(CANCER RESEARCH 52. .1443-3448. June 15. 1992)
Photofrin and Light Induces Microtubule Depolymerization in Cultured Human
Endothelial Cells1
Lee Ann Sporn2 and Thomas H. Foster
Departments of Medicine [L. A. S.] and Radiology [T. H. F.], University of Rochester School of Medicine and Dentistry, Rochester, New York 14642
ABSTRACT
Endothelial cells were cultured from human umbilical veins and incu
bated with Photofrin (1 MR/ml).Cells were then exposed to light, and
cytoplasmic microtubule (MT) status was monitored by immunofluorescence microscopy using a-tubulin antibody. As early as 15 min following
irradiation, a light dose-dependent depolymerization of MT was observed.
At sublethal light doses, this effect was transient, with MT repolymerizing within 2-3 h. Cellular ATP levels were monitored to determine
whether diminished ATP levels were correlated with MT depolymeriza
tion. No correlation was found, since ATP levels remained at a constant
value near 50% of unirradiated controls during a time interval in which
transient MT depolymerization was observed. Cell viability was moni
tored by trypan blue exclusion. Transient MT depolymerization occurred
at photodynamic doses that produced essentially no decrease in cell
viability, while at higher doses, irreversible MT depolymerization was
observed prior to loss of viability. Since MT are unstable at intracellular
calcium levels >1 ¿tM,
we postulate that MT depolymerization results
from increases in intracellular calcium caused by photodynamic insult.
MT are important in maintaining cell shape. Disruption of MT in
endothelial cells due to photodynamic therapy could result in or contribute
to exposure of the thrombogenic subendothelium or could alter vascular
permeability in the treatment area.
INTRODUCTION
Tumor response to PDT'
apparently
involves a complex
combination of effects at the level of the tumor cell and of the
tumor microvasculature. Evidence for the latter is significant
and includes both direct and indirect experimental data. Hen
derson et al. (1) concluded that PDT inactivates tumor cells by
a mechanism other than direct photodynamic cytotoxicity. This
conclusion was based on the evidence that in vivo PDT which
was optimal for tumor response did not lead to an immediate
reduction in tumor cell clonogenicity. It was postulated that
additional factors are required for tumor response that are
provided by the posttreatment tumor environment, presumably
vascular changes. Several investigators have observed changes
in the microcirculation, including reduced blood flow, vasocon
striction, platelet aggregate and thrombus formation, and endo
thelial damage (2-6). While the presence of a vascular response
to PDT is established, the detailed mechanisms through which
this response occurs remain the subject of research. Various
investigators have suggested that the photodynamic stimulation
of platelets (7), mast cells, and macrophages (8), and/or the
endothelial cells (7, 9-11) may be responsible for the reported
observations.
Received 12/18/91; accepted 4/7/92.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported in part by USPHS Grants CA368S6, HL07152,
HL43711, and HL30616, awarded by the National Cancer Institute and the
National Heart, Lung, and Blood Institute, N1H, Bethesda, MD.
2To whom requests for reprints should be addressed, at Hematology Unit,
P.O. Box 610, University of Rochester Medical Center, 601 Elmwood Avenue,
Rochester, NY 14642.
'The abbreviations used are: PDT, photodynamic therapy; CMTC, cyto
plasmic microtubule complex; MT, microtubule(s); HEPES, A'-(2-hydroxyethyl)piperazine-A"-2-ethane-sulfonic acid; vWf, von Willebrand factor.
Recent work in our laboratory has emphasized the response
of the vascular endothelial cell to photodynamic insult. We
have previously shown that human endothelial cells exhibit a
radiation dose-dependent release of vWf following photosensitization with Photofrin (12). Furthermore, release of vWf was
accompanied by a similarly dose-dependent influx of calcium
into the cells. Since the polymerization state of the cellular
microtubules is extremely sensitive to the intracellular concen
tration of free calcium, we have extended our studies to include
the response of these structures to photodynamic stimulation.
In nonmitotic cells, microtubules are seen throughout the
cytoplasm originating from microtubule-organizing centers in
an array termed the CMTC. This cellular structure, along with
the actin-containing cytoskeleton (microfilaments), is impor
tant in maintaining cell shape. Changes in endothelial cell shape
leading to cell contraction and "gap" formation are normal
occurrences in the vasculature and are important in controlling
the thrombogenic properties of the vessel wall. When endothe
lial cells in culture or in situ are exposed to physiological agents
such as thrombin, a reversible cell shape change is observed
(13-15) which transiently exposes the subendothelium to flow
ing blood, thus rendering the vessel wall thrombogenic.
