first published online 21 July 2005)
Corresponding author P. R. Brink: Department of Physiology and Biophysics, SUNY at Stony Brook, Stony Brook, NY 11794, USA. Email: peter.brink{at}sunysb.edu
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Endogenous and exogenous siRNAs, 20–30 nucleotides in length, can profoundly affect gene expression (Elbashir et al. 2001; Caplen & Mousses, 2003; Miller & Grollman, 2003; Xu et al. 2004). Hence siRNA is important in regulating cellular function and has potential therapeutic applications (Hampton, 2004). In plants, siRNA can travel from cell to cell (Yoo et al. 2004), resulting in system-wide silencing of specific genes. The intercellular path in plants that is exclusive of the extracellular space is the plasmodesmata with a pore diameter of 100–200 nm, a size more than adequate for the passage of siRNA. It is not found in either invertebrates or vertebrates.
The analogous structure in animals is the gap junction. In invertebrates, the subunit proteins of gap junction channels are the innexins, while in vertebrates they are the connexins and pannexins (White et al. 2004; Bruzzone et al. 2005). Gap junction channels have pore diameters between 1.0 and 1.5 nm. This is close to the minor diameter (width) of siRNA, so it is possible that siRNA could permeate gap junction channels, but there has been no direct demonstration of this in vertebrates or invertebrates. For example, in the invertebrate C. elegans, ingestion of siRNA resulted in systemic effects (Kittle & Buchholz, 2003), but the mechanism by which the siRNA was delivered to cells not lining the alimentary canal has not been defined and does not appear to involve the transmembrane protein SID-1 that mediated transport of RNA interfering (RNAi) (Feinberg & Hunter, 2003).
Endogenous and exogenous siRNAs survive and remain functional for hours or days (Alisky & Davidson, 2004). Given their prolonged survival, if siRNAs permeate gap junction channels then they would influence not only the cell in which they were produced, but also adjacent and perhaps even distant cells of a syncytium. Thus a small group of cells could potentially use this mechanism to alter organ function. An extension of this idea is that a small group of cells implanted in a tumour could use this mechanism to inhibit tumour growth. Recent reports have demonstrated successful targeting of genetically engineered mesenchymal stem cells to tumours where they produced interferon (Studney et al. 2004). Thus, the ability to deliver siRNA to the interior of a cell in a target tissue exclusive of the extracellular space has significant therapeutic potential. The purpose of the present study was to determine whether siRNA can permeate gap junctions and thus allow one cell to regulate gene expression in another cell.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
We used dual whole cell patch clamp to assess junctional conductance (Neyton & Trautmann, 1985). To determine the permeability of the oligonucleotides we employed the methods of Valiunas et al. (2002). Cells were bathed in a solution containing 150 mM NaCl, 10 mM KCl, 2 mM CaCl2, 5 mM Hepes (pH 7.4) and 5 mM glucose. Pipettes contained 120 mM potassium aspartate, 10 mM NaCl, 3 mM MgATP, 5 mM Hepes (pH 7.2) and 10 mM EGTA. Oligonucleotides were added to the pipette solution to concentrations of 0.25–0.5 mM. We calibrated the camera system for the morpholinos as we have previously reported for Lucifer Yellow (Valiunas et al. 2002). The effective range was 300 nM to 0.5 mM for the cell types used.
Cell lines and culture conditions
The mouse embryonic fibroblast line Mß16tsA (Gu et al. 1994), rat kidney line (NRK), mouse neuroblastoma line (N2A) and HeLa cells were purchased from the American Type Culture Collection (Manassas, VA, USA). All cells were grown as monolayers in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, at 34°C (Mß16tsA) or 37°C (NRK, HeLa and N2A) in a 5% CO2 humidified air atmosphere. Human mesenchymal stem cells were purchased from Clonetics/BioWhittaker (Walkersville, MA, USA) and cultured as previously described (Potapova et al. 2004).
The vector, pH 1P-pgkneoB (Polosina et al. 2004), directs expression of a short hairpin RNA (shRNA) that has been shown to decrease the level of DNA polymerase ß (pol ß) RNA greater than 90% (less than 10% pol ß remaining). pH 1P-pgkneoB (1 µg) was transfected into NRK cells using FUGENE according to the manufacturer's protocol (Roche) and clones were selected with geneticin (G418). Individual clones were isolated and the levels of pol ß mRNA and protein were determined by semiquantitative RT-PCR and immunoblotting. The clone with the lowest level of pol ß (NRK-kcdc) was selected for the experiments described. Pol ß knockdown Mß16tsA cells (Mß16tsA-kcdc) were made as described by Polosina et al. (2004). The siRNA for pol ß is a 22-mer with a length of slightly more than 7.0 nm.
