Download Role of Cytoskeleton Changes and Expression

[CANCER RESEARCH 46, 5923-5932, November 1986]
Role of Cytoskeleton Changes and Expression of the H-ray Oncogene during
Promotion of Neoplastia Transformation in Mouse Epidermal JB6 Cells
Kiyoshi Takahashi, Ursula I. Heine,1 James L. Junker, Nancy H. Colburn, and Jerry M. Rice
Laboratories of Comparative Carcinogenesis fK. T., U. I. H..J.L. ]., 1. M. R.¡and Viral Carcinogenesis [N. H. C.J, National Cancer Institute-Frederick Cancer Research
Facility, Frederick, Maryland 21701-1013
ABSTRACT
The relationship between cytoskeletal changes and oncogene expres
sion in initated cells during exposure to a tumor promoter was investigated
in the phorbol ester-sensitive murine epidermis-derived cell line JB6 (P*
cells) and its promotion-insensitive
variant (P~ cells) using immunocytochemical methods, soft agar assays, and tumorigenicity tests in nude
mice. Cytoskeletal changes in P* and P~ cells induced by short-term
incubation with 12-0-tetradecanoylphorbol-13-acetate
(TPA) were sim
ilar. Prolonged incubation with TPA allowed P cells to regain their
original appearance and resulted in growth inhibition; however, the
extended presence of TPA produced in P* cells persistent alterations in
the distribution of actin, vinculin, and fibronectin. P* cells proceeded to
develop multilayered foci. Using monoclonal antibodies, we detected the
lì-raxoncogene-encoded
M, 21,000 protein (p21) exclusively in focusforming cells. Both the observed morphological changes and the expres
sion of p21 were reversible in !" cells when TPA exposure was terminated
soon after foci had developed. In order for TPA-treated P* cells to grow
as tumors in nude mice, multiple cycles of exposure to TPA in conjunction
with clonal expansion in agar were necessary. The results indicate that
there exists during promotion of the I" JB6 cells a relationship between
expression
of the 11-rav gene product p21 and enhanced
proliferation
with focus formation and that both expression of p21 and focus formation
depend on the continuous presence of the promoting agent.
INTRODUCTION
According to the initiation-promotion theory of Carcinogen
esis, initiation is an irreversible, genetic alteration that can be
effected by a single exposure to a low dose of carcinogen that
by itself causes no (or few) tumors. In contrast, for tumors to
develop from initiated cells, repeated treatment with tumor
promoters is necessary, and it has been suggested that, during
this time span, initiated cells are induced to give rise to new
phenotypes that are first expanded as clones which ultimately
produce tumors. Because tumor promoters are not mutagenic
and many of their effects during promotion are reversible
(reviewed in Refs. 1 and 2), altered gene regulation mechanisms
have been suggested for promotion. In order to overcome
normal growth restrictions during the promotion process, ini
tiated cells must undergo certain changes that result in the loss
of cell contacts and the acquisition of elevated growth potential.
The reduction of cell-cell and cell-substrate contacts is usually
intimately associated with both the rearrangement of cytoskel
etal structures, especially actin and vinculin (3, 4), and the loss
of extracellular matrix proteins, such as fibronectin (5). Such
morphological changes, coupled with stimulation of growth
and cell division, may result from expression of the protein
products of certain oncogenes, as has been suggested for the M,
60,000 phosphoprotein src oncogene product (Ref. 6; reviewed
in Ref. 7) and for the Harvey ras p21 protein (8, 9). In support
of this, it has been reported that primary papillomas as well as
Received 1/20/86; revised 7/24/86; accepted 8/4/86.
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.
1To whom requests for reprints should be addressed, at the Ultrastructural
Studies Section, Laboratory of Comparative Carcinogenesis, NCI-Frederick Can
cer Research Facility. Bldg. 538. Frederick, MD 21701-1013.
squamous cell carcinomas induced in mice by an initiationpromotion protocol contain the activated H-ras oncogene (10,
11).
It has recently been documented that tumor promoters en
hance the transformation of cell cultures transfected with an
activated c-H-ras ocogene (12, 13). Thus, as has been suggested
by these authors and others (14, 15), it is especially attractive
to consider that, during multistage Carcinogenesis, tumor pro
moters might interact synergistically with activated or cellular
oncogenes, either by altering the quantitative and temporal
expression of such genes or by inducing cooperative genes (16,
17) to increase cellular proliferation and bring about neoplastic
transformation.
