| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 275, Issue 44, 34407-34414, November 3, 2000
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the Departments of
Received for publication, May 8, 2000, and in revised form, July 27, 2000
Given the roles proposed for gap junctional
intercellular communication in neuronal differentiation and
growth control, we examined the effects of connexin43 (Cx43) expression
in a neuroblastoma cell line. A vesicular stomatitis virus G protein
(VSVG)-pseudotyped retrovector was engineered to co-express the green
fluorescent protein (GFP) and Cx43 in the communication-deficient
neuro-2a (N2a) cell line. The 293 GPG packaging cell line was
used to produce VSVG-pseudotyped retrovectors coding for GFP, Cx43, or
chimeric Cx43·GFP fusion protein. The titer of viral supernatant, as
measured by flow cytometry for GFP fluorescence, was approximately
2.0 × 107 colony form units (CFU)/ml and was
free of replication-competent retroviruses. After a 7-day treatment
with retinoic acid (20 µM), N2a transformants (N2a-Cx43
and N2a-Cx43·GFP) maintained the expression of Cx43 and Cx43·GFP.
Expression of both constructs resulted in functional coupling, as
evidenced by electrophysiological and dye-injection analysis.
Suppression of cell growth correlated with expression of both Cx43 or
Cx43·GFP and retinoic acid treatment. Based on morphology and
immunocytochemistry for neurofilament, no difference was observed in
the differentiation of N2a cells compared with cells expressing Cx43
constructs. In conclusion, constitutive expression of Cx43 in N2a cells
does not alter retinoic acid-induced neuronal differentiation but does
enhance growth inhibition.
Gap junctions are intercellular plasma membrane channels which
provide direct cytoplasmic continuity between adjacent cells as well as
coordination of the function of individual cells (1). These channels
mediate direct exchange of ions and small molecules (less than 1.2 kDa)
including second messengers and electrical current among adjacent cells
(2, 3). The fundamental structural unit of the gap junction is the
connexin (Cx)1 subunit. To
clarify the role of gap junctional intercellular communication (GJIC)
in neural development, the temporal and cellular expression of
connexins has been studied. Cx43 is present in neurons in
vivo (4) and in vitro (5-7). Several studies have
demonstrated that neuronal GJIC decreases during differentiation (8).
In addition, we have shown that Cx43-mediated GJIC decreases with neuronal differentiation (7, 9, 10).
Given the multiple roles proposed for Cx43-mediated GJIC in development
and differentiation, many studies have been undertaken to alter Cx43
expression both in vitro through transfection (11-13) and
in vivo through transgenesis (14, 15). The major limitations to transfection approaches have been efficiency and time required to
obtain stable gene expression. The recent advent of retroviral vectors
to efficiently deliver genes for stable expression overcomes these
limitations (16, 17). Retroviral vectors enable efficient gene transfer
and stable gene expression in cells that are not readily susceptible to
transfection, such as primary cells, cells in vivo, and
neuronal cell lines (18). However, the titer of most current retroviral
supernatants is too low (<107 particles/ml) to achieve
acceptable clinical results (19). To overcome this deficit, more
recently developed retrovectors pseudotyped with the vesicular
stomatitis virus G (VSVG) protein have become the gene delivery tool of
choice (16). VSVG-pseudotyped retrovectors have advantages over the
current viral vectors in terms of high titer, complement resistance,
particle stability and tumor specificity (19).
In this study, the VSVG-pseudotyped retrovector enabled efficient
stable expression of Cx43 and Cx43·GFP. Cell growth was suppressed
but neuronal differentiation was unaffected.
Cell Lines and Culture Conditions--
The C6 astrocytoma cell
line (American Type Culture Collection (ATCC), Manassas, VA; CCL-107)
and neuro-2a mouse neuroblastoma cell line (ATCC, CCL-131) were
maintained as monolayer cultures in Dulbecco's modified minimal
essential medium (DMEM) (Life Technologies, Inc., Burlington, ON)
supplemented with 10% (v/v) fetal calf serum (Life Technologies,
Inc.), penicillin (100 units/ml) and streptomycin (100 µg/ml; Life
Technologies, Inc.) at 37 °C, in a humidified atmosphere containing
95% air and 5% CO2.
The 293 GPG retroviral packaging cell line (20) was a generous gift
from Dr. Richard C. Mulligan (Children's Hospital, Boston, MA). 293 GPG cells were maintained as monolayer cultures in 293 medium
consisting of DMEM supplemented with 10% heat-inactivated fetal calf
serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 0.3 µg/ml
G418 (Mediatech, Herndon, VA), 2.0 µg/ml puromycin (Sigma Chemical
Co.), and 1.0 µg/ml tetracycline (Fisher Scientific, Nepean, Ontario,
Canada) at 37 °C, in a humidified atmosphere containing 95% air and
5% CO2.
Retrovectoral Construction and Synthesis--
The AP2 retroviral
vector (19) was used in these studies. Two genes including an inserted
cDNA and the enhanced green fluorescent protein reporter
gene can be expressed from this single bicistronic, nonsplicing murine
plasmid retrovector (Fig. 1A)
(19). The cDNA fragment coding for IRES and EGFP was removed
from the plasmid backbone by single endonuclease digestion with
NotI, and the resulting fragment was religated together as
outlined below. The resulting plasmid was designated NAP2 (Fig.
1B).
The Cx43 cDNA (1.45 kilobases) (43) (kindly provided by Dr.
E. Beyer, Washington University, School of Medicine, St. Louis, MO) and
the Cx43·GFP chimeric cDNA (2.25 kilobases) was removed from the
pEGFPN1Cx43 plasmid (44) was inserted into the NAP2 vector. The
resulting plasmids were designated NAP2Cx43 and NAP2Cx43·GFP (Fig. 1,
C and D).