The effects of photodynamic therapy on the cytoskeletal
status of the cell could be responsible for similar gap formation
and could, therefore, potentiate the formation of occlusive
vascular thrombi in the treatment area. Here, we explore the
effects of Photofrin and light on the CMTC in cultured endo
thelial cells.
MATERIALS AND METHODS
Endothelial Cell Culture. Endothelial cells were harvested from 3-5
human umbilical veins as previously described (16, 17), pooled, and
cultured in McCoy's 5A medium (Flow Laboratories, McLean, VA)
with 20% fetal bovine serum. At confluence, cultures were passaged in
the presence of 50 Mg/ml endothelial cell growth supplement (Bioméd
ical Technologies, Inc., Stoughton, MA), 100 Mg/m' heparin (Sigma
Chemical Co., St. Louis, MO), and 25 Mg/m' insulin (Sigma) so that
subsequent passages reached confluence in approximately 5 days. Typ
ically, passage 2 cells were used for experimental protocols.
Drug Treatment and Irradiation. Cells were incubated for 2 h at 37*C
with Photofrin (Quadra Logic Technologies, Inc., Vancouver, British
Columbia, Canada) at a concentration of 1 Mg/m' in serum-free medium
supplemented with Nutridoma-HU (Boehringer Mannheim Biochemicals, Indianapolis, IN). Cells were then washed twice with serum-free
medium, and complete culture medium was replaced. Photoradiation
was performed with the unfiltered output from a pair of fluorescent
lamps so that the incident photoradiation power density at the level of
the cells was 0.2 mW/cm2, unconnected for the absorbance of the culture
medium. The mitochondria! energy inhibitor oligomycin (650 n\i) and
deoxy-D-glucose (10 mivi) were purchased from Sigma and were dis
solved in complete culture medium prior to incubation with cell
cultures.
Fluorescence Staining. For studies requiring fluorescence staining,
cells were seeded directly onto 12-mnr glass coverslips and cultured to
near confluence. Following photodynamic treatment, cells were fixed
for 20 min in 3.7% formaldehyde in phosphate-buffered saline and then
permeabilized for 15 min in 0.5% Triton X-100 in phosphate-buffered
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PHOTOFRIN PDT INDUCES MICROTUBULE DEPOLYMERIZAT1ON
saline. Fluorescence staining for tubulin was performed as previously
described (16) using monoclonal anti-tubulin antibody (culture super
natant), kindly provided by Dr. Joanna Olmsted, University of Roch
ester, used at a 1:10 dilution for 30 min, and then covered for 30 min
with a 1:10 dilution of rhodamine-conjugated goat antibody to mouse
IgG. Status of microtubule polymerization was quantitated by randomly
choosing visual Fields and scoring cells in the fieli) as either polymerized
(MT extending to cell edges) or depolymerized (absence of MT or
paucity of MT not extending to cell edges) until 100 cells were scored.
Staining of cellular F-actin was performed using rhodamine-phalloidin
(Molecular Probes, Eugene, OR) at a dilution of 1:10 for 30 min.
Determination of Cellular ATP Levels. Cells used for ATP determi
nations were cultured to confluence in 6-well tissue culture plates which
contained approximately 8 x IO5cells. Following photodynamic treat
ment, cells were lifted with 0.5 ml 0.025% trypsin-0.1% EDTA in
Hanks' balanced salt solution, 1 ml complete culture medium was
added, and the cell suspension was frozen in liquid nitrogen and stored
overnight at —¿70*C.
Cell extracts were prepared as described previously
(18) with slight modification. Briefly, frozen cell suspensions were
thawed by addition of 1 ml 5% trichloroacetic acid-3 HIM EDTA at
room temperature. The solution was then diluted with 5 ml ice-cold 25
mM HEPES-25 mM MgS04 buffer, pH 8.0-1 ml Hanks' balanced salt
solution-270 n\ l N NaOH. The final pH of the extract was 7.2-7.4.