The most active known tumor promoter, isolated from croton
oil, is TPA.2 TPA exerts a wide range of biological and bio
chemical effects, including loss of actin cables (18), decrease in
fibronectin (19, 20) and procollagen (21), and interruption of
intercellular communication due to the loss of gap junctions
(22), thus modulating the original phenotype to assume in many
respects a transformed appearance. However, because these
effects are not specific for initiated cells, are often transient in
the presence of TPA, and are often reversible upon removal of
TPA, it is not clear which of the TPA-mediated effects are
essential to promote the latent, initiated cells.
To examine possible interactions between a tumor promoter,
phenotypic modulation, and oncogene expression during pro
motion, we used cloned cells from the JB6 mouse epidermisderived cell line (23, 24). Clones of this cell line are especially
useful for such work because they include the promotioncompetent (P+) cells (clone 41) that can be irreversibly trans
formed by treatment with TPA alone and therefore are thought
to represent initiated or postinitiated cells. A promotion-incom
petent (P~) clone (clone 30) served as control. Cells from this
clone do not transform in response to TPA and thus are thought
to be earlier preneoplastic cells. Moreover, as previously re
ported (25), transfection of JB6 P~ cells with activated viral Hras DNA transformed these cells to anchorage independence.
Thus, these JB6 cells presumably do not express activated p21.
A second control consisted of permanently transformed cells,
clone RT 101, a TPA-transformed derivative of JB6 P+ clone
41. It is noteworthy that while TPA has many effects on many
kinds of cells, JB6 cells retain specificity for promoters of
squamous epithelium and fail to respond to promoters of, e.g.,
hepatic parenchyma! neoplasia (26).
In the present study, we investigated structural alterations
such as rearrangement of cytoskeletal components, including
filamentous actin, microtubules, and vinculin, and changes in
cell substrate behavior, as shown by an altered pattern of
fibronectin distribution, due to short-term exposure to TPA in
P+ and P~ cells. We compared the results to those obtained in
long-term studies in which P+ cells were repeatedly exposed to
TPA in semisolid medium (clonal expansion) and the resulting
2 The abbreviations used are: TPA, 12-O-tetradecanoylphorbol-13-acetate;
FITC, fluorescein isothiocyanate; PKC, protein kinase C; PBS, phosphate-buff
ered saline; p 21, M, 21,000 protein.
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CYTOSKELETAL
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anchorage-independent clones were used for further propaga
tion. Cell cultures developed by this regimen were tested for
their capacity to show anchorage independence in TPA-free
medium and to produce tumors in nude mice. The expression
of the H-ras oncogene product p21 was monitored in both
short-term and long-term studies.
Our findings show that the morphological alterations result
ing from short-term exposure to TPA persist in P+ cells cultured
in the continued presence of TPA; however, under the same
experimental conditions, these effects are only transient in P~
cells. We show also that continuous exposure of P* cells to
TPA results, after confluence, in the formation of numerous
multilayered foci, whereas P~ cells show density-dependent
inhibition of growth even in the continued presence of TPA.
We detected the H-ras gene product p21 only in focus-forming
P+ cells cultured in the continuous presence of TPA; the onco
gene product was not detected in flat cells of the TPA-treated
monolayers or in permanently transformed P+ cells produced
by repeated exposure to TPA. These findings relate the expres
sion of an oncogene-encoded protein specifically to focus for
mation that results from exposure to a tumor promoter.
The results of our long-term studies show that the irreversible
expression of the transformed phenotype proceeds stepwise and
involves permanent reduction in actin-containing stress fibers
and cellular fibronectin. Progression to the transformed cell
type depends on repeated exposure to TPA in conjunction with
clonal expansion in agar and leads to increased anchorage
independence and, finally, tumorigenicity. In each subclone,
after confluence, the formation of foci in the presence of TPA
was associated with the transient expression of H-ras p21, thus
indicating a requirement for the transient expression of the
oncogene product during promotion, but not in maintenance of
the resulting tumor cell phenotype.