Western blot analysis
Cell lysates were prepared from confluent monolayer cells as described by Biade et al. (1998). Equal amounts of cellular protein (70 or 50 µg) were resolved by 10 or 12% SDS-PAGE and transferred to nitrocellulose or Immum-Blot PVD (Bio-Rad) membranes. Blots were incubated with one of the following antibodies: 18S monoclonal anti-pol ß (NeoMarkers, Fremont, CA, USA, 669P201), anti-actin (Santa Cruz, Santa Cruz, CA, USA, sc-7210), anti-Cx43, anti-Cx26 and anti-Cx32 (Zymed Laboratories Inc., San Francisco, CA, USA, 71-0700, 71-0500 and 71-0600, respectively). Immunoblots were carried out with secondary antibody conjugated to horseradish peroxidase (Santa Cruz), detected by a MB chemiluminescence kit (Pierce, Rockford, IL, USA).
RT-PCR analysis
RNA was isolated from cells using RNeasy MinElute (Qiagen, Valencia, CA, USA, catalogue no. 74204). RT-PCR was performed using SuperScript One-Step RT-PCR with Platinum Taq kit (Invitrogen, Carlsbad, CA, USA, catalogue no. 10928-042) with 1 µg of RNA as template and products subjected to electrophoresis in a 1% agarose gel. Primers for pol ß were (5'-GACATGCTCACAGAACTCG-3', 5'-CGGATGGTGTACTCATTGAT5-3'), and for actin they were (5'-ACAGATCATGTTTGAGACC-3', 5'-CCACCGATCCACACAGAGTA-3').
Flow cytometry
To determine how pol ß mRNA levels of wild type (wt) were altered due to contact with siRNA-expressing cells (kcdc), wt and kcdc cells were grown together for several days and then separated by flouorescence activated cell sorting (FACS). Before cells were mixed, either the wt or kcdc cells were stained using the Vybrant CFDA SE Cell Tracer Kit according to the manufacturer's protocol (Molecular Probes). Wt and kcdc cells were mixed in proportion 1:1 or 1:2 for NRK, 1:6 for Mß16tsA, 1:2 for N2A/Mß16tsA-kcdc and NRK/Mß16tsA-kcdc, and 1:1 for NRK-kcdc/N2A. The mixed cultures were grown together for 6–8 days. Cells were trypsinized and resuspended in cold PBS containing 2 mM EDTA. Separation of unlabelled, non-fluorescent cells from positive, highly fluorescent cells was performed on a Becton Dickinson FACS Vantage model flow cytometer equipped with an air-cooled argon laser emitting 488 nm laser light. Fluorescence signals were collected using a 530 nm wavelength filter with a 30 nm bandpass. Cells with low fluorescence intensities were discarded to ensure that both the non-fluorescent and highly fluorescent cells were not significantly contaminated with the other cell type. RNA was isolated from the sorted cells and control cells that had been grown separately and analysed by RT-PCR.
Morpholino modelling
A tri-guanine base morpholino model was generated using the Dundee ProDrg server (Schuettelkopf & van Aalten, 2004). The model was energy minimized using the Gromacs (v3.2: Berendsen et al. 1995; Lindahl et al. 2001; van der Spoel et al. 2004) molecular simulation package with molecular topology files generated by the ProDrg server. The starting Prodrg structure was first solvated with water molecules using the program GENBOX. Following a 1 ps (picosecond) minimization run using the MDRUN program and the Gromacs force field, ffGMX, the solvated system was equilibrated with a 20 ps minimization run in which positional restraints were placed on the morpholino. The solvated morpholino was then subjected to a 50 ps simulated annealing run at room temperature. For comparison, the coordinates of DNA and RNA were obtained from the Protein Data Bank (PDB) and Nucleic acid Database (NDB), respectively, at Rutgers University. All structures were visualized with PYMOL (pymol.sourceforge.net).