Extracts were kept on ice until assay of ATP. The luciferin-luciferase
assay of ATP levels in cell extracts was performed using an assay kit
purchased from Calbiochem Biochemicals (San Diego, CA). The luci
ferin-luciferase was diluted to a concentration of 5 mg/ml with the
HEPES buffer (pH 7.75) provided in the kit and was kept in the dark
on ice for l h to allow the enzyme to stabilize before use. For assay, 50
ill of the cell extract was added to 1 ml 25 mM HEPES-25 mM MgS04
buffer, pH 8.0, in a glass scintillation vial. To initiate the light reaction,
50 //I (0.25 mg/sample) of the luciferin-luciferase solution was added
to the vial, which was swirled gently by hand and placed in the
scintillation counter. A 1900 TR liquid scintillation analyzer was used
(Packard Instrument Co., Downer's Grove, IL), preset for single photon
counting and modified by disengaging the static control ring. Counting
was initiated 30 s after the addition of the enzyme and was performed
for 30 s. Samples were run in triplicate, and values were averaged (error
among triplicate measurements was <20%). ATP levels were then
obtained from a standard curve prepared by using pure ATP.
Cell Viability Studies. Endothelial cell viability was assessed by
determining their ability to exclude trypan blue. At various times
following irradiation, cells were trypsinized and incubated for 1 min
with 0.2% trypan blue stain (Sigma). The percentage of viable cells was
determined using a hemocytometer.
RESULTS
The polymerization state of the CMTC of human umbilical
vein endothelial cells was studied following photosensitization
with Photofrin and subsequent irradiation. Following light
treatment, cells were incubated at 37°Cfor 15 min, fixed,
permeabilized, and stained by fluorescence using a-tubulin an
tibody. Unirradiated cells preincubated with Photofrin exhib
ited a polymerized CMTC with MT extending to the cell edges
(Fig. la). Following a 2-min light exposure (24 mJ/cm2, broad
band), the number of cytoplasmic MT was greatly reduced (Fig.
\b), and the remaining MT did not extend completely to the
cell edges. At higher light doses (5 min, 60 mJ/cnr), cells were
virtually devoid of MT (Fig. le). In these cells, the polymeri
zation state of the actin-containing cytoskeleton (microfilaments) was unaffected (Fig. Id).
The light dose dependence of MT depolymerization was
scored by microscopic visualization of the status of MT polym
erization. In seven independent trials, each using separate endo
thelial cell pools, a light dose-dependent depolymerization was
observed (Fig. 2). Depolymerization was observed with light
exposures as short as 1 min (12 mJ/cnr) but generally occurred
following irradiation for 2 or 3 min. A very low percentage of
cells (average, 8%) contained polymerized MT following a 5min light exposure. The time at which initial depolymerization
occurred varied slightly among experiments but generally oc
curred between 5 and 15 min following irradiation.
When the state of polymerization of the CMTC was moni
tored over 6 h, MT depolymerization occurring in response to
relatively low light doses was found to be transient. The dose
and time dependence of this transient effect on MT status is
illustrated by the graph in Fig. 3. In this representative experi
ment, little effect on the CMTC polymerization state was
observed following a 1-min light treatment at any time point
tested. Two-min irradiation resulted in rapid depolymerization
of the CMTC, which returned to near normal by l h and
Fig. 1. Immunofluorescence
staining of
endothelial cell cytoskeletal components fol
lowing photodynamic stimulation. Endothelial
cells were cultured on glass coverslips and in
cubated with Photofrin and then irradiated
(0.2 mW/cm2, broad band) for 0 (a), 2 (*), or
5 min (e and d). At 15 min following light
exposure, cells were fixed, permeabilized, and
stained by fluorescence using a-tubulin anti
body («<•)
or rhodamine-phalloidin (ill Bar,
10 t/m; magnification, X 1150.
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PHOTOFRIN
Q
UJ
PDT INDUCES MICROTUBULE
DEPOLYMERIZAT1ON
100
1,2, or 3 min (12, 24, or 36 mJ/cm2). Some loss of cell viability
80
occurred in response to an irradiation of 2 or 3 min but only at
24 h following irradiation. Significant loss of viability occurred
in response to a 5-min light treatment at all time points tested
and was most pronounced at 24 h postlight exposure.
60
DISCUSSION
40
UJ
Ü
U.
O
20
5?