Production of VSVG-pseudotyped Retrovirus--
To produce
pseudotype retroviral vectors, 293 GPG cells were plated at 2 × 106 cells per 60-mm dish the night before
transfection in 293 media. They were then transiently
transfected with AP2, NAP2Cx43, and NAP2Cx43·GFP plasmid retrovectors
by the LipofectAMINE PLUSTM (Life Technologies, Inc.)
procedure according to the manufacturer's instructions. Serum-free
DMEM medium with tetracycline (1 µg/ml) was used for all of the
transfection processes. After a 6-h incubation of cells in the
incubator, an additional 2 ml of the 293 medium was added for
transfection. The following morning, the medium was replaced with fresh
293 medium. On the next day, the 293 medium was removed from cells and
replaced with 3 ml of DMEM with 10% fetal calf serum. On the next day,
the supernatants were collected about every 12-24 h for 1 week. All
culture supernatants were filtered through 0.45-µm syringe-mounted
filters (Gelman Sciences, Ann Arbor, MI). The filtered culture
supernatants were used as infectious viral stock for subsequent
experiments. Aliquots of 1.0 ml were also frozen at Titration of Retroviral Supernatant--
Flow cytometric
analysis was performed to determine the titer of the viral supernatant
as measured by GFP fluorescence. In brief, 2 × 105 C6
cells/well were plated in 6-well tissue culture plates the night before
infecting the cells with the retrovirus. The next day, cells from 3 wells were trypsinized and counted to determine the average number of
cells per well at the time of exposure to retrovirus. Serial dilutions
(1:10) of the viral supernatant in a final volume of 1 ml of DMEM, 10%
fetal bovine serum were prepared and added to each well. The following
day, 2.0 ml of medium were added, and cells were incubated for 2 more
days. Three days after viral infection, the transduced C6 cells were
trypsinized and resuspended in 2 ml of DMEM for flow cytometric
analysis assay. Analysis was performed on a FACStarPlusTM
(Becton Dickinson). Live C6 cells were gated based on scatter/side scatter profile and analyzed for GFP fluorescence to determine the
percentage of GFP-positive cells. Data acquisition and analysis were
performed using CELLQuestTM software (Becton Dickinson).
The titer (colony-forming units (CFU) per ml) was calculated as: (% GFP-positive cells × cell number at initial viral exposure)/viral
volume (ml) applied when transduction was not saturated (19).
Replication Competent Retrovirus (RCR) Assay--
C6 cells
infected with GFP were passed in culture for 6 weeks (15 passages) to
allow for spread of RCR that might be present. 3 ml of culture
supernatant from these C6 cells was then used to infect 106
naïve C6 cells, and 48 h later, the newly infected
naïve cells were evaluated by detecting GFP-fluorescent cells.
Protein Isolation and Western Blot Analysis--
Both
undifferentiated and differentiated N2a wild-type and transformant cell
cultures were washed twice with PBS and scraped off the plates in lysis
buffer (0.05 M Tris, pH 6.8, 0.1% SDS) with a rubber
policeman. The protein concentration of the cell lysate was determined
in each case using the Bio-Rad protein assay kit. Protein samples from
an equal number of cells (2.5 × 105 each) were
subjected to 10% SDS-polyacrylamide gel electrophoresis. The gel was
transferred to nitrocellulose membranes (Bio-Rad) at 5 watts overnight.
Membranes were stained with Ponseau S to control for protein loading
and transfer. The membranes were blocked in 5% (w/v) nonfat dry milk
(NFDM) in PBS for 1 h at room temperature, incubated with
monoclonal anti-Cx43 primary antibody overnight at 4 °C in PBS with
1% NFDM, washed with PBS, and incubated 1 h with
horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibody in PBS with 1% NFDM at room temperature. Finally, membranes were incubated with Supersignal substrate working solution (Pierce) for 5 min and exposed to autoradiographic film for 2-10 min.
Immunocytochemistry--
For this study the following primary
antibodies were used: 1) affinity-purified rabbit polyclonal anti-Cx43
CT-360 (21) (5 µg/ml); 2) affinity-purified mouse monoclonal
anti-Cx43 (1 µg/ml, Chemicon, Temecula, CA); 3) affinity-purified
mouse monoclonal anti-neurofilament 200 (phosphorylated and
non-phosphorylated, 1 µg/ml, Sigma). Secondary antibodies were
obtained from Dimension Laboratories (Mississauga, ON), and included
goat anti-rabbit IgG conjugated to fluorescein isothiocyanate (1 µg/ml) and goat anti-mouse IgG conjugated to Cy3 (1 µg/ml).
Cells were grown to confluence on glass, fixed in 4% paraformaldehyde
(w/v) for 10 min at 4 °C, washed with PBS, pH 7.4, permeabilized with 0.1% Triton X-100 for 10 min at room temperature, blocked in PBS
with 10% (v/v) normal goat serum (Dimension Laboratories) and 1.0%
(w/v) bovine serum albumin for 30 min, and then incubated for 1 h
with primary antibody(s) in PBS with 1% (w/v) bovine serum albumin.
The coverslips were washed with PBS and then incubated for 1 h
with the secondary antibody(s) in PBS with 1% (w/v) bovine serum
albumin. Coverslips were again washed and were mounted on slides in PBS
containing 50% (v/v) glycerol and 1% (w/v)
p-phenylenediamine (Fisher Scientific) to minimize quenching
of the fluorescent signal. Fluorescence was visualized on a Zeiss
Axiophot photomicroscope (Zeiss, North York, ON). Samples incubated
without primary antibody served as negative controls.
Current Injection--
Monolayers of undifferentiated and
differentiated N2a, N2a-Cx43, and N2a-Cx43·GFP cells grown on
coverslips were placed in a bath chamber containing culture medium at
room temperature (~22 °C) and viewed with a Leica DMIRB inverted
microscope with a × 20 phase-contrast objective. Glass
microelectrodes were prepared from microcapillary tubing (World
Precision Instruments (WPI), Kwik-Fill, no. 1B100F-3) with a Narishige
vertical microelectrode puller. After backfilling with 1 M
KCl, the electrodes had tip resistances of 40-80 megohm. They were
mounted on mechanical micromanipulators (Leica) and connected by
chlorinated silver wires to electrometers (WPI, Intra 767). The bath
was grounded using a copper wire connected to the bath chamber. Two
independent electrodes were used, one to pass current into a cell and
the other to record changes in membrane potentials at various
distances, i.e. 25, 100, and 400 µm respectively.