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
To test the hypothesis that rod-shaped molecules of the size range of siRNA are permeable to gap junction channels, we used oligonucleotides (constructed by Morpholino Inc.) of three lengths, 12-, 16- and 24-mer. Their sequences had subunits consisting of a nucleic acid base, a morpholine ring and a phosphoramidate linker. All three lengths were constructed to ensure that they would not readily hybridize or be degraded by cellular nucleases (Mudziak et al. 1996; Summerton & Weller, 1997). Each probe was labelled with carboxyfluorescein on its 3' end and was subjected to HPLC to separate any unattached carboxyfluorescein from the labelled oligonucleotide. Based on previous research (e.g. Summerton & Weller, 1997) it is unlikely that degradation of the morpholino and the removal of the tag occurred in the cytoplasm. We also ran HPLC on our nucleotides in the pipette before and after using them in experiments and could find no free tag.
The three sequences used in our study are: 12-mer, 5'-CCTCTTACCTCA-3'; 16-mer, 5'-CCTCTTACCTCAGTTA-3'; and 24-mer, 5'-CCTCTTACCTCACCTCTTACCTCA-3'. All three oligonucleotides had minor diameters of 
1.0 nm and length ranging from 3.8 (12-mer) to 7.6 nm (24-mer). The minor diameter and approximate lengths were determined as described in the Methods. The minimized morpholino model has a single stranded width of 10.7 Å and base stacking distance of 4 Å. In comparison, the width of a single stand of B-DNA (PDB identification no. 1DPN) is 11 Å and that of single-stranded RNA (NDB identification no. 472d) is 10 Å. The base stacking distances of RNA and DNA are 4 and 3 Å, respectively.
In cells, siRNA can exist in either single-stranded or double-stranded forms, which will comprise an equilibrium distribution. Since the single-stranded siRNA has the smaller minor diameter, it would be expected to permeate gap junction channels more easily than the double-stranded form. To test this, a compliment to the 12-mer was synthesized and combined with our original 12-mer, resulting in the formation of a double-stranded oligonucleotide whose permeability could be tested in the same fashion as that of the single-stranded form. We used HeLa cells expressing Cx43 (Valiunas et al. 2004), Mß16tsA-kcdc and Mß16tsA cells expressing Cx26/Cx32, or human mesenchymal stem cells expressing Cx43 (Valiunas et al. 2004) to probe for oligonucleotide transfer of the 12-, 16- and 24-mers.
Figure 1A shows an example of oligonucleotide transfer in HeLa cells. The transfer is visualized from source cell to recipient cell over a 12 min interval for the 12-mer. The inset in the right-hand panel shows the junctional current measured in response to a voltage step for the same cell pair. The measured conductance was 40 nS. Figure 1B shows a lack of transfer for a Mß16tsA-kcdc cell pair after a 20 min interval. The junctional conductance for this pair was 30 nS. These cells express Cx32 and Cx26 but not Cx43 (see Fig. 3). For both Mß16tsA-kcdc (n = 5) and Mß16tsA (wt) cells (n = 5) no transfer of 12-mer was observed. In four cases (2 Mß16tsA-kcdc and 2 Mß16tsA (wt) cells) where junctional conductance was greater than 25 nS (range 25–40 nS), no transfer of 12-mer was observed over 20–30 min intervals. The data shown in Fig. 1B are from one of these experiments. Oligonuclotide transfer was also observed between human mesenchymal stem cells (Fig. 1C) where the junctional conductance was 17 nS. Figure 1D illustrates the transfer of the 24-mer 40 min after injection. The junctional conductance was 35 nS. In Fig. 1E the source cell has been removed, leaving the recipient cell. Hela and hMSCs cells have been shown to express Cx43 (Valiunas et al. 2004). These data suggest that synthetic nucleotides selectively pass through Cx43 but not Cx26 or Cx32 gap junction channels, and they suggest the hypothesis that siRNA might behave similarly.
|
|
. As the length of the single-stranded oligonucleotide increased, the permeability decreased. The hybridized 12-mer had significantly lower permeability than the single strand, indicating that the width of the molecule also influences the rate of transfer from cell to cell. This finding is expected for molecules whose minor diameter is close to the pore diameter (Goldberg et al. 1999, 2004; Valiunas et al. 2002).
