[CANCER RESEARCH 51, 2677-2682. May 15. 1991]
Vacuole Formation and Cytokeratin Rearrangement of Hepatoma Cells Induced by
Teleocidin Are Not Associated with Down-Regulation of Protein Kinase C
Yoshiyasu Kaneko, Ayumi Tsukamoto, and Kiyoshi Kurokawa
First Department of Medicine, The University of Tokyo, Faculty of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan
ABSTRACT
PLC/PRF/5 human hepatoma cells cultured with teleocidin reduced
the rate of cell proliferation and were transformed into large cells with
many vacuole-like subcellular structures. In these vacuolated cells, the
protein content per cell increased without changing the total cellular
protein synthesis. Cytokeratin was one of the proteins which increased
quantitatively. This intermediate filament formed fibrous network struc
tures throughout the enlarged cytoplasm. The assembly of other cytoskeletal proteins such as actin, tubulin, and vimentin was not altered
remarkably, suggesting that teleocidin morphologically transformed the
hepatoma cells by changing the assembly of Cytokeratin protein selec
tively. On the other hand, the alterations of cell proliferation, cell
morphology, and Cytokeratin assembly induced by teleocidin were not
associated with either down-regulation of protein kinase C or reduced
number of epidermal growth factor receptors. In addition, these teleocidin
effects were not mimicked by the protein kinase C agonist l-oleoyl-2acetylglycerol or inhibited by the protein kinase C inhibitor l-(5-isoquinolinylsulfonyl)-2-methylpiperazine. From these results it can be specu
lated that the morphological transformation and reduced cell proliferation
induced by teleocidin may be mediated by still unknown mechanisms
unrelated to protein kinase C.
INTRODUCTION
Teleocidin purified from Streptomyces is a phorbol ester-like
tumor promoter (1). Its action is mediated by protein kinase C
and subsequent protein phosphorylation (1-3). Enhanced nu
clear protein phosphorylation leads to an activation of cellular
oncogenes like c-fos, which may promote chemical carcinogenesis (2, 3).
In addition to the tumor-promoting effects, phorbol esters
and teleocidin can alter cellular differentiation state (4, 5). As
reported previously, teleocidin induces characteristic differen
tiation markers in human hepatoma cells, while phorbol esters
change HL60 leukemia cells into mature cells (4, 5). Activation
and translocation of protein kinase C are among the early
biochemical events that trigger cell differentiation. Neverthe
less, these two events alone are not sufficient to induce cell
differentiation (5). In addition, dissociation of protein kinase C
activation from phorbol ester-induced maturation of leukemia
cells was reported and the effects of teleocidin on PLC/PRF/5
hepatoma cells were not antagonized by protein kinase C inhib
itors (4-6). Therefore, in the HL60 leukemia and the hepatoma
cells, an ambiguity remains about whether the long-term effects
of phorbol esters, such as the induction of cell differentiation,
are mediated by protein kinase C and protein phosphorylation.
Phorbol ester and protein kinase C have negative feedback
effects in addition to the stimulatory actions (3). The negative
feedback mechanisms, such as down-regulation of protein ki
nase C and inactivation of phospholipase C and EGF' receptor,
Received 8/20/90; accepted 3/8/91.
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.
1The abbreviations used are: EGF, epidermal growth factor; PMSF, phenylmethylsulfonyl fluoride; OAG, 1-oleoyl-2-acetylglycerol; H7, l-(5-isoquinolinylsulfonyl)-2-methylpiperazine; TCA, trichloroacetic acid; SDS. sodium dodecyl
sulfate.
may be related with the inhibition of cell proliferation and the
induction of cellular differentiation by tumor promoters (3).
However, the hypothetical sequential action of tumor pro
moters, that is, positive short-term activation of protein kinase
C, followed by a negative action, has not been completely
substantiated. Agents that persistently activate protein kinase
C, such as phorbol esters, may promote more complex and
fundamental changes in its activity (3). Also, a possibility that
the long-term effects of the tumor promoters are mediated by
a still unknown mechanism cannot be ruled out completely.
To clarify whether the induction of cell differentiation by
tumor promoters is mediated by negative actions such as downregulation of protein kinase C and EGF receptors, we analyzed
the mechanism of action of teleocidin on PLC/PRF/5 hepa
toma cells which were morphologically transformed by this
tumor promoter (4). The results show that the morphological
transformation of PLC/PRF/5 cells was brought about prob
ably by a quantitative increase and an enhanced assembly of
Cytokeratin. In addition, the long-term effects of teleocidin,
such as the antiproliferative effect, vacuole formation, and
rearrangement of the intermediate filament, were not associated
with down-regulation of protein kinase C.