LIGHT DOSE (minutes)
Fig. 2. Light dose dependence of MT depolymerization induced by photodynamic stimulation. Following Photofrin incubation, endothelial cells receiving
irradiations of 0-5 min were stained by fluorescence using «-tubulinantibody and
then scored by microscopic visualization as to the polymerization state of the
CMTC. Cells were scored as "polymerized" if they possessed numerous microtubules extending to the cell edges. One hundred cells were scored for each light
dose. Points, means (bars, ±SE)of percentage of cells possessing polymerized
microtubules from seven independent trials.
appeared identical with unirradiated controls by 3 h postlight
exposure. A 3-min irradiation also resulted in rapid MT depo
lymerization; however, at this light dose the effect was not
transient. At these higher light doses, no recovery of the CMTC
was observed even at 6 h postirradiation. The time required for
recovery of the CMTC at light doses causing transient MT
depolymerization varied among experiments but normally oc
curred within 2-3 h of initial depolymerization. Fig. 4 shows
transient MT depolymerization in cells stained by fluorescence
with anti-tubulin antibody.
Cellular ATP levels were monitored at various times follow
ing photodynamic insult to determine whether lowering of ATP
levels correlated with the observed MT effects. Results of 3 or
4 independent experiments demonstrated a gradual, light dosedependent decrease in ATP levels over the observed 6 h when
values were normalized to unirradiated controls (Fig. 5). Actual
ATP values calculated as fmol ATP/cell are also presented in
Table 1. A decrease in ATP levels was evident as early as 15
min following light exposure, and at 6 h, was an average of 27
and 22% of control levels following 2- and S-min irradiations,
respectively. MT polymerization was monitored in parallel by
immunofluorescence microscopy. Data from a representative
experiment is illustrated in Fig. 6, which shows both cellular
ATP levels and MT status in Photofrin-treated cells over a 2-h
period following a 3-min irradiation. Transient MT depoly
merization occurred over this time interval, while the ATP
levels remained approximately constant at a value near 50%
that of unirradiated controls (Fig. 6a). Therefore, there ap
peared to be no correlation between cellular ATP levels and
cell MT status. This notion is further supported by the obser
vation that reducing cellular ATP levels to even lower values
(20-30% of unirradiated controls) by treatment with a combi
nation of the mitochondria! energy inhibitor oligomycin and
deoxy-D-glucose resulted in no decrease in the percentage of
cells with polymerized MT (Fig. 6b).
Trypan blue exclusion studies were conducted to determine
the light doses and times following light exposure that produced
loss of viability (Fig. 7). Minimal loss of cell viability occurred
over a 6-h period postlight exposure in cultures irradiated for
Treatment of human umbilical vein endothelial cells with
Photofrin and light resulted in light dose-dependent depoly
merization of the CMTC (Fig. 2) as monitored by immunoflu
orescence microscopy (Fig. 1). This effect usually occurred
between 5 and 15 min following light exposure, and at certain
light doses, the CMTC was seen to repolymerize within several
hours (Fig. 3). Higher light doses (36 mJ/cnr greater) also
resulted in depolymerization of the CMTC; however, at these
fluences the effect was irreversible. Incubation of cells with
Photofrin in the absence of irradiation had no apparent effect
on the state of polymerization of the CMTC.
The effect of Photofrin and light on the CMTC could not be
correlated with a decrease in cellular ATP levels. When MT
status was monitored in parallel with ATP levels following
photodynamic insult, ATP levels had decreased to near 50% of
control values at both 1 and 2 h following treatment. MT,
however, were seen to be depolymerized at 1 h but had repolymerized at 2 h following light exposure. Furthermore, cells
incubated with the inhibitors of energy metabolism oligomycin
and deoxy-D-glucose had similarly reduced cellular ATP levels
but exhibited no change in the polymerization state of the
CMTC. The transient MT change observed was also not due
to general collapse of the cellular cytoskeleton, since cellular
actin remained polymerized in stress fibers even under condi
tions resulting in complete depolymerization of MT (Fig. 1).
Cell viability was compromised only at light doses most often
resulting in irreversible depolymerization of the CMTC (36
mJ/cm2 or greater) and was most prominent at 24 h following
light exposure (Table 1). Thus, the transient MT depolymeri
zation observed following irradiation of Photofrin-treated cells
was a sublethal effect. The irreversible MT depolymerization
occurring at higher light doses appeared to occur prior to loss
of cell viability even if lethal cell damage had been sustained.