|
Cell-to-cell transfer of siRNA
To test whether siRNA would transit gap junction channels, we used three cell lines: (1) NRK cells that endo-genously express Cx43 (Musil & Goodenough, 1991; Hand et al. 2002); (2) mouse embryonic fibroblasts, Mß16tsA cells, which express Cx32 and Cx26 but no Cx43 (see Fig. 3A); and (3) connexin-deficient N2A cells (Veenstra et al. 1995). We do not provide a western blot demonstrating Cx43 expression in NRK cells because their endogenous expression of this connexin has been reported previously (Musil & Goodenough, 1991; Hand et al. 2002). For NRK and Mß16tsA cells the mean ± S.D. junctional conductance was 18.2 ± 10 (n = 7) and 18.1 ± 9 nS (n = 16), respectively (N2A parental cells are communication deficient and so no measurement of junctional conductance was made; Veenstra et al. 1994).
Dual whole cell patch clamp illustrates gap junction-mediated coupling in Mß16tsA and NRK cells (Fig. 3B). The data shown in Fig. 3B represent examples of junctional conductances, 4 and 10 nS, respectively, which demonstrate voltage dependence. N2A cells are communication deficient and we show no records as a consequence (Brink et al. 1997). We generated a mouse embryonic fibroblast cell line that stably expressed siRNA against pol ß (Mß16tsA-ß kcdc). Polosina et al. (2004) have reported a Mß16tsA knockdown in which pol ß is 5–10% of normal levels. We also generated an NRK cell line that stably expressed an siRNA that targets pol ß. The western blot for NRK-kcdc has 30% of wild-type protein levels of pol ß, as shown in Fig. 4A. The NRK-kcdc cell line also showed an increased sensitivity to the DNA methylating agent methylmethane sulphonate (MMS, Fig. 4B), the hallmark of a pol ß deficiency (Sobol et al. 1996). Lastly, NRK-kcdc cells were stained with a vital dye (see Methods) and co-cultured with NRK-wt cells for 6–8 days. Mß16tsA-kcdc cells were stained and co-cultured with Mß16tsA-wt, N2A cells or NRK-wt cells for 6–8 days. Prior to co-culture, all wt cells expressed normal pol ß levels. After co-culturing the mixed populations were separated via fluorescence-activated cell sorting (FACS). Pol ß mRNA levels were measured by semiquantitative RT-PCR. Figure 5 shows that pol ß expression was reduced in the NRK-wt cells that were co-cultured with NRK-kcdc cells, but was unaffected in the Mß16tsA-wt, N2A and NRK-wt cells after co-culture with Mß16tsA-kcdc cells. Figure 5A shows RT-PCR results for co-culturing of NRK-wt and NRK-kcdc cells (left-hand panel) where FACS demonstrates knockdown in the wt cells. Co-cultured Mß16tsA-wt and Mß16tsA-kcdc cells (right-hand panel) failed to show a knockdown in the wt cells after FACS. This result demonstrates that cells which are electrically coupled with Cx32 and Cx26 are incapable of effective transfer of siRNA. Figure 5B shows the lack of knockdown, from left to right, for N2A cells co-cultured with Mß16tsA-kcdc cells, NRK-wt cells co-cultured with Mß16tsA-kcdc cells or N2A cells co-cultured with NRK-kcdc cells. In this case there should be no electrical coupling (since N2A cells are connexin deficient) and there is no siRNA transfer. The data presented in Fig. 5B eliminate the possibility that an extracellular pathway mediates the transfer of siRNA based on the expectation of little or no cell-to-cell coupling mediated by gap junction channels in these cases (Brink et al. 1997; Gemel et al. 2004). One significant feature associated with successful siRNA transfer to NRK-wt cells from NRK-kcdc cells is the presence of Cx43. This connexin was absent in Mß16tsA-wt and Mß16tsA-kcdc mouse fibroblasts expressing Cx32 and Cx26. This suggests that siRNA can permeate through gap junction channels composed of Cx43 but not those composed of Cx32 or Cx26.
