1
Department of Biology, Imperial College of Science, Technology and Medicine,
Sir Alexander Fleming Building, London, SW7 2AZ, UK
2
Department of Cell Biology, Institute of Molecular Biology, Jagiellonian
University, 31-120 Krakow, Poland
*
Author for correspondence (e-mail:
m.djamgoz{at}ic.ac.uk
)
Accepted April 12, 2001
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SUMMARY |
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Key words: Cancer, Metastasis, Voltage-gated Na+ channel, Prostate, Dunning, Rat
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INTRODUCTION |
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A variety of motile cells, from protozoan to mammalian, also respond to an
externally applied direct-current (DC) electric field by changing the
orientation of their movement (Erickson and Nucitelli,
1984; Robinson,
1985
; Ferrier et al.,
1986
; Frank and Gruler,
1990
; Nishimura et al.,
1996
; McCaig and Zhao,
1997
). This property, known as
`galvanotaxis', is involved in a number of basic biological processes, such as
embryonic development (Jaffe and Nucitelli,
1977
; McCaig,
1989a
; McCaig,
1989b
; McCaig and Dover, 1989)
and can also manifest itself under pathophysiological conditions, as in wound
healing (e.g. Chiang et al.,
1992
). In the related
phenomenon of `galvanotropism', external electric fields can facilitate
cellular process extension, again in both normal conditions (e.g. Hotary and
Robinson, 1990
) and
pathological situations (e.g. bone healing; Zhuang et al.,
1997
) and nerve regeneration
(Borgens et al., 1981
).
However, it is not known whether cancer cells are galvanotactic. Also, it is
not known whether there is any relation between galvanotaxis and metastatic
potential. Nevertheless, metastasis can involve specific tissue invasion; for
example, rat prostate cancer MAT-LyLu cells metastasise specifically to lymph
nodes and lungs (Isaacs et al.,
1986
). Furthermore, metastasis
can originate from sites in the body (e.g. epithelial ducts, skin) where local
DC electric fields would occur. In the case of the rat prostate gland, a
transepithelial potential of some 10 mV has been recorded (Szatkowski
et al., 2000
). Interestingly,
electro-imaging of mammary and cervical tissues has been used in clinical
detection of malignancy (Fukuda et al.,
1996
; Faupel et al.,
1997
; Cuzick et al.,
1998
), although its basis is
not well understood.
Cells can react to an applied weak DC electric field in different ways,
moving towards the cathode or the anode, and it is possible that a variety of
membrane mechanisms are involved in such responses (e.g. Nuccitelli,
1988; Robinson,
1985
; Soong et al.,
1990
). It has been observed
that, under the influence of externally applied DC electric fields,
Ca2+ concentration increased significantly and was maintained for
the duration of exposure (Onuma and Hui,
1988
). In this situation, the
cell would move away from the end where intracellular Ca2+ rises
and contractile activity occurs (Cooper and Keller,
1984
). Accordingly, in the
presence of Ca2+ channel blockers (e.g. D-600, verapamil), the
directionality of migration can be disturbed and replacement of external
Ca2+ by Mg2+ can also reverse the electrotactic response
(Cooper and Schliwa, 1986
;
Onuma and Hui, 1988
;
Nuccitelli and Smart, 1989
).
Importantly, however, Ca2+-independent control of galvanotaxis has
also been found (e.g. Brown and Loew,
1994
; Palmer et al.,
2000
), and it is not clear
whether other ionic mechanisms also play a role in galvanotactic responses of
cells. We have shown previously that there are distinct electrophysiological
differences between strongly and weakly metastatic cells of rat and human
prostate carcinoma (Grimes et al.,
1995
; Laniado et al.,
1997
; Smith et al.,
1998
; Foster et al.,
1999
). In particular,
functional voltage-gated Na+ channels (VGSCs) occurred specifically
in the highly metastatic cells (Grimes et al.,
1995
; Laniado et al.,
1997
; Grimes and Djamgoz,
1998
). Indeed, VGSC expression
and invasiveness in vitro were correlated in numerous rat and human prostate
cancer cell lines (Smith et al.,
1998
).