Electrotonic potentials were generated by passing hyperpolarizing
pulses of current (50 nA; 100 ms) down one electrode from a
stimulus-isolating instrument (WPI, A310 Accupulser). Membrane and
electrotonic potentials were recorded on a Gould RS 3200 two channel recorder.
In cell coupling experiments, each measurement of the electrotonic
voltage at a given interelectrode distance required that both the
electrodes obtain and maintain the full cell membrane potential. Upon
insertion of a microelectrode into a cell, a sharp drop in the
electrometer reading (identified as the resting membrane potential) was
noted. Current pulsing was initiated shortly after gaining a stable
membrane potential at the injecting electrode; after 4-5 pulses, the
electrode was removed. Upon removal of the electrode from the cell, the
measured potential returned to zero: recordings that did not return to
within 10% of the baseline were assumed to be ruptured cells, and
these recordings were discarded. Three interelectrode distances were
used: 25 µm (the distance to an adjacent cell), 100 µm, and 400 µm.
Dye Injection--
Clusters of N2a wild-type and N2a
transformants that had been infected with retrovector encoding Cx43 or
Cx43·GFP were pressure injected with 10 mM
carboxyfluorescein (Molecular Probes, Eugene, OR) in distilled water
using an Eppendorf microinjection system (Eppendorf, Hamburg, Germany).
In all cases, it took up less than one minute for carboxyfluorescein to
pass to adjacent cells after 0.5-s pressure injection. To be regarded
as coupled, at least one adjacent cell received the dye from the
injected donor cell.
In Vitro Growth Analysis--
N2a wild-type and transformants
from confluent cultures were seeded at 2.0 × 105
cells per 6-well culture plate. The growth analysis was carried out in
the absence and present of retinoic acid (RA) (20 µM). The cell viability was determined by trypan blue staining. At day 0, 3, 5, and 12 after plating, triplicate plates were dissociated with 0.25%
trypsin (Life Technologies, Inc.) and 1 mM EDTA
(BDH) in PBS, pH 7.4 and counted in a COULTER COUNTER® Z1
Series Particle Counter (Coulter Electronics, Burlington, ON).
Statistical Analysis--
Statistical analysis involving testing
for significance of differences for quantitative observations was
performed. First, all sets of data were subjected to a one-way analysis
of variance to determine significant difference between any two sets.
Subsequently, the sets of data were subjected to Student's
t test to determine whether significant differences existed
between the individual samples. The data was analyzed with Prism
(Graphpad Inc. Software, San Diego, CA).
Fluorescence Microscopy and Image Analysis--
Cells were
evaluated with a Zeiss Axioskop microscope equipped with a filter set
for fluorescein (exciter filter BP 485, banner filter 515-565 nm) and
for Cy3 (exciter filter BP 546, banner filter BP 590) using × 40 or × 63 Plan-Neufluor objectives. The images were captured using
Sensicontrol 4.02 (The Optikon Corporation Ltd., Kitchener, ON). Once
all of the digital images were captured, they were imported and
compiled into final figures using Adobe Photoshop 5.0 (Adobe Systems
Incorporated, San Jose, CA) and Corel Draw 8.0 (Corel Co., Ottawa, ON).
Effectiveness of Retroviral Infection of C6 Glioma Cells--
C6
cells were chosen for titering retrovector supernatant as C6 cells
divide rapidly and are easily infected. Using GFP as a reporter protein
allowed the rapid titration of retroviral supernatant. The supernatant
containing AP2 retrovector was sequentially collected, filtered, and
serially diluted in a final volume of 1 ml and added to 3.28 × 105 C6 cells/well in a 6-well plate. Three days after a
single application of retrovector, the infected C6 cells were analyzed
by flow cytometric analysis to determine the percentage of cells
expressing the GFP reporter protein (Fig.
2). The titer extrapolated from these
experiments was about 2.0 × 107 CFU/ml, calculated as
described under "Experimental Procedures." This titer is at least
1,000 times higher than that of NIH 3T3-based retroviral packaging cell
lines (22).
Stability of Retroviral Gene Delivery and Replication Competent
Retrovirus Assay--
C6 cells infected with viral supernatant
containing AP2 retrovectors demonstrated strong GFP fluorescence for at
least 15 passages over a 6-week period (Fig.
3). Naturally or because of recombination
events, viruses that contain all of the cis-acting viral elements and
genes coding for necessary viral structural proteins are
"replication-competent" (18). To test for RCR, 3 ml of
culture supernatant from these C6 cells was then used to infect
106 naïve C6 cells. 48 h later the newly
infected naïve cells were evaluated by detecting
GFP-fluorescent cells. Retroviruses produced from 293 GPG packaging
cells were free of detectable RCR upon long term cultivation as
demonstrated by the failure of GFP fluorescence to be transferred from
virally transduced C6 cells to naïve C6 cells (data not
shown).
Expression of Cx43 and Cx43·GFP in Differentiated N2a
Cells--
Communication-deficient N2a cells were infected with
retrovector encoding for Cx43 and Cx43·GFP to determine their ability to express full-length Cx43 and Cx43·GFP chimeric protein after differentiation. Wild-type N2a and N2a-GFP cells were used as controls.
The synthesis of Cx43 and Cx43·GFP protein was investigated by
Western blot analysis of total cell lysates from wild-type N2a cells,
N2a-GFP, N2a-Cx43, and N2a-Cx43·GFP transformants under differentiated conditions.
Differentiated N2a-Cx43 and N2a-Cx43·GFP cells expressed Cx43 protein
(42-44 kDa) and Cx43·GFP protein (72-74 kDa) (Fig.