|
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
2–4 kDa, minor diameters of 1.0–1.1 nm and lengths of 7.6 nm, are able to diffuse from cell to cell via Cx43 channels. As far back as 1971 Kolodny suggested that 3T3 cells could transfer mRNA via gap junctions (Kolodny, 1971). Our data strongly suggest that even though these cells make Cx43 (Carystinos et al. 2003), his result was rather a demonstration of transfer of single nucleotides or possibly small oligonucleotides. In addition, siRNA for pol ß can be effectively transferred. In all cases the likely forms would be non-hybridized and to a lesser extent hybridized forms since they are less permeant. It is unlikely that hairpin forms of siRNA are transferred based on the effective action of Dicer enzymes (Shankar et al. 2005), which cleave hairpin siRNA leaving only non-hybridized and hybridized forms. In addition, the shape of the hairpin siRNA should limit permeation. Quantitative analysis of the co-culture of NRK-kcdc (knockdown cells) with NRK-wt shows a reduction in pol ß expression in the NRK-wt for the 1:1 ratio (wild-type to kcdc) which was not statistically significant but was highly significant when the ratio was one NRK-wt to two NRK-kcdc (1:2). The potential for transfer of oligonucleotides via the extracellular space seems unlikely because Cx43 was the only connexin that was permeable to morpholinos and siRNA for pol ß. This is unlike other connexin-induced extracellular pathways, where Cx43, Cx26 and Cx32 all trigger extracellular-mediated cell-to-cell communication via the opening of hemichannels (Tran Van Nhieu et al. 2003). Another unlikely possibility requires that the Cx43-expressing cells have higher rates of pinocytosis relative to cells expressing other connexins.
For the morpholinos, degradation resulting in the release of the tag on the 3' end seems unlikely based on previous studies (Summerton & Weller, 1997), and if there were cytoplasmic-dependent breakdown into the cytoplasm then the flux from source cell to recipient cell would be expected to be independent of morpholino length. This was not the case.
Obviously, this is not an all-inclusive study and there may be other connexins that allow passage of siRNA. The implication is that, through regulation of connexin expression, a tissue can regulate its pattern of gene expression. This potential role for gap junctions has not been appreciated previously. In principle, a small group of cells or possibly a single cell within a synctium has the potential to affect gene expression in a larger group of cells or possibly an entire organ. Moreover, this phenomenon suggests the possibility of introducing cells expressing specific siRNAs to therapeutically modify gene expression in a target organ or a tumour. The data presented here suggest that siRNA against pol ß will transfer from a pol ß knockdown cell to a wild type cell through gap junction channels composed of Cx43 but not Cx32/Cx26. The length (22-mer) of siRNA against pol ß is typical of most other siRNAs, so if it can move from cell to cell, other RNA molecules should do so as well. To test this hypothesis, fluorescently labelled oligonucleotides, simulating siRNAs, were generated (12-mer, 16-mer and 24-mer). They too were found to permeate gap junction channels composed of Cx43 but not Cx26/Cx32. These data support the novel hypothesis that gap junction channels have connexin-specific permeability to siRNA.
These experiments also suggest that cellular delivery of siRNA might have potential as a therapeutic tool (Lieberman et al. 2003; Berkhout, 2004). The major barrier to clinical application has been a means for in vivo delivery of exogenous siRNA to the interior of the target cell (Zhang et al. 2004). We have recently shown that human mesenchymal stem cells make connexins (including Cx43) and form gap junctions in vitro and in vivo (Valiunas et al. 2004; Potapova et al. 2004), raising the possibility of their use as an autologous cell-based delivery system for gene suppression. Data presented here suggest that Cx43-expressing mesenchymal stem cells will transfer siRNA to other Cx43-expressing cells. At this stage, we have identified only Cx43 channels as being permeable to siRNA. Cx43 is ubiquitously expressed in vertebrates. More extensive studies may identify other connexins that form siRNA-permeable channels. Another potential limitation is the rate of degradation of the siRNA. The extent to which siRNA can diffuse through a syncytium depends on both the rate of cell-to-cell transfer through gap junctions and the rate of intracellular degradation. Because of its relatively large size, siRNA diffuses slowly through gap junctions formed from Cx43, thus the rate of degradation would have to be slow to allow extensive diffusion. However, in invertebrates, siRNA-recipient cells have the ability to self-replicate the siRNA (Fire et al. 1998; Lipardi et al. 2001). If a similar mechanism exists in mammals, then siRNA-producing cells introduced into a syncytium could trigger an effective propagation of the siRNA throughout that tissue. Many questions remain, but our data clearly show that siRNA can be delivered from the interior of one cell to that of another independent of the extracellular space and influence gene expression in the recipient cell.
|
Footnotes |
|---|
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Barr
L, Dewey
M
&
Berger
W (1965). Propagation of action potentials and the structure of the nexus in cardiac muscle. J General Physiol
48, 797–823.