Taking the available evidence together, the possibility arises that
metastatic cells could be galvanotactic and that membrane ion channel (VGSC)
activity could play a role in this process. The present study aimed to
evaluate this overall hypothesis. In order to test the possible involvement of
VGSC activity in galvanotactic reaction, tetrodotoxin (TTX), a highly specific
blocker of VGSCs, was used. The working concentration of TTX was 1 µM,
which would effectively block the TTX-sensitive VGSCs present in the strongly
metastatic MAT-LyLu line (Grimes and Djamgoz,
1998; Diss et al.,
2001
). A further, `opposite'
test was carried out using veratridine (a VGSC `opener'), which potentiates
VGSC activity by lowering the threshold of the activation voltage (Eskinder et
al., 1993
). Finally, possible
involvement of voltage-gated Ca2+ channels was tested using
verapamil as a general blocker (Cooper and Schliwa,
1986
).
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MATERIALS AND METHODS |
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Galvanotaxis
Migration was assayed using a galvanotaxis apparatus described in detail by
Korohoda et al. (Korohoda et al.,
2000). Essentially, this was
made up from a glass observation chamber comprising a sandwich of two glass
coverslips (with the cells free to move in between) mounted in a Plexiglas
holder. Direct current was applied for 6 hours, through Ag/AgCl reversible
electrodes (of 6 cm2 surface area) immersed in wells filled with
cultured medium (RPMI + 5% FCS). Each well was connected by an agar bridge to
a neighbouring pool, which was continuous with the observation chamber. The
cover-glasses composing the latter measured 60 mmx5 mmx0.2 mm. The
electric current flowing through the chamber was measured continuously with a
milliampermeter; the voltage gradient was calculated using Ohm's Law and
confirmed by measuring with a high-input impedance voltmeter (Korohoda and
Kurowska, 1970
; Cho et al.,
1996
). 24 hours before
starting an experiment, the cells were replated onto one of the glass covers.
At the beginning of the experiment, this was sandwiched onto a second plate
(with a 0.2 mm gap, the cells still being bathed in their normal growth
medium), sealed with silicone grease and mounted in the Plexiglas apparatus.
Observations were carried out on an inverted microscope (Olympus, IMT-2).
Cells were observed using phase contrast optics at a total magnification of
285x (corresponding to a field of view of 710x710 µm). All
experiments were done at 37°C (the temperature of the chamber was
monitored and found to remain constant). Analysis of cells' motility during
the experiment (6 hours) confirmed that the recording conditions were very
stable because there was no observable change in any parameter under the
control conditions. All experiments were carried out for 6 hours unless
otherwise stated.
Cell images were recorded with a Hitachi CCD camera, digitized and
processed with the computer programs as described previously (Korohoda and
Madeja, 1997; Korohoda et al.,
1997a
; Korohoda et al.,
1997b
). For cell motion
tracking, a series of time-lapse images of cells were acquired using a
frame-grabber device and a `slow motion VCR' program (VCR 1.0). The cell
trajectories were constructed from 72 successive cell centroid positions
recorded over 6 hours with a time interval of 5 minutes (30 seconds in some
experiments). For each experimental condition, the trajectories of at least 50
cells were analysed. Usually, 15-20 cells were examined in one experiment and
data from three or four experiments were pooled. Detailed analyses of the data
were performed using the program `Mathematica' (Wolfram Research Inc.,
Champaign, IL; Korohoda and Madeja,
1997
; Korohoda et al.,
1997a
).
Parameters
The following parameters characterising different aspects of cell
locomotion were computed and analysed for each cell or cell population
(Korohoda et al., 2000).