120
100
o
m
t!
OC
UJ
80
60
40
UJ
O
20
1.0
2.0
3.0
4.0
5.0
6.0
TIME (hours)
Fig. 3. Light dose and time dependence of microtubule depolymerization.
Percentage of cells possessing polymerized microtubules was determined by
microscopic visualization of cells stained by fluorescence using n-tubulin antibody
at 5 min to 6 h following irradiations of 0-3 min (0-36 mJ/cm2). For each
condition, 100 cells were scored.
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PHOTOFRIN PDT INDUCES MICROTUBULE DEPOLYMERIZATION
Fig. 4. Immunofluorescence staining of the
CMTC under conditions resulting in transient
depolymerization. Photofrin-treated cells were
irradiated for O min (a and c) or 2 min (h and
</). then fixed, permeabilized, and stained us
ing a-tubulin antibody 1 (a and b) and 2 h (c
and d) postirradiation. Bar, 10 UM; magnifi
cation, x 1150.
1.0
E
i
0.8
0.6
0.4
0.2
0.0
1.0
2.0
3.0
4.0
5.0
6.0
TIME (hours)
Fig. 5. Normalized cellular ATP levels following photodynamic insult. 1 mio
thelial cells cultured in 6-well plates were incubated with Photofrin and then
exposed to light for 0, 2, and 5 min (0.2 mW/cm2). Cells (approximately 8 x 10'
cells/sample) were harvested and ATP levels determined by the luciferin-luciferase
assay at IS min to 6 h. Data (points) from a single experiment were normalized
to unirradiated controls and SD (bars) of these normalized values from four
independent trials are shown graphically.
Table 1 Cellular ATP levels following photodynamic insult
Actual ATP values obtained from the study described in Fig. 5 are presented
as fmol ATP/cell and are expressed as mean values ±SD.
(min)22.2
dose
Time following
irradiation
(h)0.25
2
3
603.5
±0.9
2.7 ±0.5
3.5 ±1.3
3.0 ±1.2Light
±0.8
1.8 ±0.3
1.4 ±0.6
0.7 ±0.351.5
±0.4
0.7 ±0.2
0.7 ±0.3
0.5 ±0.1
Previous investigators have focused on deleterious effects of
photodynamic therapy on the mitotic spindle (which is com
posed of microtubules) and the resultant effect on tumor cell
multiplication. Berg and Moan (19) demonstrated that Photof
rin and light resulted in an increase in the mitotic index of a
human cervix carcinoma cell line (NHIK 3025), apparently due
to disruption of the organization of the spindle apparatus.
Christensen (20), also using NHIK 3025 cells, reported a block
in mitosis induced by hematoporphyrin dihydrochloride and
light exposure. Other photoactivatable drugs have been shown
to affect microtubule polymerization. The synthetic porphyrin
meÃ-0-tetra(4-sulfonatophenyl)porphine
inhibited assembly of
purified microtubules even in the absence of photoactivation,
possibly as a direct result of tubulin binding (21). This and
related compounds affected intracellular microtubules, how
ever, only following photoactivation (22). An increase in the
mitotic index of cultured NHIK 3025 cells was observed follow
ing treatment with these agents which presumably resulted from
disruption of microtubules. Additionally, Wieman et al. (23)
reported that Photofrin PDT at considerably higher dosages
(25 i/g/ml) than those used in the present study progressively
and irreversibly altered microfilament distribution in cultured
endothelial cells.
Disassembly of the CMTC observed in response to Photofrin
and light is likely a result of increased intracellular calcium
concentration caused by singlet oxygen-mediated injury. Oxidant injury to cells induced by exposure to hydrogen peroxide
has been shown to disrupt calcium homeostasis leading to a
rapid increase in intracellular calcium which precedes loss of
cell viability (24). Previously, we have shown that oxidant injury
induced by Photofrin and light results in an influx of calcium
and vWf storage granule (Weibel-Palade body) release (12).
Using Chinese hamster ovary cells preincubated with the photosensitizer chloroaluminum phthalocyanine, Ben-Hur et al.