4). As expected Cx43 was not detected in
wild-type N2a cells or N2a-GFP expressing cells. The C6-13 cells which
were transfected with Cx43 were used as a positive control, and contain
multiple bands of immunoreactivity, which represents phosphorylated and
unphosphorylated species of Cx43 (23). The low amount of phosphorylated
species in N2a cells infected with wild-type Cx43 or Cx43·GFP
cDNAs may reflect poor post-translational processing of Cx43 in
this cell line.
Localization of Cx43 and Cx43·GFP in Undifferentiated and
Differentiated N2a Cells--
To determine the localization of Cx43 in
N2a-Cx43, N2a-Cx43·GFP, N2a-GFP, or wild-type N2a cells,
differentiated or undifferentiated cells were either immunolabeled with
anti-Cx43 antibodies or directly examined for GFP fluorescence (Fig.
5).
In both undifferentiated and differentiated N2a-Cx43 and N2a-Cx43·GFP
cells, Cx43 was localized to areas of cell-cell contact as well as in
punctate structures within the cytoplasm (Fig. 5, A,
B, E, and F). As control, GFP
fluorescence was located throughout the cytoplasm in N2a-GFP cells
(Fig. 5, C and G) and no fluorescence was
detected in wild-type N2a cells immunolabeled for Cx43 (Fig. 5,
D and H). In all cases, the undifferentiated N2a
and N2a transformants were not neurofilament positive (data not shown).
An induction of neurofilament protein (200 kDa, phosphorylated and
non-phosphorylated) was apparent in processes of wild-type N2a cells
and all three N2a transformants (N2a-GFP, N2a-Cx43 and N2a-Cx43·GFP)
after 20 µM RA treatment for 7 days (Fig. 5,
I, J, K, and L). On
average, 90% of the cells were neurofilament positive after exposure
to 20 µM RA for 7 days. There was no apparent difference
in neurofilament staining between wild-type N2a, N2a-Cx43 and
N2a-Cx43·GFP cells.
Gap Junctional Intercellular Electrical Coupling Up-regulation in
Cx43- and Cx43·GFP-expressing N2a Cells--
To determine whether
Cx43 and Cx43·GFP formed functional gap junction channels in N2a
cells before and after differentiation, communication-deficient N2a
cells and N2a-Cx43 and N2a-Cx43·GFP transformants were examined for
electrical coupling. For undifferentiated N2a-Cx43 cells, electrotonic
spread of injected current, resulting in a membrane potential change at
the second electrode, could be detected 400 µm from the injecting
electrode (n = 8). For undifferentiated N2a-Cx43·GFP
cells, potential deflections were detected at 25 µm
(n = 8) but only twice in six attempts at 100 µm and
never at 400 µm (n = 8). Conversely, undifferentiated
N2a cells displayed no electrical coupling at any distance tested
(n = 9). In differentiated N2a-Cx43 cells, potential
defections were detected at 400 µm (n = 4). Also,
potential deflections were detectable in differentiated N2a-Cx43·GFP
cells at 25 µm (n = 3) but not at 100 µm
(n = 2) or 400 µm (n = 4). Thus, both
N2a-Cx43 and N2a-Cx43·GFP cells formed gap junctional channels that
were electrically coupled. In addition, the N2a-Cx43·GFP cells
appeared less adherent (i.e. they were easily pulled off the
coverslip by the microeletrodes, which may account for the differences
in coupling between Cx43 and Cx43·GFP cells.
Gap Junctional Intercellular Dye Coupling Is Up-regulated When Cx43
or Cx43·GFP Is Expressed in N2a Cells--
To examine the
dye-coupling capacity of Cx43 or Cx43·GFP-mediated GJIC, both
undifferentiated and differentiated N2a wild-type and transformants
were microinjected with 10 mM carboxyfluorescein (CF).
Undifferentiated and differentiated N2a-Cx43·GFP cells (Fig. 6, A and B,
asterisk), N2a-Cx43 cells (Fig. 6, E and
F, asterisk) and N2a wild type (Fig. 6,
I and J, asterisk) were injected with CF. CF spreads rapidly to adjacent cells in both differentiated and
undifferentiated N2a-Cx43·GFP and N2a-Cx43 cells within a minute (Fig. 6, A, B, E, and
F). The gap junctional plaques formed by Cx43·GFP were
seen as punctate fluorescent spots between adjacent cells (Fig. 6,
A and B, arrows). N2a-Cx43 and
N2a-Cx43·GFP cells effectively transferred CF to neighboring cells in
more than 70% of cases under undifferentiated conditions and in
greater than 50% after neuronal differentiation (Table
I).
Furthermore, CF was extensively transferred to at least the 6th order
of neighboring undifferentiated Cx43 or Cx43·GFP expressing cells
(Fig. 6, A and E), although this was reduced to
the 2nd to 3rd order when the same cells were differentiated with
retinoic acid (Fig. 6, B and F). As a control,
communication-deficient wild-type N2a cells exhibited no dye coupling
before or after differentiation (Fig. 6, I and
J).
N2a Cells Expressing Cx43 or Cx43·GFP Display in Low but
Significant Reduction in Cell Growth After RA-induced
Differentiation--
Under the normal culture condition, Cx43 and
Cx43·GFP exhibited no growth inhibition in N2a cells (data not
shown). In the presence of 20 µM of RA, however, the
growth rate of N2a-Cx43 and N2a-Cx43·GFP cells was significantly
decreased (Fig. 7) at every time point
when compared with wild-type N2a or N2a-GFP cells. There were no
significant differences between N2a-Cx43 and N2a-Cx43·GFP transformants or between wild-type N2a and N2a-GFP cells (Fig. 7).
By virtue of its intrinsic fluorescence, GFP is readily detected
in living cultured cells, and the efficiency of transduction can be
rapidly and directly determined. GFP fluorescence provides a visual
assessment for rapidly determining the viral titer by flow cytometric
analysis, which is consistent with a direct test of viral-mediated
transfer of drug resistance to host cells (24). In this study, the
titer of the AP2 retrovector determined by flow cytometric analysis of
GFP fluorescence was about 2.0 × 107 CFU/ml which is
similar to other studies (19). The efficient gene transduction of high
titer viral supernatant eliminates the need to generate stable
transformed cell lines, which requires months of selection and
characterization. Therefore, the AP2 retroviral gene delivery system is
well suited for quickly examining expression of exogenous gene(s)
in vitro.