(1) Total length of cell trajectory (TLT). This was essentially the `true' length of the path (in µm) travelled by the cell. The value of TLT was calculated from the sequence of n straight-line segments, each corresponding to a cell-centroid translocation between two successive images.
(2) Average speed of cell locomotion (ASL). This was defined as the total length of cell trajectory (TLT) ÷ time of recording (6 hours).
(3) Total length of cell displacement (TLD). This was the distance (in µm) from the starting point direct to the final position of the cell.
(4) Average rate of cell displacement (ARD). This was defined as the total length of cell displacement from the starting point to the final cell position (TLD) ÷ time of recording (6 hours).
(5) Coefficient of movement efficiency (CME). This was defined as the ratio
of total cell displacement (TLD) to total cell trajectory length (TLT). The
value of CME would be 1 for cells moving persistently along one straight line
in one direction, and 0 for random movement (Friedl et al.,
1993; Korohoda et al.,
1997b
).
(6) Average directional cosine (ADC
). The angle
was
defined as the directional angle between the x axis (parallel to the
electric field) and a vector AB, A and B being the original and each
subsequent positions of the cell, respectively. This parameter would equal +1
for a cell moving towards the cathode, -1 for a cell moving in the direction
of the anode and 0 for random movement (Gruler and Nuccitelli,
1991
; Korohoda et al.,
1997b
). This parameter was
used generally to quantify the directionality of movement.
(7) Average directional cosine ß (ADCß). The angle ß was
defined as the directional angle between the x axis (parallel to the
electric field) and a vector AB, A and B being two successive positions of the
cell, respectively. This parameter would equal +1 for a cell moving towards
the cathode, -1 for a cell moving in the direction of the anode and 0 for
random movement (Gruler and Nuccitelli,
1991; Korohoda et al.,
1997b
). This parameter was
particularly useful in testing the reversibility of effects of electric field
or a given pharmacological agent.
Pharmacology
In experiments involving pharmacological treatment of the cells under
investigation, a given test compound was introduced into the observation
chamber and recording started immediately. In some experiments, it was
possible to re-perfuse the cells with the control medium while recording to
check the reversibility of the effect, if any. The following compounds were
used: TTX (citrate free), veratridine and verapamil. All chemicals were
obtained from Sigma-Aldrich.
Data analysis
Data are presented as means ± s.e.m. Statistic significance was
determined using the Mann-Whitney test. Values of P < 0.01 were
assumed to represent significance.
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RESULTS |
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The electric field effects seen on the motility of the MAT-LyLu cells were enhanced as the applied voltage gradient was made stronger (Fig. 3A,B). Thus, as the electric field was increased from 0.1 V cm-1 to 4.0 V cm-1, both the directionality of movement and the overall length of cell displacement increased steadily. These effects were reversible. Thus, upon extinguishing or reversing the field, the cells' corresponding responses were rapidly abolished or reversed, respectively (Fig. 4). On the whole, however, there was some heterogeneity in the cells' response to the electric fields, with small, rounded cells appearing more sensitive than larger cells with prominent extensions.
|
|
We have shown previously that only strongly metastatic cells express
functional VGSCs (Grimes et al.,
1995; Laniado et al.,
1997
; Smith et al.,
1998
). Therefore, the possible
role of VGSC activity in the cells' response to electric field was
investigated by pharmacological manipulation of VGSC activity (Grimes and
Djamgoz, 1998
; Fraser et al.,
2000
; Figs
5,6,7;
Tables 1,
2). An additional parameter,
Pc (percentage of cells crossing a criterion boundary of
200 µm from the position occupied just before application of the field
during the 6-hour recording period) was used to compare the effects of the
drugs on the extent of the cells' directional migration
(Fig. 7). Under control
conditions, only very few (
2%) MAT-LyLu cells crossed the barrier; this
was greatly facilitated by the electric field
(Pc=24.0±2.9%; P=0.001)
(Fig. 7), consistent with the
demonstration above. None of the AT-2 cells migrated to this extent
(Fig. 2C;
Fig. 5E). Treatment of the
MAT-LyLu cell cultures with verapamil (1-10 µM) produced no effect (not
illustrated). However, the specific VGSC blocker TTX at 1 µM noticeably
reduced the percentage of MAT-LyLu cells migrating over the 200 µm
criterion distance (Pc=7.6±4.3%), whereas 2.5-5
µM TTX blocked the effect of the field completely
(Pc=0%) (Fig.