MATERIALS
AND METHODS
Materials. Teleocidin was a generous gift from Fujisawa Pharmaceu
tical Co. (Osaka, Japan). PMSF, DNase 1, H7, and OAG were obtained
from Sigma Chemical Co. (St. Louis, MO). [3H]Leucine was purchased
from New England Nuclear (Boston, MA). 125I-EGF(>900 Ci/mmol)
was obtained from Amersham (United Kingdom). Monoclonal antibody
to cytokeratins 1 to 19 (anti-cytokeratin pan) was obtained from Boehringer Mannheim (West Germany). Rabbit antibodies against tubulin,
actin, and vimentin were obtained from Transformation Research, Inc.
(Framingham, MA). Fluorescein isothiocyanate-conjugated anti-rabbit
IgG was obtained from Organon Teknika Corp. (PA). Monoclonal
antibody against protein kinase C was obtained from Amersham. Biotinylated anti-mouse and anti-rabbit IgG, avidin, and biotinylated peroxidase were obtained from Vector Laboratories, Inc. (Burlingame,
CA). Streptavidin-alkaline phosphatase conjugate, nitroblue tetrazolium, 5-bromo-4-chloro-3-indolylphosphate,
and prestained protein
molecular weight standards were obtained from Bethesda Research
Laboratories (Gaithersburg, MD). 3,3'-Diaminobenzidine tetrahydrochloride was obtained from Wako Pure Chemical Industries, Ltd.
(Tokyo, Japan).
Cell Culture and Fractionation of Nuclear Proteins. PLC/PRF/5
hepatoma cells (5 x IO5) were cultured in RPMI 1640 medium plus
5% fetal bovine serum with or without teleocidin. Cells were dissolved
in a nuclear buffer (10 mivi Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM
MgCI2) containing 0.5% Nonidet P-40 and 0.1 mM PMSF. The nuclei
were separated from the cytosol by centrifugation and were washed 3
times in the nuclear buffer.
Cellular DNA and Protein. DNA was measured by the diphenylamine
method (7). Protein was assayed by the method of Lowry et al. (8). To
measure the protein synthesis, the cells cultured for 3 days with or
without 10 ng/ml of teleocidin were incubated for further 3 h with 1
MCi/ml of ['H]Ieucine. The radioactivity incorporated into proteins was
measured by TCA precipitation method (4).
Analysis of Cytoskeletal Proteins. For immunofluorescence micro
scopic study, the cells were cultured on coverslips for 3 days with or
2677
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1991 American Association for Cancer Research.
EFFECTS OF TELEOCIDIN
ON CYTOKERATIN OF HEPATOMA CELLS
without 10 ng/ml of teleocidin. The cells were fixed with ethanol and
then with acetone. After fixation, cells were incubated successively with
10% skim milk for 30 min, with anti-cytoskeletal protein antibody for
l h at 37°C,and with FITC-conjugated second antibody for l h at
37°C.To analyze actin and tubulin by immunoblotting, the cells were
dissolved in a buffer containing 1% SDS and 1% ft-mercaptoethanol,
and were boiled for 3 min. For the immunoblot analysis of cytokeratin
and vimentin, these intermediate filaments were partially purified as
reported (9). Briefly, the cells were lysed in a buffer containing 1%
Triton X-100, 0.6 M KC1, and 0.5 mivi PMSF, treated with 0.5 mg/ml
of DNase I for 1 min at 37'C, and centrifuged in Eppendorf microcen
trifuge tube at 15,000 rpm for 10 min. Triton X-100-insoluble fractions
were suspended in a buffer containing 2% SDS, 10% /32-mercaptoethanol, and 8 M urea, and were boiled for 5 min. These samples were run
on a 0.1% SDS-12.5% polyacrylamide gel (10), and were blotted onto
a nitrocellulose filter (11). The filter was preincubated with 10% skim
milk for 30 min, and incubated with the first antibodies for 15 h at 4°C
and with biotinylated second antibody for 60 min at 24°C.Color was
developed by incubating the filter with avidin-alkaline phosphatase
conjugate for 60 min, and with nitroblue tetrazolium and 5-bromo-4chloro-3-indolylphosphate (12).
Immunoblot Analysis of Protein Kinase C. Cytosol and membrane
fractions were separated by an established procedure (13). Briefly, the
cells were lysed in 0.5% Nonidet NP-40 solution containing 0.1%
PMSF and nuclei were removed by centrifugation. Postnuclear super
natant was spun at 100,000 x g for 1 h. Resulting pellet and supernatant
were used as membrane and cytosol fraction, respectively. Cytosol and
membrane proteins (50 /¿g)
were subjected to immunoblot analysis as
described above (10, 11). The first and the second antibodies used were
mouse monoclonal anti-protein kinase C antibody and biotinylated
anti-mouse IgG antibody, respectively. Color was developed by incu
bating with avidin-biotinylated peroxidase complex, and with 11:<)_•
and
3,3-diaminobenzine tetrahydrochloride, successively (14).