(25) recently demonstrated a transient increase in intracellular
calcium from about 0.2-1 ¿IM
occurring within 5 min of irra
diation. Microtubules are stable at calcium concentrations <1
fiM but become unstable at calcium concentrations >l-4 UM
(26). Such calcium sensitivity is incurred both through direct
interaction of calcium with tubulin (27-29) and through a
calmodulin-mediated effect regulated by the presence of microtubule-associated proteins (30-32). Calcium-induced depoly
merization of microtubules begins at the cell periphery and
proceeds toward the cell center and is readily reversible when
intracellular calcium levels are allowed to return to baseline
levels (33). Both the transient nature and the pattern of micro-
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PHOTOFRIN
PDT INDUCES MICROTUBULE
100
DEPOLVMERIZATION
100,
Q
HI
IT
UJ
i
2
uu
O
u.
O
a?
12345
LIGHT DOSE (minutes)
Fig. 7. Effect of photodynamic insult on endothelial cell viability. Cell viability
was monitored by trypan blue exclusion at various times (IS min to 24 h) at
irradiation doses of 0-5 min.
è
4
o
60
>
40
•¿3
UJ
LU
U
20
1.00
insensitive to intracellular calcium concentration and which
participate in the translocation process. In a study of tubulin
purified from bovine brain, it was found that calmodulin incurs
calcium sensitivity on tubulin only in the presence of MTassociated proteins (36). It is also possible that the translocation
component of the release process occurs rapidly following stim
ulation, prior to MT depolymerization.
It is part of the normal function of the vascular endothelial
cell to regulate both thrombogenic and permeability properties
of the vessel wall. For example, in response to various cytokines,
the endothelial cell can transiently alter the procoagulant prop
erties of the cell surface (37). In response to other physiological
mediators such as thrombin or histamine, the cell can release
vWf from intracellular stores to aid in platelet adhesion and
may undergo transient cell shape change to expose the throm
bogenic subendothelium and increase vessel wall permeability.
Therefore, even in the absence of lethal endothelial cell damage,
the action of PDT could produce significant effects on the
vasculature. We previously reported that Photofrin and light
induces vWf release from Weibel-Palade bodies of endothelial
cells prior to cell lysis or death. Here, we demonstrate disrup
tion of the endothelial cell CMTC induced by photodynamic
insult. Such an effect or combination of effects could underlie
or contribute to some of the vascular changes associated with
PDT.
60
2.00
TIME (hours)
Fig. 6. MT status and ATP levels following photodynamic insult. Shown are
data from a representative experiment in which ATP levels were determined and
MT status was monitored on cells grown in parallel on glass coverslips. a,
Photofrin-treated cells at various times following a 3-min irradiation; h. cells
treated with a combination of the mitochondria! inhibitor oligomycin (650 TIM)
and deoxy-D-glucose (10 HIM).MT data are presented as percentages of cells (100
cells scored per condition) possessing polymerized MT.
tubule depolymerization that we observe in response to Photofrin and light are consistent with such a calcium-dependent
mechanism.
Evidence exists to suggest that MT play a role in the WeibelPalade body release process. Sinha and Wagner (34) reported
that Weibel-Palade body release, which occurs by vesicle translocation to the cell surface followed by granule fusion with the
cell membrane, is inhibited by pretreatment of cells with colchicine, an inhibitor of MT polymerization. It was postulated
that cytoplasmic MT are required in the release process to serve
as "tracks" for Weibel-Palade body translocation. Others have
reported inconsistent and variable effects of MT inhibitors on
the secretory process (35). The involvement of MT in the
Weibel-Palade body release process is certainly not well under
stood and may be quite complex. Transient MT depolymeri
zation occurs during the course of the Weibel-Palade body
release process induced by the physiological secretagogue,
thrombin, or the calcium ionophore, A23187,4 suggesting that
ACKNOWLEDGMENTS
We wish to thank Dr. Victor Marder and Dr. Russell Hilf for critical
reading of the manuscript, Melissa Primavera and Donna Hartley for
excellent technical assistance, and Carol Weed for help in preparation
of the manuscript.
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MT depolymerization may actually be involved in the release
process. There could also exist a subset of MT which are
* L. A. Sporn and T. H. Foster, unpublished observation.
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3448
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1992 American Association for Cancer Research.
Photofrin and Light Induces Microtubule Depolymerization in
Cultured Human Endothelial Cells
Lee Ann Sporn and Thomas H. Foster
Cancer Res 1992;52:3443-3448.
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