Bennett MVL (1977). Electrical transmission: a functional analysis and comparison to chemcial transmission. In The Nervous System, vol. 1, Cellular Biology of Neurons, part 1, ed. Brookhart J, Moutcastle V, Kandel E & Geiger S, pp. 357–416. Am Physiol Soc, Bethesda, Maryland.
Berendsen HJC, van der Spoel D & van Drunen R (1995). GROMACS: a message-passing parallel molecular dynamics implementation. Comp Phys Comm 91, 43–56.[CrossRef]
Berkhout B (2004). RNA interference as an antiviral approach: targeting HIV-1. Curr Opin Mol Ther 6, 141–145.[Medline]
Bevans
CG
&
Harris
AL (1999). Direct high affinity modulation of connexin channel activity by cyclic nucleotides. J Biol Chem
274, 3720–3372.
Biade
S, Sobol
RW, Wilson
SH
&
Matsumoto
Y (1998). Impairment of proliferating cell nuclear antigen-dependent apurinic/apyrimidinic site repair on linear DNA. J Biol Chem
273, 898–902.
Brink PR, Cronin K, Banach K, Peterson E, Westphale E, Seul KH, Ramanan SV & Beyer EC (1997). Evidence for heteromeric gap junction channels formed from rat Connexin43 and human connexin37. Am J Physiol 273, C1386–C1396.[Medline]
Bruzzone R, Barbe MT, Jakob NJ & Monyer H (2005). Pharmacological properties of homomeric and heteromeric pannexin hemichannels expressed in Xenopus oocytes. J Neurochem 92, 1033–1043.[CrossRef][Medline]
Caplen
NJ
&
Mousses
S (2003). Short interfering RNA (siRNA) -mediated RNA interference (RNAi) in human cells. Ann N Y Acad Sci
1002, 56–62.
Carystinos
GD, Kandouz
M, Alaoue-Jamali
MA
&
Batist
G (2003). Unexpected induction of the human connexin 43 promoter by the ras signaling pathway is mediated by a novel putative promoter sequnce. Mol Pharmocol
63, 821–831.
Elbashir
SM, Lendeckel
W
&
Tuschl
T (2001). RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev
15, 188–200.
Elfgang
C, Eckert
R, Lichtenberg-Rate
H, Butterweck
A, Taub
O, Klein
RA, Hulser
DF
&
Willecke
K (1995). Specific permeability and selective formation of gap junction channels in connexin-transfected HeLa cells. J Cell Biol
129, 805–817.
Feinberg
EH
&
Hunter
CP (2003). Transport of dsRNA into cells by the transmembrane protein SID-1. Science
301, 1545–1547.
Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE & Mello CC (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811.[CrossRef][Medline]
Gemel
J, Valiunas
V, Brink
PR
&
Beyer
EC (2004). Connexin43 and connexin26 form gap junctions, but not heteromeric channels in co-expressing cells. J Cell Sci
117, 2469–2480.
Goldberg G, Lampe PE & Nicholson BJ (1999). Selective transfer of endogenous metabolites through gap junctions composed of different connexins. Nature Cell Biol 7, 457–459.
Goldberg G, Valiunas V & Brink PR (2004). Selectivity permeability of gap junction channels. Biochem Biophys Acta 662, 96–101.
Gu
H, Marth
JD, Orban
PC, Mossmann
H
&
Rajewsky
K (1994). Deletion of a DNA polymerase ß gene segment in T cells using cell type-specific gene targeting. Science
265, 103–106.
Hampton
T (2004). With RNA interference, silence is golden. JAMA
291, 2803–2804.
Hand GM, Muller DJ, Nicholson BJ, Engels A & Sosinsky GE (2002). Isolation and characterization of gap junctions from tissue cultured cells. J Mol Biol 315, 587–600.[CrossRef][Medline]
Kittler R & Buchholz F (2003). RNA interference: gene silencing in the fast lane. Seminars Cancer Biol 13, 259–265.[CrossRef]
Kolodny GM (1971). Evidence for transfer of macromolecular RNA between mammalian cells in culture. Exp Cell Res 65, 313–324.[CrossRef][Medline]
Lieberman J, Song E, Lee S & Shankar P (2003). Interfering with disease: opportunities and roadblocks to harnassing RNA interference. Trends Mol Med 9, 397–403.[CrossRef][Medline]
Lindahl E, Hess B & van der Spoel D (2001). GROMACS 3.0: a package for molecular simulation and trajectory analysis. J Mol Mod 7, 306–317.