5A,B; Fig. 7). The
effects of TTX were reversed by washing the cells with the control medium
(Fig. 6). TTX had very little
effect on the average speed of movement of the MAT-LyLu cells
(Table 1) or any of the
measured parameters of the AT-2 cells
(Table 2;
Fig. 5E,F).
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Potentiating VGSC activity with veratridine (VER) had the opposite effect
to TTX, enhancing the specific actions of the electric field treatment on the
MAT-LyLu cells (Fig. 5C,D;
Fig. 7). Thus, in the presence
of 1 µM VER, the coefficient of movement efficiency increased significantly
from the normal value of 0.34 to 0.62 without any effect on the average speed
of cell movement (Table 1), and
30.0 ± 10% of cells crossed the 200 µm criterion barrier
(Fig. 7). The effects of VER
were dose-dependent (Table 1;
Fig. 7). The value of
ADC was enhanced significantly at 5 µM, the cells increasing their
directional response further than with application of the field alone. At the
highest concentration of VER used (10 µM),
Pc=44.0±2.0% (P=0.002 compared with
electric field without VER) (Fig.
7). Cell viability, assessed by trypan blue staining, was not
affected by VER (10 µM) or TTX (5 µM). However, VER treatment again had
relatively little effect on the AT-2 cells
(Table 2), consistent with
functional VGSCs being absent in these cells.
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DISCUSSION |
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The galvanotactic responses of various cell types, including neurons and
epithelia, have been found to be cathodal (McCaig and Zhao,
1997; Palmer et al.,
2000
), as shown here for the
MAT-LyLu cells. The effect is unlikely to be due to any asymmetry in the
distribution of VGSC protein over the cell surface because this was found to
be uniform on MAT-LyLu cells (not shown). Furthermore, any gross
redistribution of VGSCs (or other protein) in the membrane by electrophoresis
would also seem to be unlikely because this would take hours (Jaffe,
1977
), or at least several
minutes (Fang et al., 2000), whereas the electric field produced an observable
morphological change and directional response in the cells within 30 seconds.
The following possibilities can be considered as underlying mechanisms.
Basic transmembrane Ca2+ influx
It has been observed that, under the influence of externally applied DC
electric fields, intracellular Ca2+ concentration increased
significantly and was maintained for the duration of exposure (Onuma and Hui,
1988; Perret et al.,
1999
). In the simplest case,
of a cell devoid of voltage-gated (depolarization-activated) Ca2+
channels (VGCCs), passive influx of Ca2+ and contraction would on
the anodal side and the cell would move towards the cathode. In cells with
VGCCs, however, Ca2+ influx would (also) occur on the cathodal,
depolarised, side; thus, the direction of movement might depend on the balance
between the two sites of intracellular Ca2+ rise. These
possibilities are unlikely to be directly applicable here for several reasons.
(1) Any simple passive Ca2+ influx should have affected the two
cell types similarly but this was not seen
(Fig. 2B,D). (2) Verapamil (a
general blocker of Ca2+ channels) was found not to affect the
galvanotactic response of the cells. (3) The membrane depolarisation caused
directly by the electric field should have facilitated Ca2+ channel
activation directly without involving VGSCs; that is, TTX should not have had
any effect. (4) Our preliminary patch-clamp recordings and intracellular
Ca2+ measurements suggest, in fact, that the MAT-LyLu cells do not
possess voltage-gated Ca2+ channels (or significant
Na+-Ca2+ exchanger activity).