Many connexins are differentially expressed during neuronal
differentiation. During development, neuroblasts are highly coupled, with a loss or reduction of GJIC during terminal differentiation (8,
25). A similar decrease in the connexin expression level and GJIC has
been reported during in vitro differentiation of two
neuronal stem cells, i.e. the P19 mouse embryonal carcinoma cell line (9) and the NT2 human teratocarcinoma cell line (6, 7).
In our study, N2a neuroblastoma cells provide an ideal in
vitro model to examine the putative role of Cx43-mediated GJIC in neuronal differentiation and growth control because they are of neuronal origin, do not express any known connexins (26), and exhibit
no detectable gap junctional intercellular coupling. Retroviral gene
transduction, Western blot analysis, and immunocytochemistry revealed
that N2a cells were able to maintain the expression of Cx43 and
Cx43·GFP protein after neuronal differentiation. Cx43 and Cx43·GFP
were localized as punctate staining in the cytoplasm and in the
membrane between apposed cells. About 90% of the N2a cells
overexpressing Cx43 and Cx43·GFP could be differentiated into mature
neurons, similar to wild-type cells. These results suggest that
Cx43-mediated GJIC does not alter neuronal differentiation of N2a
cells. It is possible that in their undifferentiated state, N2a cells
have already gone through a possible GJIC-dependent differentiation stage because undifferentiated N2a cells express a
certain level of the neuronal marker microtubule-associated protein 2 (MAP2) (27).
Gap juctions are known to provide a channel for the intercellular flow
of essential electrical signals and small molecules (28). The current
flow through gap junctions is important in the rapid and synchronous
activation of excitable tissues (29). In our studies, all N2a-Cx43 and
N2a-Cx43·GFP cells were electrically coupled both before and after
neuronal differentiation. However, the cells expressing Cx43·GFP
exhibited lower levels of electrical coupling than cells expressing
Cx43. Because the carboxyl-terminal region of Cx43 is presumably
involved in the regulation of channel gating (1), fusion of GFP to this
portion of the connexin may interfere with channel function. Using dual
whole-cell patch clamp recording, Bukauskas et al. (30) have
reported some alterations in transjunctional voltage gating for N2A
cells transfected with Cx43 or Cx43·GFP but offered no explanation
for this difference. We have also noted that N2A cells transfected with
Cx43 or Cx43·GFP are less dye coupled after differentiation. This
result is consistent with the in vivo studies, which have
indicated that electrotonic junctions are wide spread between mammalian
neurons in many areas, including neocortex, hippocampus, inferior
olive, locus coeruleus, hypothalamus, striatum, and retina (3, 31,
32).
From our dye-coupling analysis, undifferentiated N2a-Cx43 and
N2a-Cx43·GFP cells were found to be coupled on average to 6th order
neighbors. After differentiation, however, the coupling decreased on
average to 2nd or 3rd order. One possibility is that the differentiated
cells bear long processes, and their spatial arrangement differs from
undifferentiated cells. Therefore, the dye must pass a similar distance
but involves fewer cells. It is also possible that the functional
dye-coupling capacity of Cx43 and Cx43·GFP does decrease with
neuronal differentiation. Our studies demonstrate for the first time
highly coupled differentiated neuronal cells, which provide a suitable
model to further investigate the electrotonic junction between mature
neurons both in vitro and in vivo.
One of the major characteristics of neoplastic cells is their
uncontrolled rapid growth. Extensive aspects of tumor growth have been
studied, including the implication that loss or lack of gap junctional
communication is associated with tumor development (33-37). The
introduction and overexpression of connexin cDNAs in tumor cell
lines by transfection has demonstrated a reduced growth rate (38-40).
C6 cells transfected with Cx43 cDNA exhibited a decrease in cell
growth both in vitro and in vivo (11-13, 41). In
contrast to C6-Cx43 cells which grow slower than the parental C6 cells,
the N2a-Cx43 and N2a-Cx43·GFP transformants demonstrated no
significant decrease in growth rate in vitro when
undifferentiated. It appears that the establishment of GJIC in
communication-deficient tumor cells does not always lead to growth
inhibition. It has been demonstrated that certain connexin genes exert
a growth control effect whereas others do not (38). In addition, gap
junctions composed of different connexins demonstrate some molecular
transport selectivity (42). In the presence of retinoic acid (20 µM), however, the growth of N2a-Cx43 and N2a-Cx43·GFP was significantly inhibited. This finding suggests that growth inhibition in N2a transformants may be because of the RA treatment and related
transjunctional molecule(s) rather than Cx43 itself.
This study constitutes the first report of an in vitro
generation of highly coupled mature neurons obtained using retroviral delivery of connexin constructs. Recent therapeutic applications have
employed retroviral delivery of the Herpes simplex virus thymidine kinase gene to brain tumors, with limited success (19). Given
the potential tumor suppressor effects of connexins, as well as the
role of GJIC in mediating some aspects of the "bystander effect"
associated with Herpes simplex virus thymidine kinase expression and
combined pro-drug (i.e. ganciclovir) treatment, we propose
that retroviral delivery of connexin genes may be therapeutically relevant.
*
This work was supported by the Medical Research Council of
Canada.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Published, JBC Papers in Press, August 2, 2000, DOI 10.1074/jbc.M003917200
The abbreviations used are:
Cx, connexin;
GJIC, gap junctional intercellular communication;
VSVG, vesicular stomatitis
virus G protein;
GFP, green fluorescent protein;
N2a, neuro 2a cell
line;
RA, retinoic acid;
DMEM, Dulbecco's minimum essential medium;
CFU, colony form unit;
RCR, replication-competent retrovirus;
PBS, phosphate-buffered saline;
NFDM, nonfat dry milk;
CF, carboxyfluorescein.