Binding and Nuclear Accumulation of I25I-EGF. The binding and
nuclear accumulation of 125I-EGFwere measured as reported previously
( 15-17). Briefly, the cells cultured for 3 days with or without teleocidin
(10 ng/ml) were incubated with 30,000 cpm (approximately 50 pg/ml)
of '"I-EGF. At this concentration, '"I-EGF binds to the high affinity
3 3h
=
unlabeled EGF (1 Mg/ml) was added to some of the culture to assess
the amount of nonspecific binding.
RESULTS
1
234
days in culture
0 0.01 0.1 1 10
teleocidin(ng/ml)
Fig. 1. Effect of teleocidin on the proliferation of PLC/PRF/5 hepatoma cells.
Left, time course. PLC/PRF/5 hepatoma cells (5 x 10s) were cultured for various
lengths of time (abscissa) with (•)or without (O) 10 ng/ml of teleocidin. Right,
dose response. The cells (5 x 10*) were cultured for 3 days with different
concentrations of teleocidin (abscissa). Points, mean of triplicate experiments;
bars, SD.
receptors as reported previously ( 15). The incubation was continued for
l h at 24'C to measure the surface binding of '"I-EGF, while nuclear
accumulation of EGF was measured by incubating the cells with '•'"!EGF for 2 h at 37°Cin the presence of chloroquine (50 /IM). Chloroquine (50 >IM)was added to the reaction mixture to prevent possible
degradation of I251-EGF by lysosomal enzyme (16). A vast excess of
0
B
Fig. 2. Effect of teleocidin on the morphology of PLC/PRF/5 hepatoma cells.
PLC/PRF/5 hepatoma cells (5 x 10') were cultured for 3 days with (B) or without
(A) 10 ng/ml of teleocidin. x 700.
indicates that the DNA content per cell was not altered by
treating the cells with teleocidin. In contrast, the amounts of
cellular and nuclear proteins became approximately 2-fold when
the cells were cultured with teleocidin (Fig. 3). The cytosol
protein of control cells increased remarkably for the first 24 h
of culture, and then decreased in association with the increase
in cell number. On the other hand, the protein of cells cultured
with teleocidin remained at a high level (Fig. 3A). This teleo
cidin effect was observed at 0.1 ng/ml and was maximum at 10
ng/ml (Fig. 35). Similarly, the nuclear protein was increased
quantitatively in those cells cultured with teleocidin (Fig. 3, C
and D). To investigate whether the increased protein content
of cells cultured with teleocidin may be due to an increased
cellular protein synthesis, we measured ['Hjleucine incorpora
tion into TCA precipitates of cells. The TCA-precipitable ra
dioactivity in control cells was 5331 ±1438 cpm/106 cells,
Teleocidin inhibited the proliferation of PLC/PRF/5 hepa
toma cells (Fig. 1). The inhibitory effect of teleocidin was
recognized at 0.1 ng/ml and reached maximum at 10 ng/ml
(Fig. 1). In association with the reduced cell proliferation, the
morphological appearance of PLC/PRF/5 cells changed re
markably. As shown in Fig. 2, cells were enlarged and their
cytoplasm became flattened. In addition, numerous vacuolelike subcellular structures were formed, which sometimes oc
cupied the whole cytoplasm (Fig. 2). The effect of teleocidin on
the cell morphology was not mimicked by 10 to 100 ^M OAG,
a stimulant of protein kinase C, or antagonized by 10 to 100
(¿MH7, an inhibitor of protein kinase C.
Since the size of PLC/PRF/5 hepatoma cells cultured with
teleocidin was apparently larger than that of cells in control
culture, we measured the amounts of DNA and protein of cells
while that of cells cultured with 10 ng/ml of teleocidin was
cultured for 3 days with or without 10 ng/ml of teleocidin.
DNA of cells cultured with and without teleocidin was 12.7 ± 4730 ±998 cpm/106 cells. The result indicates that a continual
2.17 (SD) and 13.8 ±0.09 Mg/106 cells, respectively. This
increase in protein synthesis is not required to maintain the
2678
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1991 American Association for Cancer Research.
EFFECTS OF TELEOCIDIN
(B)
(A)
= 300
Jì
2
-
ON CYTOKERATIN OF HEPATOMA CELLS
200
•Ay^2'
100,
'/
)L
'^J,
<J 100,^
123-1
days in culture
0 001 0.1 1 10
teleocidin(ng/ml)
150
(D)
100
50
0 0.01 0.1 1 10
1234
teleocidin(ng/ml)
days in culture
Fig. 3. Protein content of PLC/PRF/5 hepatomacellsculturedwith teleocidin.