Lipardi C, Wei Q & Paterson BM (2001). RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs. Cell 107, 297–307.[CrossRef][Medline]
Loewenstein
WR (1981). Junctional intercellular communication: the cell to cell membrane channel. Physiol Rev
61, 829–913.
Miller H & Grollman AP (2003). DNA repair investigations using siRNA. DNA Repair 2, 759–763.[Medline]
Mudziak RM, Barofsky E, Barofsky DF, Weller DL, Huang SB & Weller DD (1996). Resistance of morpholino phosphorodiamidate oligomers to enzymatic degradation. Antisense Nuc Acid Drug Dev 6, 267–272.[Medline]
Musil
LS
&
Goodenough
DA (1991). Biochemcial analysis of connexin43 intracellular transport, phosphorylation, and assembly into gap junctional plaques. J Cell Biol
115, 1357–1374.
Neijssen J, Herberts C, Drijfhout JW, Reits E, Janssen L & Neefjes J (2005). Cross-presentation by intercellular peptide transfer through gap junctions. Nature 434, 83–88.[CrossRef][Medline]
Neyton J & Trautmann A (1985). Single channel currents of an intercellular junction. Nature 317, 331–335.[CrossRef][Medline]
Pitts JD (1998). The discovery of metabolic co-operation. Bioessays 20, 1047–1051.[CrossRef][Medline]
Polosina YY, Rosenquist TA, Grollman AP & Miller H (2004). ‘Knock down’ of DNA polymerase ß by RNA interference: recapitulation of null phenotype. DNA Repair 3, 1469–1474.[Medline]
Potapova
I, Plotnikov
A, Lu
Z, Danilo
P, Valiunas
V, Qu
J, Doronin
S, Zuckerman
J, Shlapakova
I, Gao
J, Pan
Z, Herron
A, Robinson
RB, Brink
PR, Rosen
M
&
Cohen
IS (2004). Human mesenchymal stem cells as a gene delivery system to fabricate a biological pacemaker. Circ Res
94, 952–959.
Schuettelkopf AW & van Aalten DMF (2004). PRODRG – a tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallogr, Biol Crystallogr 60, 1355–1363.[CrossRef]
Shankar
P, Manjunath
W
&
Liebermann
J (2005). The prospect of silencing disease using RNA interference. JAMA
293, 1367–1373.
Simpson
I, Rose
B
&
Loewenstein
WR (1977). Size limit of molecules permeating the junctional membrane channels. Science
195, 294–296.
Sobol RW, Horton JK, Kuhn R, Gu H, Singhal RK, Prasad R, Rajewsky K & Wilson SH (1996). Requirement of mammalian DNA polymerase-ß in base-excision repair. Nature 379, 183–186.[CrossRef][Medline]
Studney
N, Marini
FC, Dembinski
JL, Zompetta
C, Cabreira-Hansen
M, Nebiyou Bekele
B, Champlin
RE
&
Andreeff
M (2004). Mesenchymal stem cells: potential precursors for tumor stoma and targeted-delivery vehicles for anticancer agents. J Nat Cancer Inst
96, 1593–1603.
Summerton J & Weller D (1997). Morpholino antisense oligmers: design, preparation and properties. Antisense Nucl Acid Drug Dev 7, 187–195.[Medline]
Tran Van Nhieu G, Clair C, Bruzzone R, Mesnil M, Samsonetti P & Combettes L (2003). Connexin-dependent inter-cellular communication increases invasion and dissemination of Shigella in epithelial cells. Nature Cell Biol 5, 720–727.[CrossRef][Medline]
Tsien
RW
&
Weingart
R (1976). Inotropic effect of cyclic AMP in calf ventricular muscle studied by a cut end method. J Physiol
260, 117–142.
Valiunas
V, Beyer
EC
&
Brink
PR (2002). Gap junction channels show a quantitative difference in selectivity. Circ Res
91, 104–111.
Valiunas
V, Doronin
S, Valiuniene
L, Potapova
I, Zuckerman
J, Walcott
B, Robinson
RB, Rosen
MR, Brink
PR
&
Cohen
IS (2004). Human mesenchymal stem cells make cardiac connexins and form functional gap junctions. J Physiol
555, 617–626.
van der Spoel D, Lindahl E, Hess B, van Buuren AR, Apol E, Meulenhoff PJ, Tielman DP, Sijbers ALTM, Feenstra KA, van Drunen R & Berendsen HJC (2004). Gromacs User Manual, version 3.2. http://www.gromacs.org.