Neuronal Differentiation and Growth Control of Neuro-2a Cells
After Retroviral Gene Delivery of Connexin43*
,,, and
Anatomy and Cell Biology and
§ Medical Biophysics, Child Health Research Institute,
University of Western Ontario, London, Ontario, Canada N6A 5C1 and the
¶ McGill Centre for Translational Research in Cancer, Lady
Davis Institute, McGill University, Montreal, QC, Canada H3T 1E2
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (24K):
[in a new window]
Fig. 1.
Schematic representations of plasmid
retrovectors. A, AP2 plasmid retrovector is a
bicistronic murine retroviral vector allowing the insertion of a
cDNA sequence in multiple cloning sites upstream of the IRES and
the EGFP cassette. B, NAP2 plasmid retrovector resulted from
cutting the IRES and EGFP fragment of the AP2 plasmid retrovector.
C, the NAP2-Cx43 plasmid retrovector resulted from insertion
of Cx43 cDNA into the NotI-ClaI cloning sites
of NAP2. D, the NAP2-Cx43·GFP retrovector resulted from
insertion of Cx43·GFP cDNA into the
BglII-NotI sites.
80 °C for
later use.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (36K):
[in a new window]
Fig. 2.
Titration of retroviral supernatant using GFP
intrinsic fluorescence. Retroviral supernatant was serial diluted
in a final volume of 1 ml and added to 3.28 × 105 C6
cells per well in a 6-well culture plate. Three days after a single
application of AP2 retrovector, C6 cells were analyzed for GFP
fluorescence by flow cytometry. The percentage of GFP-positive cells is
indicated in each histogram figure. Titer extrapolated from these
experiments was about 2.0 × 107 CFU/ml. M1 determined
the positive threshold for GFP fluorescence.
View larger version (135K):
[in a new window]
Fig. 3.
Stability of GFP expression using retroviral
gene delivery. GFP expression in C6 cells detected by fluorescent
microscopy (× 40, oil) at the time of first passage three days after
infection (A) and the 15th passage (B).
Comparison with the corresponding phase contrast images (C
and D) revealed that more than 98% of C6 cells in the
population remained GFP positive during this 6-week period.
Magnification is × 600.
View larger version (46K):
[in a new window]
Fig. 4.
Western blot analysis of the expression of
Cx43 and Cx43·GFP protein in differentiated N2a transformants.
Protein lysates from equal number of cells were resolved by
SDS-polyacrylamide gel electrophoresis and immunolabeled with anti-Cx43
monoclonal antibody. The differentiated N2a-Cx43 and N2a-Cx43·GFP
express Cx43 protein (42-44 kDa) and Cx43·GFP proteins (72-74 kDa).
No detectable bands corresponding to Cx43 protein could be seen in
wild-type N2a and N2a-GFP cells. The C6-13 cells which were
transfected with Cx43 were used as a positive control. D,
differentiated.
View larger version (45K):
[in a new window]
Fig. 5.
Cx43 and Cx43·GFP expression in N2a
wild-type and transformants before and after neuronal
differentiation. In undifferentiated transformants, the Cx43 and
Cx43·GFP proteins appeared mostly as punctate staining (A
and B, arrows) on the cell surface against a
diffuse fluorescent background. In differentiated transformants, Cx43
and Cx43·GFP protein are located in the processes, cell body, and
cell surface (E and F, arrowheads). No
Cx43 or Cx43·GFP was detected in N2a wild-type and N2a-GFP cells
(C, D, G, and H). As
control, GFP fluorescence is located throughout the cytoplasm in
N2a-GFP cells (C and G). A and
E were immunolabeled with anti-Cx43. B,
C, F, and G show only GFP
fluorescence. A monoclonal neurofilament 200 antibody was used in
panels I-L. In all cases, the undifferentiated cells were
neurofilament negative (data not shown). After neuronal
differentiation, about 90% of all the RA-treated cells were
neurofilament positive (I, J, K, and
L). Magnification is × 1,000.
View larger version (57K):
[in a new window]
Fig. 6.
Dye-coupling assay of N2a wild-type and
transformants both before and after differentiation. The
undifferentiated N2a-Cx43 and N2a-Cx43·GFP cells were coupled on
average to 6th order neighbors (A and E). After
differentiation, however, the coupling decreased on average to 2nd to
3rd order (B and F). CF spreads rapidly from
injected cells (A, B, E, and
F; asterisk) to adjacent cells when compare with
phase contrast images (C, D, G, and
H). The direct visualization of Cx43·GFP during
microinjection is shown in A and B
(arrows). N2a wild type and N2a-GFP exhibited no dye
coupling to adjacent cells either before or after differentiation
(I and J) when compared with phase contrast
images (K and L). Magnification is × 850.
Summary of dye injection assay
View larger version (52K):
[in a new window]
Fig. 7.
In vitro growth analysis in the
presence of RA (20 µM). N2a
wild-type and derived transformants from confluent cultures were seeded
at 2.0 × 105 per 6-well culture plate. 3, 5, and 12 days after plating in the presence of RA, triplicate plates were
dissociated into individual cell suspensions and counted. The
statistical significance (p < 0.05, 0.01, and 0.001)
are represented by 1, 2, or 3 asterisks, respectively.
n = 6; bars, mean ± S.E.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
FOOTNOTES
To whom correspondence should be addressed: Dept. of Anatomy
and Cell Biology, Medical Sciences Building, University of Western Ontario, London, ON, Canada N6A 5C1. Tel.: 519-661-4067; Fax: 519-661-3936; E-mail: cnaus@julian.uwo.ca.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Bruzzone, R.,
White, T. W.,
and Paul, D. L.
(1996)
Eur. J. Biochem.
238,
1-27
2.
Bruzzone, R.,
and Ressot, C.
(1997)
Eur. J. Neurosci.
9,
1-6
3.
Dermietzel, R.,
and Spray, D. C.
(1993)
Trends Neurosci.