Time course: PLC/PRF/5 hepatoma cells (5 x 10*)were cultured for various
lengths of time (abscissa) with (•)or without (O) 10 ng/ml of teleocidin. A,
cytosol protein; C, nuclear protein. Dose response: The cells (5 x IO5)were
culturedfor 3 dayswith differentconcentrationsof teleocidin(abscissa).B,cytosol
protein; D, nuclear protein. Cytosol and nuclear proteins were measured as
described in "Materials and Methods." Points, mean of triplicate experiments;
bars.SD.
Fig. 5. Effects of teleocidin on the structure of actin, tubulin. and vimentin of
PLC/PRF/5 hepatoma cells. The cells (5 x 10') were cultured for 3 days with or
without 10 ng/ml of teleocidin. The cells were fixed and immunostained by using
anti-cytoskeletal protein antibody and fluorescein isothiocyanate-conjugated sec
ond antibody. A, actin of control cells; B, actin of teleocidin-treated cells; C,
tubulin of control cells: D. tubulin of teleocidin-treated cells: E, vimentin of
control cells; F, vimentin of teleocidin-treated cells, x 700.
D
B
1 2
-50
-42
Fig. 6. Immunoblot analysis of cytoskeletal proteins of PLC/PRF/5 hepatoma
cells cultured with teleocidin. The cells (5 x 10*)were cultured for 3 days with or
without 10 ng/ml of teleocidin. Cell lysates of 1 x 10* cells were subjected to
immunoblotting to analyze actin (A) and tubulin (Hi. Triton \ 100 insoluble
fractions from 1x10' cells were used to analyze vimentin (C) and cytokeratin
(D). I, control cells; 2, cells cultured with teleocidin. Ordinale, molecular weight
in thousands.
Fig. 4. Effect of teleocidin on the structure of cytokeratin of PLC/PRF/5
hepatoma cells. The cells (5 x 10*) were cultured for 3 days with (B and C) or
without (A) 10 ng/ml of teleocidin. The cells were fixed and immunostained by
using anti-cytokeratin antibody and fluorescein isothiocyanate-conjugated second
antibody, x 740.
increased protein content of PLC/PRF/5 cells cultured with
teleocidin.
Next, we tried to identify the proteins which increased selec
tively in those enlarged and vacuolated cells after the long-term
exposure to teleocidin. Cytoskeletal proteins could be candi
dates since the cell morphology is regulated by these proteins.
Immunofluorescence microscopic study using specific antibod-
ies was carried out to analyze the state of polymerization of
cytoskeletal proteins (Figs. 4, 5, and 6). Fig. 4 demonstrates
the effect of teleocidin on the cytokeratin assembly. In control
cells, thin fibrous structures of assembled cytokeratin were
barely detectable in the cytoplasm near the nucleus, and some
of them extended toward the cell boundary (Fig. 4A). In those
cells cultured with teleocidin, a remarkable assembly of cyto
keratin was observed. Network of assembled cytokeratin fibers
was detected throughout the enlarged cytoplasm (Fig. 4B).
Thick bundle-like structure was formed around the vacuole-like
structures (Fig. 4C). Fig. 5 demonstrates the immunofluorescence patterns of actin, tubulin, and vimentin of PLC/PRF/5
cells. In control cells these cytoskeletal proteins were observed
as having diffuse and vague immunofluorescence patterns. On
the contrary, in teleocidin-treated cells they were weakly stained
as halo-like structures around the vacuoles, and fibrous or
network structures were not remarkable (Fig. 5). Fig. 6 dem
onstrates the immunoblot analysis of the cytoskeletal proteins
2679
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1991 American Association for Cancer Research.
EFFECTS OF TELEOCIDIN ON CYTOKERAT1N OF HEPATOMA CELLS
of the hepatoma cells cultured with or without teleocidin. One
discrete band was recognized when the filter was incubated with
anti-actin, anti-tubulin, or anti-vimentin antibody (Fig. 6, A, B,
and C). The intensity of the signal was not remarkably different
between the cells cultured with and without teleocidin. On the
other hand, at least 6 bands were recognized when the filter
was incubated with anti-cytokeratin antibody (Fig. 6D). Teleo
cidin did not alter the banding pattern, but the signal was more
intense in those cells cultured with teleocidin (Fig. 6D).