Veenstra
RD, Wang
HZ, Beblo
DA, Chilton
MG, Harris
AL, Beyer
EC
&
Brink
PR (1995). Selectivity of connexin-specific gap junctions does not correlate with channel conductance. Circ Res
77, 1156–1165.
Veenstra
RD, Wang
HZ, Beyer
EC, Ramanan
SV
&
Brink
PR (1994). Connexin37 forms high conductance gap junction channels with subconductance state activity and selective dye and ionic permeabilities. Biophys J
66, 1915–1928.
Walcott B, Moore LC, Birzgalis A, Claros N, Valiunas V, Ott T, Willecke K & Brink PR (2002). The role of gap junctions in fluid secretion of lacrimal glands. Am J Physiol 282, C501–C507.
White TW, Wang H, Mui R, Litteral J & Brink PR (2004). Cloning and functional expression of invertebrate connexin from Halocynthia pyroformis. FEBS 577, 42–48.[CrossRef][Medline]
Xu Y, Linde A, Larsson O, Thormeyer D, Elmen J, Wahlestedt C & Liang Z (2004). Functional comparison of single- and double-stranded siRNAs in mammalian cells. Biochem Biophys Res Comm 316, 680–687.[CrossRef][Medline]
Yoo
B, Kragler
F, Varkonyi-Gasic
E, Haywood
V, Archer-Evans
S, Lee
YM, Lough
TJ
&
Lucasa
WJ (2004). A systemic small RNA signaling system in plants. Plant Cell
16, 1979–2000.
Zhang G, Song YK, Vollmer R, Stolz DB, Gasiorowski JZ, Dean DA & Liu D (2004). Hydroporation as a mechanism of hydrodynamic delivery. Gene Therapy 11, 675–682.[CrossRef][Medline]
|
Acknowledgements |
|---|
This article has been cited by other articles:
|
|
 |
|
  M. E Dinger, T. R Mercer, and J. S Mattick RNAs as extracellular signaling molecules J. Mol. Endocrinol., April 1, 2008; 40(4): 151 - 159. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Kanaporis, G. Mese, L. Valiuniene, T. W. White, P. R. Brink, and V. Valiunas Gap Junction Channels Exhibit Connexin-specific Permeability to Cyclic Nucleotides J. Gen. Physiol., March 31, 2008; 131(4): 293 - 305. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. M. Oxford, H. Musa, K. Maass, W. Coombs, S. M. Taffet, and M. Delmar Connexin43 Remodeling Caused by Inhibition of Plakophilin-2 Expression in Cardiac Cells Circ. Res., September 28, 2007; 101(7): 703 - 711. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. W. Yum, J. Zhang, V. Valiunas, G. Kanaporis, P. R. Brink, T. W. White, and S. S. Scherer Human connexin26 and connexin30 form functional heteromeric and heterotypic channels Am J Physiol Cell Physiol, September 1, 2007; 293(3): C1032 - C1048. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. S. Duffy, I. Iacobas, K. Hotchkiss, B. J. Hirst-Jensen, A. Bosco, N. Dandachi, R. Dermietzel, P. L. Sorgen, and D. C. Spray The Gap Junction Protein Connexin32 Interacts with the Src Homology 3/Hook Domain of Discs Large Homolog 1 J. Biol. Chem., March 30, 2007; 282(13): 9789 - 9796. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Rackauskas, M. M. Kreuzberg, M. Pranevicius, K. Willecke, V. K. Verselis, and F. F. Bukauskas Gating Properties of Heterotypic Gap Junction Channels Formed of Connexins 40, 43, and 45 Biophys. J., March 15, 2007; 92(6): 1952 - 1965. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H.-Z. Wang, P. R. Brink, and G. J. Christ Gap junction channel activity in short-term cultured human detrusor myocyte cell pairs: gating and unitary conductances Am J Physiol Cell Physiol, December 1, 2006; 291(6): C1366 - C1376. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. R. Rosen Are Stem Cells Drugs?: The Regulation of Stem Cell Research and Development Circulation, October 31, 2006; 114(18): 1992 - 2000. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), 24-mer (
) and 12-mer double-stranded oligonucleotides (