16,
186-192
4.
Nadarajah, B.,
Thomaidou, D.,
Evans, W. H.,
and Parnavelas, J. G.
(1996)
J. Comp. Neurol.
376,
326-342
5.
Rozental, R.,
Mehler, M. F.,
Morales, M.,
Andrade-Rozental, A. F.,
Kessler, J. A.,
and Spray, D. C.
(1995)
Dev. Biol.
167,
350-362
6.
Belliveau, D. J.,
Bechberger, J. F.,
Rogers, K. A.,
and Naus, C. C.
(1997)
Dev. Genet.
21,
187-200
7.
Bani-Yaghoub, M.,
Bechberger, J. F.,
and Naus, C. C.
(1997)
J. Neurosci. Res.
49,
19-31
8.
Kandler, K.,
and Katz, L. C.
(1995)
Curr. Opin. Neurobiol.
5,
98-105
9.
Bani-Yaghoub, M.,
Underhill, T. M.,
and Naus, C. C.
(1999)
Dev. Genet.
24,
69-81
10.
Bani-Yaghoub, M.,
Bechberger, J. F.,
Underhill, T. M.,
and Naus, C. C.
(1999)
Exp. Neurol.
156,
16-32
11.
Naus, C. C.,
Zhu, D.,
Todd, S. D.,
and Kidder, G. M.
(1992)
Cell. Mol. Neurobiol.
12,
163-175
12.
Zhu, D.,
Caveney, S.,
Kidder, G. M.,
and Naus, C. C.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
1883-1887
13.
Zhu, D.,
Kidder, G. M.,
Caveney, S.,
and Naus, C. C.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
10218-10221
14.
Juneja, S. C.,
Barr, K. J.,
Enders, G. C.,
and Kidder, G. M.
(1999)
Biol. Reprod.
60,
1263-1270
15.
Reaume, A. G.,
De Sousa, P. A.,
Kulkarni, S.,
Langille, B. L.,
Zhu, D.,
Davies, T. C.,
Juneja, S. C.,
Kidder, G. M.,
and Rossant, J.
(1995)
Science
267,
1831-1834
16.
Burns, J. C.,
Friedmann, T.,
Driever, W.,
Burrascano, M.,
and Yee, J. K.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
8033-8037
17.
Aboody-Guterman, K. S.,
Pechan, P. A.,
Rainov, N. G.,
Sena-Esteves, M.,
Jacobs, A.,
Snyder, E. Y.,
Wild, P.,
Schraner, E.,
Tobler, K.,
Breakefield, X. O.,
and Fraefel, C.
(1997)
Neuroreport
8,
3801-3808
18.
Frederick, M. A.,
Roger, B.,
and Robert, E. K.
(1993)
Current Protocols in Molecular Biology
, pp. 9.9-9.14, John Wiley & Sons, Inc., New York
19.
Galipeau, J.,
Li, H.,
Paquin, A.,
Sicilia, F.,
Karpati, G.,
and Nalbantoglu, J.
(1999)
Cancer Res.
59,
2384-2394
20.
Ory, D. S.,
Neugeboren, B. A.,
and Mulligan, R. C.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
11400-11406
21.
Laird, D. W., and Revel, J. P. (1990) J. Cell
Sci. 97, Part 1, 109-117
22.
Pear, W. S.,
Nolan, G. P.,
Scott, M. L.,
and Baltimore, D.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
8392-8396
23.
Musil, L. S.,
Cunningham, B. A.,
Edelman, G. M.,
and Goodenough, D. A.
(1990)
J. Cell Biol.
111,
2077-2088
24.
Bagley, J.,
Aboody-Guterman, K.,
Breakefield, X.,
and Iacomini, J.
(1998)
Transplantation
65,
1233-1240
25.
Cepeda, C.,
Walsh, J. P.,
Peacock, W.,
Buchwald, N. A.,
and Levine, M. S.
(1993)
Cereb. Cortex
3,
95-107
26.
Veenstra, R. D.,
Wang, H. Z.,
Westphale, E. M.,
and Beyer, E. C.
(1992)
Circ. Res.
71,
1277-1283
27.
Wang, L. J.,
Colella, R.,
Yorke, G.,
and Roisen, F. J.
(1996)
Exp. Neurol.
139,
1-11
28.
Spray, D. C.,
Harris, A. L.,
and Bennett, M. V.
(1981)
J. Gen. Physiol.
77,
77-93
29.
Spray, D. C.,
and Bennett, M. V.
(1985)
Annu. Rev. Physiol.
47,
281-303
30.
Bukauskas, F. F.,
Jordan, K.,
Bukauskiene, A.,
Bennett, M. V.,
Lampe, P. D.,
Laird, D. W.,
and Verselis, V. K.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
2556-2561
31.
Dudek, F. E.,
Andrew, R. D.,
MacVicar, B. A.,
Snow, R. W.,
and Taylor, C. P.
(1983)
in
Basic Mechanisms of Neuronal Hyperexcitability
(Jasper, H.
, and van Gelder, N., eds)
, pp. 31-73, A. R. Liss, New York
32.
Cook, J. E.,
and Becker, D. L.
(1995)
Microsc. Res. Tech.
31,
408-419
33.
Loewenstein, W. R.,
and Kanno, Y.
(1966)
Nature
209,
1248-1249
34.
Mesnil, M.,
and Yamasaki, H.
(1993)
Mol. Carcinogen.
7,
14-17
35.
Holder, J. W.,
Elmore, E.,
and Barrett, J. C.
(1993)
Cancer Res.
53,
3475-3485
36.
Ruch, R. J.
(1994)
Ann. Clin. Lab. Sci.
24,
216-231
37.
Yamasaki, H.,
and Naus, C. C.
(1996)
Carcinogenesis
17,
1199-1213
38.
Mesnil, M.,
Krutovskikh, V.,
Piccoli, C.,
Elfgang, C.,
Traub, O.,
Willecke, K.,
and Yamasaki, H.
(1995)
Cancer Res.