To investigate whether the effects of teleocidin described
above are associated with down-regulation of protein kinase C,
we measured the protein kinase C in the cytosol and membrane
fraction by immunoblotting. Fig. 1A demonstrates that the
amounts of protein kinase C in the cytosol and membrane
fraction did not differ significantly between the cells cultured
for 3 days with and without teleocidin. In addition, the time
course study revealed that protein kinase C was not downregulated after the exposure of the cells to teleocidin (Fig. 7, B
and C). Protein kinase C was detected in the membrane fraction
of PLC/PRF/5 cells which were not stimulated with teleocidin,
and the possible increase of protein kinase C expected from its
translocation from the cytoplasm to the plasma membrane after
teleocidin treatment was not apparent in this experimental
system (Fig. 7).
Many of the actions of teleocidin are considered to be me
diated by activation of protein kinase C and subsequent protein
phosphorylation. Phorbol esters are known to phosphorylate
and down-regulate EGF receptor on the cell surface (3). Since
the down-regulation of EGF receptor may be a cause of inhi
bition of cell proliferation and induction of cell differentiation,
we measured I25I-EGF binding to the cells cultured with or
without teleocidin. The amount of surface-bound and nuclearaccumulated 125I-EGF in those cells cultured for 3 days with
teleocidin was approximately 2.2- and 2.1 -fold ofthat of control
cells, respectively (Table 1). This increase appeared to be asso
ciated with the increase in the total cellular and nuclear proteins
of cells cultured with teleocidin (Fig. 3).
DISCUSSION
As shown in the present study, teleocidin inhibited cell pro
liferation and altered morphological appearance of PLC/PRF/
5 hepatoma cells. Since these changes were associated with an
enhanced assembly of cytokeratin, teleocidin was considered to
induce the morphological transformation of the hepatoma cells
by changing this cytoskeletal protein. The results are consistent
with the recent reports showing that an induction of differen
tiation of cells is associated with an increase and a reorganiza
tion of intermediate filaments (18, 19).
A prominent morphological change induced by teleocidin
was the appearance of many vacuole-like subcellular structures.
The contents of the vacuoles are probably water and electro
lytes, since the histochemical staining using hematoxylin and
eosin, p-aminosalicylic acid and Sudan III, etc., failed to prove
the presence of proteins, carbohydrates, and lipids. There are
several explanations for the vacuole formation. First, an in
creased pinocytosis may be responsible, since electron micro
scopic study revealed the presence of pinocytotic vesicles at the
cell surface.2 However, teleocidin did not always enhance the
uptake of every radioactive material added to the culture me
dium (4). Therefore, the pinocytosis appears to be not a major
cause of vacuole formation. The second possibility is that the
1 Unpublished data.
IA I
klla
-80
(B)
k Da
-RO
iC l
I
2
3
•.
4
x „..*
IcDa
Fig. 7. Effect of teleocidin on the protein kinase C of the cytosol and membrane
fractions of PLC/PRF/5 hepatoma cells. A, protein kinase C of the cytosol and
membrane fraction of PLC/PRF/5 hepatoma cells cultured for 3 days with or
without 10 ng/ml of teleocidin. Lane 1, cytosol of control cells; Lane 2, cytosol
of teleocidin-treated cells; Lane 3, membrane of control cells; Lane 4, membrane
of teleocidin-treated cells. B, protein kinase C of the membrane fraction of PLC/
PRF/5 hepatoma cells treated with 10 ng/ml of teleocidin for different lengths
of time. Lane I, O min; Lane 2, 30 min; Lane 3, 60 min; Lane 4, 3 h; Lane 5, 24
h. C, protein kinase C of the cytosol fraction of PLC/PRF/5 hepatoma cells
treated with 10 ng/ml of teleocidin for different lengths of time. Lane 1, O min;
Lane 2, 30 min; Lane 3, 60 min; Lane 4. 3 h: lane 5. 24 h. Ordinate, molecular
weight in thousands.
Table 1 Effects of teleocidin on the binding and nuclear accumulation of
'"I-EGF by PLC/PRF 15 hepatoma cells
PLC/PRF/5 hepatoma cells cultured for 3 days with teleocidin (10 ng/ml)
were incubated with 30,000 cpm of '"I-EGF. The amounts of '"I-EGF bound to
cell surface membrane and accumulated into the nuclei were measured as de
scribed in "Materials and Methods." Values are mean ±SD of triplicate experi
ments.