Surface charge
The `direct' effect of the electric field on the membrane potential could
be compounded by the presence of a negative surface charge, which is known to
occur in cancer cells and to increase in line with metastatic potential
(Abercrombie and Ambrose, 1962;
Carter and Coffey, 1988
; Carter
et al., 1989
; Price et al.,
1987
). It is therefore
possible that, in the presence of the exogenous electric field, the charge
`cloud' would be asymmetrical, and this could generate a spatial variation in
membrane potential (Gross,
1988
; Heberle et al.,
1994
; Scherrer,
1995
) that is different
between the two cell types. In the case of the MAT-LyLu cells, the negative
charges could accumulate on the anodal surface, thereby depolarising the cell
membrane. This could then simply increase the passive influx of
Ca2+, leading to contraction on the anodal side and propulsion of
the cells towards the cathode, as has been observed (Bedlack et al.,
1992
; Olivotto et al.,
1996
). In addition, the VGSCs
could interact directly with the cytoskeleton (Sheng and Kim,
1996
), possibly by the
membrane depolarisation inducing repeated conformational changes in the VGSC
proteins. Furthermore, VGSC ß-subunits possess a cell-adhesion motif
(e.g. Isom et al., 1995
) and
this could facilitate interaction with the extracellular matrix. We should
also note that control of galvanotaxis by Ca2+ released from
internal stores (Rosado and Sage,
2000
) and other more elaborate
secondary messenger have also been proposed (McCaig and Zhao,
1997
). However, how these
could involve VGSC activity is not clear at present. We also do not know
whether electric field treatment could have any long-term effect. Further work
is required to evaluate these possibilities.
The hitherto unknown role of VGSC activity in galvanotaxis that we have
demonstrated could have important implications in both cellular physiology and
pathophysiology. First, as regards normal biological functioning, a variety of
basic processes do involve directional or patterned growth, including
target-specific axonal migration and patterning of regional synaptic
connectivity. These processes have frequently been shown to be blocked by TTX
treatment, so would appear to depend on VGSC activity (Dubin et al.,
1986; Catalano and Shatz,
1998
; Meyer,
1982
; Penn et al.,
1998
; Shatz,
1990
). Accordingly, VGSC
expression and activity in cells exposed to endogenous electric fields could
facilitate directional growth in vivo.
Second, assuming that our findings are also applicable to the situation in
vivo (Fig. 8), especially
because VGSC protein is produced in clinical tumours at levels correlated with
pathological grading (Stewart et al.,
1999), it would follow that
the directional migration of prostate cells during the early stages of
metastasis could be influenced significantly by endogenous transepithelial
potentials (TEPs). We have recently found that rat prostate epithelia have a
lumen potential of about -10 mV (Szatkowski et al.,
2000
). Such a lumen potential
would correspond to transepithelial voltage gradient of 5 V cm-1,
assuming that the cellular thickness of the prostatic ducts is 20 µm
(Fig. 8). Such a voltage
gradient is comparable to the DC electric field strengths used to induce
galvanotaxis in the present study. If a similar situation occurs in the human
prostate epithelium in vivo then it would follow that presumed premetastatic
cells with VGSC activity would tend to migrate into the lumen and be
detectable in the semen (Gardiner et al.,
1996
; Barren et al.,
1998
). Subsequently, as
metastatic behaviour progressed, accompanied by deformation of the epithelia,
the negative TEP would degrade, cellular migration into lumen would slow down
and might even reverse, encouraging invasion of the surrounding tissue.
Furthermore, the transendothelial potential (Revest et al.,
1993
) could similarly
influence extra- and intravasation of circulating metastatic cells, which
might be a critical step in metastasis (Wyckoff et al.,
2000
).
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ACKNOWLEDGMENTS |
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