55,
629-639
39.
Rose, B.,
Mehta, P. P.,
and Loewenstein, W. R.
(1993)
Carcinogenesis
14,
1073-1075
40.
Eghbali, B.,
Kessler, J. A.,
Reid, L. M.,
Roy, C.,
and Spray, D. C.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
10701-10705
41.
Naus, C. C.,
Elisevich, K.,
Zhu, D.,
Belliveau, D. J.,
and Del Maestro, R. F.
(1992)
Cancer Res.
52,
4208-4213
42.
Elfgang, C.,
Eckert, R.,
Lichtenberg-Frate, H.,
Butterweck, A.,
Traub, O.,
Klein, R. A.,
Hulser, D. F.,
and Willecke, K.
(1995)
J. Cell Biol.
129,
805-817
43.
Beyer, E. C.,
Paul, D. L.,
and Goodenough, D. A.
(1987)
J. Cell Biol.
105,
2621-2629
44.
Jordan, K.,
Solan, J. L.,
Dominquez, M.,
Sia, M.,
Hand, A.,
Lampe, P. D.,
and Laird, D. W.
(1999)
Mol. Biol. Cell
10,
2033-2050
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  S. Langlois, K. N. Cowan, Q. Shao, B. J. Cowan, and D. W. Laird Caveolin-1 and -2 Interact with Connexin43 and Regulate Gap Junctional Intercellular Communication in Keratinocytes Mol. Biol. Cell, March 1, 2008; 19(3): 912 - 928. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. M. L. Hutnik, C. E. Pocrnich, H. Liu, D. W. Laird, and Q. Shao The Protective Effect of Functional Connexin43 Channels on a Human Epithelial Cell Line Exposed to Oxidative Stress Invest. Ophthalmol. Vis. Sci., February 1, 2008; 49(2): 800 - 806. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Langlois, A. C. Maher, J. L. Manias, Q. Shao, G. M. Kidder, and D. W. Laird Connexin Levels Regulate Keratinocyte Differentiation in the Epidermis J. Biol. Chem., October 12, 2007; 282(41): 30171 - 30180. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. P.K. Lai, J. F. Bechberger, R. J. Thompson, B. A. MacVicar, R. Bruzzone, and C. C. Naus Tumor-Suppressive Effects of Pannexin 1 in C6 Glioma Cells Cancer Res., February 15, 2007; 67(4): 1545 - 1554. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Kalra, Q. Shao, H. Qin, T. Thomas, M. A. Alaoui-Jamali, and D. W. Laird Cx26 inhibits breast MDA-MB-435 cell tumorigenic properties by a gap junctional intercellular communication-independent mechanism Carcinogenesis, December 1, 2006; 27(12): 2528 - 2537. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. Belliveau, M. Bani-Yaghoub, B. McGirr, C. C. G. Naus, and W. J. Rushlow Enhanced Neurite Outgrowth in PC12 Cells Mediated by Connexin Hemichannels and ATP J. Biol. Chem., July 28, 2006; 281(30): 20920 - 20931. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Thomas, K. Jordan, J. Simek, Q. Shao, C. Jedeszko, P. Walton, and D. W. Laird Mechanisms of Cx43 and Cx26 transport to the plasma membrane and gap junction regeneration J. Cell Sci., October 1, 2005; 118(19): 4451 - 4462. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. T. Fu, J. F. Bechberger, M. A. Ozog, B. Perbal, and C. C. Naus CCN3 (NOV) Interacts with Connexin43 in C6 Glioma Cells: POSSIBLE MECHANISM OF CONNEXIN-MEDIATED GROWTH SUPPRESSION J. Biol. Chem., August 27, 2004; 279(35): 36943 - 36950. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. I. Veitch, J. E. I. Gittens, Q. Shao, D. W. Laird, and G. M. Kidder Selective assembly of connexin37 into heterocellular gap junctions at the oocyte/granulosa cell interface J. Cell Sci., June 1, 2004; 117(13): 2699 - 2707. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Thomas, D. Telford, and D. W. Laird Functional Domain Mapping and Selective Trans-dominant Effects Exhibited by Cx26 Disease-causing Mutations J. Biol. Chem., April 30, 2004; 279(18): 19157 - 19168. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Rukstalis, A. Kowalik, L. Zhu, D. Lidington, C. L. Pin, and S. F. Konieczny Exocrine specific expression of Connexin32 is dependent on the basic helix-loop-helix transcription factor Mist1 J. Cell Sci., August 15, 2003; 116(16): 3315 - 3325. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Qin, Q. Shao, S. A. Igdoura, M. A. Alaoui-Jamali, and D. W. Laird Lysosomal and Proteasomal Degradation Play Distinct Roles in the Life Cycle of Cx43 in Gap Junctional Intercellular Communication-deficient and -competent Breast Tumor Cells J. Biol. Chem., August 8, 2003; 278(32): 30005 - 30014. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. E. I. Gittens, A. A. Mhawi, D. Lidington, Y. Ouellette, and G. M. Kidder Functional analysis of gap junctions in ovarian granulosa cells: distinct role for connexin43 in early stages of folliculogenesis Am J Physiol Cell Physiol, April 1, 2003; 284(4): C880 - C887. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Qin, Q. Shao, H. Curtis, J. Galipeau, D. J. Belliveau, T. Wang, M. A. Alaoui-Jamali, and D. W. Laird Retroviral Delivery of Connexin Genes to Human Breast Tumor Cells Inhibits in Vivo Tumor Growth by a Mechanism That Is Independent of Significant Gap Junctional Intercellular Communication J. Biol. Chem., August 2, 2002; 277(32): 29132 - 29138. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Li, S. Brodsky, S. Kumari, V. Valiunas, P. Brink, J.-I. Kaide, A. Nasjletti, and M. S. Goligorsky Paradoxical overexpression and translocation of connexin43 in homocysteine-treated endothelial cells Am J Physiol Heart Circ Physiol, June 1, 2002; 282(6): H2124 - H2133. [Abstract] |