Control
Teleocidin
Surface-bound '"I-EGF
(cpm/10'cells)
Nuclear-accumulated '"I-EGF
(cpm/10«cells)
1226± 132
2774 ±98
578 ±1.5
1208± 248
secretion of fluids and electrolytes may be suppressed by teleo
cidin. Since intracellular transport is regulated by cytoskeletal
proteins, teleocidin may interfere with this process by changing
the cytoskeletal structure (19-21). The present study disclosed
that teleocidin caused excessive formation of cytokeratin net
work (Fig. 4). This cytokeratin assembly may inhibit the intra
cellular transport of the vacuoles, at least partly (Fig. 4). An
other possibility is that teleocidin may enhance the secretion of
water and electrolytes into the preexisting small vacuoles. Re
sulting swelling of vacuoles may bring changes in the relative
staining of some of the cytoskeletal proteins around the vacu
oles (Figs. 4 and 5). However, further studies are necessary to
determine whether the cytoskeletal reorganization may be a
cause or a result of the formation of vacuole-like structures.
As shown in the present study, the enhanced assembly of
cytokeratin is associated with the morphological transforma
tion and the appearance of vacuole-like structures. Assembly
and disassembly of cytokeratin may be regulated by phosphorylation-dephosphorylation
reactions. It has been reported that
phosphorylation of intermediate filaments in vitro results in
their disassembly (22). If similar mechanisms are operating in
intact cells as well, the assembly of cytokeratin is expected to
be enhanced by dephosphorylation. Teleocidin is a stimulator
of protein kinase C and is usually considered to enhance protein
phosphorylation (1, 2). However, a possibility that teleocidin
could enhance cytokeratin assembly by activation of protein
kinase C and subsequent selective dephosphorylation of cyto-
2680
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1991 American Association for Cancer Research.
EFFECTS OF TELEOCIDIN
ON CYTOKERATIN OF HEPATOMA CELLS
keratin protein cannot be ruled out completely. Thus, the
mechanism by which teleocidin modulates the state of polym
erization of cytokeratin remains to be studied.
The present study also revealed that teleocidin brought an
increase in the total cellular protein. This may be due to an
altered expression of genes or a reduced protein degradation
since the total protein synthesis per cell was not increased.
Teleocidin may enhance the expression of genes of some of the
structural proteins such as cytokeratin by stimulating phorbol
ester responsive element in their 5' promoter region or by
inducing a specific morphological signature in the nuclear ma
trix-intermediate filament scaffold (23, 24). The altered gene
expression could bring quantitative and qualitative changes of
the cellular proteins and thereby alter the state of cell prolifer
ation. Although phorbol esters have been reported to cause G2
delay in HeLa cells, G2 delay seems to be unlikely for PLC/
PRF/5 hepatoma cells since teleocidin does not increase their
DNA content (25). Gl delay or arrest appears to be most
probable, but no data are available to determine the point in
the cell cycle at which the block occurred. On the other hand,
other possibilities such that teleocidin inhibits cell proliferation
or interferes with the autophagy associated with growth cessa
tion and thereby increases cellular protein content cannot be
ruled out completely (26).
The inhibition of cell proliferation and the induction of
morphological transformation required a long-term exposure
of PLC/PRF/5 hepatoma cells to teleocidin. Although downregulation of protein kinase C is supposed to be responsible for
such long-term actions, the results of the present study (Fig. 7)
do not coincide with this supposition (3). As reported previ
ously, teleocidin decreases EGF binding to the high-affinity
binding sites of liver and hepatoma cells (15, 16). However, this
down-regulation is a transient phenomenon and the EGF bind
ing returns to the basal level after 12 h of incubation with
teleocidin (15, 16). The present study also revealed that the
EGF binding to the hepatoma cells after the prolonged exposure
to teleocidin was not decreased in comparison with that of
control cells (Table 1). Therefore, the antiproliferative and
morphological effects of teleocidin may not be explained by
down-regulation and inactivation of EGF receptors. In addition,
the teleocidin effects are not mimicked by OAG or antagonized
by H7, suggesting that the long-term effect of teleocidin may
not be mediated by phosphorylation events (4-6). On the other
hand, protein kinase C is known to have a DNA-binding domain
in the NHj-terminal region, which is produced by specific
proteolytic cleavage and translocates to the nucleus (27-30).
The accumulation of this fragment causes alterations of higher
structures of the chromosomes and changes the transcriptional
activity of genes (30, 31). The fact that protein kinase C of
PLC/PRF/5 hepatoma is not down-regulated after exposure to
teleocidin (Fig. 7) may indicate that this kinase is continuously
produced during the treatment with teleocidin and that its NHiterminal region is accumulated into the nuclei to alter gene
transcription (30-33).
The present study revealed that teleocidin induction of the
differentiation of PLC/PRF/5 hepatoma cells is associated with
an enhanced cytokeratin assembly (Fig. 4). This intermediate
filament may be involved in the formation of vacuole-like
structures, as well. The studies using this experimental system
will give insights into the molecular mechanisms of cytokeratin
assembly, intrace Ihilar transport, and cell differentiation (19,
21,34,35).
REFERENCES
1. Itai, A., Kalo, Y., Tomioka, N.. litaka. Y., Endo, Y., Hasegawa, M., Shudo.
K., Fujii, H., and Sakai, S-I. A receptor model for tumor promoters: rational
superposition of teleocidins and phorbol esters. Proc. Nati. Acad. Sci. USA,
85: 3688-3692, 1988.
2. Blumberg. P. M. Protein kinase C as the receptor for the phorbol ester tumor
promoters: Sixth Rhodes Memorial Award Lecture. Cancer Res., 48: 1-8,
1988.
3. Nishizuka, Y. The molecular heterogeneity of protein kinase C and its
implications for cellular regulation. Nature (Lond.), 334:661-665, 1988.
4. Kaneko, Y., Toda, G., and Oka, H. Effects of teleocidin on the morphology
and c-myc expression of hepatoma cells which are not inhibited by protein
kinase antagonists. Biochem. Biophys. Res. Commun., /45: 549-555, 1987.
5. Tran, P. L., LePeuch, C., and Basset. M. Effects of fluorescent derivatives of
TPA on HL60 cells: dissociation between the differentiation-induced and
protein kinase C activity. J. Cell Physiol., 139: 313-319, 1989.
6. Kreutter, D., Caldwell. A. B.. and Morin. M. J. Dissociation of protein kinase
C activation from phorbol ester-induced maturation of HL-60 leukemia cells.
J. Biol. Chem., 260: 5979-5984, 1985.
7. Burton, K. A study of the conditions and mechanism of the diphenylamine
reaction for the colorimetrie estimation of deoxyribonucleic acid. Biochem.
J., 62:315-323, 1956.
8. Lowry, O. H., Rosebrough. N. J., Fair, A. L.. and Randall. R. J. Protein
measurement with the Polin phenol reagent. J. Biol. Chem., 193: 265-275.
1951.
9. Ermich, T., Schulz, J., and Schumann, D. Pattern of cytokeratins: I. SDSelectrophoretic analysis of the frequency of cytoskeletal keratins in the normal
mucosa of mouth. Biomed. Biochim. Acta, 47: 737-742. 1988.
10. Laemmli, U. K. Cleavage of structural proteins during the assembly of the
head of bacteriophage T4. Nature (Lond.), 227: 680-685, 1970.
11. Towbin, H., Stachelin, T., and Gordon, J. Electrophoretic transfer of proteins
from polyacrylamide gels to nitrocellulose sheets: procedure and some appli
cation. Proc. Nati. Acad. Sci. USA, 76: 4350-4354, 1979.
12. Lear)',.'. J., Brigati, D. J.. and Ward, D. C. Rapid and sensitive colorimetrie
method for visualizing biotin labelled DNA probes hybridized to DNA or
RNA immobilized on nitrocellulose: bio-blots. Proc. Nati. Acad. Sci. USA,
«0:4045-4049, 1983.
13. K¡lano.T., Masayoshi, G., Kikkawa, U., and Nishizuka, Y. Assay and
purification of protein kinase C. Methods Enzymol., 124: 349-359, 1986.
14. Hsu, S. M., Raine. L., and Famger, H. The use of avidin-biotin-peroxidase
complex (ABC) in immunoperoxidase techniques: a comparison between
ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem.,
29:577-580,1981.
15. Imai, Y., Kaneko, Y., Matsuzaki, F., Endo. Y., and Oda. T. Teleocidin B
inhibits binding of epidermal growth factor to cellular receptors probably by
the same mechanism as phorbol esters. Biochem. Biophys. Res. Commun.,
97:926-931. 1980.
16. Kaneko, Y., and Imai, Y. Teleocidin, a possible naturally occurring tumor
promoter from Streptomyces, modulates actions of epidermal growth factor
on rat hepatoma cells. Life Sci., 29: 1571-1576, 1981.
17. Rapper. S. E., Burwen, S. J., Barker. M. E.. and Jons, A. L. Translocation
of epidermal growth factor to the hepatocyte nucleus during rat liver regen
eration. Gastroenterology, 92: 1243-1250, 1987.
18. Fuchs, E., Tyner, A. G., Guidice, G. J., Marchuk, D., RayChaudhury, A.,
and Rosenberg, M. The human keratin genes and their differential expres
sion. Curr. Top. Dev. Biol., 22: 5-34, 1987.
19. Bershadsky, A. D.. Ivanova, O. Y., Lyass. L. A., Pletyushkina, O. Y., Vasiliev,
J. M., and Gelfand, I. M. Cytoskeletal reorganizations responsible for the
phorbol ester-induced formation of cytoplasmic processes: possible involve
ment of intermediate filaments. Proc. Nati. Acad. Sci. USA, 87:1884-1888,
1990.
20. Kelly, R. B. Microtubules. membrane traffic, and cell organization. Cell, 61:
5-7, 1990.
21. Uemura, Y., Nishizuka, M., Kodama, F., and Shirakawa, S. Alteration of
intracellular actin levels induced by phorbol diester in human HL-60(HL60]
leukemia cells susceptible or resistant to differentiation, and the effects of
protein kinase inhibitors. Leuk. Res., 13: 545-552, 1989.
22. Inagaki, M., Nishi, Y., Nishizawa, K.. Matsuyama, M., and Sato, C. Sitespecific phosphorylation induces desassembly of vimentin filaments in vitro.
Nature (Lond.), 328:649-652, 1987.
23. Deschamps, J., Meljlink, F.. and Verma, I. M. Identification of a transcrip
tional enhancer element upstream from the proto-oncogene fas. Science
(Washington DC), 230: 1174-1177, 1985.
24. Fey, E. G., and Penman, S. Tumor promoters induce a specific morphological
signature in the nuclear matrix-intermediate filament scaffold of MadinDarby canine kidney (MDCK) cell colonies. Proc. Nati. Acad. Sci. USA, 81:
859-866, 1984.
25. Kinzel, V., Bonheim, G., and Richards, J. Phorbol ester-induced G2 delay in
HeLa cells analyzed by time lapse photography. Cancer Res., 48:1759-1762,
1988.
26. Pfeifer, U., Tessitore, L., Bonelli, G., and Baccino, F. M. Regulation of
protein turnover versus growth state. III. Growth cessation is associated with
activation of autophagy in Yoshida ascites hepatoma AH-130JAH130]. Virchows Arch. B Cell Pathol., 55: 363-369, 1988.
27. Lee, M-H., and Bell, R. M. The lipid binding, regulatory domain of protein
2681
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1991 American Association for Cancer Research.
EFFECTS OF TELEOCID1N ON CYTOKERAT1N OF HEPATOMA CELLS
kinaseC. J. Biol. Chem.. 26/: 14867-14870, 1986.
28. Hoshijima. M.. Kikuchi, A., Tanimoto, T., Kaibuchi. K.. and Takai, Y.
Formation of a phorbol ester-binding fragment from protein kinase C by
proteolytic digestion. Cancer Res.. 46: 3000-3004. 1986.
29. Huang. F. L., Arora, P. K., Kanna. E. F.. and Huang. K-P. Proteolytic
degradation of protein kinase C in the phorbol ester-induced interleukin-2
secreting thymoma cells. Arch. Biochem. Biophys., 267: 503-514, 1988.
30. Thomas, T. P., Talwar, H. S., and Anderson. W. B. Phorbol ester-mediated
association of protein kinase C to the nuclear fraction in NIH3T3 cells.
Cancer Res., 48: 1910-1919, 1988.
31. Kiss. Z.. Deli, E., and Kuo, J. F. Temporal changes in intracellular distribution of protein kinase C during differentiation of human leukemia HL60
celi induced by phorbol ester. FEBS Lett., 231:41-46. 1988.
32. Huang, F. L., Arora, P. K., Kanna, E. F., and Huang, K-P. Proleolytic
degradation of protein kinase C in the phorbol ester-induced interleukin-2
secreting thymoma cells. Arch. Biochem. Biophys.. 267; 503-514. 1988.
33. Housey. G. M.. Johnson. M. D.. Hsiao. W. L. W.. O'Brian. C. A., Murphy,
J. P., Kirschmeier, P. K., and Weinstein, I. B. Over production of protein
kinase C causes disordered growth control in rat fibroblasts. Cell, 52: 343354, 1988.
34. Albers. K., and Fuchs, E. Expression of mutant keratin cDNAs in epithelial
cells reveals possible mechanisms for initiation and assembly of intermediate
filaments. J. Cell. Biol., 108: 1477-1493, 1989.
35. Morasson, M. I., Rieber, M. S., Urbina, C., and Rieber, M. Changes in
eytoskeleton-associated molecules in cells with different degree of prolifera
tion and metastatic ability. Cell. Biol. Int. Rep., 13: 863-872, 1989.
2682
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1991 American Association for Cancer Research.
Vacuole Formation and Cytokeratin Rearrangement of
Hepatoma Cells Induced by Teleocidin Are Not Associated with
Down-Regulation of Protein Kinase C
Yoshiyasu Kaneko, Ayumi Tsukamoto and Kiyoshi Kurokawa
Cancer Res 1991;51:2677-2682.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/51/10/2677
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1991 American Association for Cancer Research.