MATERIALS AND METHODS
Chemicals. TPA (Chemicals for Cancer Research, Inc., Eden Prairie,
MN) was initially dissolved in dimethyl sulfoxide at a concentration of
1 mg/ml. The stock solution was maintained at -20°C and diluted in
culture medium before use.
Cell Culture. The origin and maintenance of BJ6 cells and their
promotion-sensitive clones have been described (23, 24). Promotable
(P+) cells (clone 41, passage 80), nonpromotable (P~) cells (clone 30,
passage 100), and TPA-transformed cells (clone RT 101) were used.
Cells were seeded at a concentration of 5 x 10s cells/60-mm Petri dish
in Eagle's minimum essential medium containing 5% heat-inactivated
fetal calf serum supplemented with penicillin and streptomycin. TPA
was used at a concentration of 0.1 Mg/ml. Cells were examined by
phase-contrast and immunofluorescence microscopy. In short-term ex
periments, examination was carried out between 2 min and 10 days
after addition of TPA, using appropriate controls.
Evaluation of Anchorage-independent Growth by Soft Agar Assay.
Cells were suspended in culture medium supplemented with 10% fetal
calf serum containing 0.33% Difco agar at 40°Cto which solvent alone
(0.01% dimethyl sulfoxide) or TPA (0.1 Mg/ml) had been added. The
suspension, 1.5 ml containing IO4 cells/dish, was layered over 0.5%
agar base in standard medium. Assays were carried out in duplicate
without refeeding; colonies consisting of 10 or more cells were counted
after 14 days.
Repeated TPA Treatment and Establishment of Subclones. I' cells in
0.33% Difco agar medium containing TPA (0.1 Mg/ml) were layered at
a concentration of 5 x IO2 cells/dish over 0.5% agar base. After
culturing for 3 weeks with refeeding, resulting colonies were isolated
and grown as monolayers in the absence of TPA. Among 20 clonal
lines derived from soft agar colonies of I' ' cells, the cell line that showed
highest anchorage-independent growth in TPA-free agar medium, des
AND H-ras P2I IN JB6 CELLS
ignated C, (implying one cycle of TPA exposure), was used for a second
treatment with TPA according to the schedule described above. Similar
selection for anchorage-independent growth yielded clonal line C2. By
3, 4, and 5 successive intervals of exposure to TPA and cloning, we
established clonal cell lines C3 to ( ..
Assay for Tumorigenicity. Cells of clones <', through C3, as well as
the original cells of clone 41 (C0), were suspended in serum-free medium
at a concentration of 1 or 5 x IO6 cells/0.2 ml and injected s.c. into
nude mice. RT 101 cells, a clone of JB6 cells consisting of permanently
transformed cells, were injected into nude mice to serve as a positive
control. The resulting tumors were examined by routine histopathological methods.
Fluorescence Cytochemistry. Cells used for fluorescence microscopy
were grown on glass coverslips. Staining was performed by inverting
the cover slips onto 30-50 til of the appropriate solution on Parafilm
(American Can Co., Greenwich, CT). To detect filamentous actin, cells
were fixed in 4% formaldehyde (TEM grade; Tousimis, Rockville, MD)
in PBS, extracted in acetone at -15"C for 5 min, and incubated with
0.17 MMfluorescein-phalloidin (Molecular probes, Inc., Junction City,
OR) at room temperature for 30 min.
For indirect immunofluorescence experiments, antibodies were di
luted with PBS containing 0.1-1 % bovine serum albumin; incubations
were for 30 min at room temperature, and washes following fixation
and antibody incubations were for 10 min each. Stained cells were
mounted in Elvanol (Monsanto, Springfield, MA) (27) and examined
on a Leitz Ortholux microscope fitted for epifluorescence. Specifics of
the various staining protocols were as follows. For fibronectin, cells
were fixed and extracted according to the actin staining protocol,
incubated with rabbit anti-human fibronectin (Seragen, Boston, MA)
diluted 1:50, followed by FITC-labeled goat anti-rabbit IgG (Cappel
Laboratories, Cochranville, PA) diluted 1:50. For vinculin, cells were
fixed with 2% formaldehyde in PBS for 5 min, followed by permeabilization with 0.2% Triton X-100 in PBS for 5 min, incubated with rabbit
anti-vinculin (Transformation Research, Inc., Framingham, MA) di
luted 1:30, followed by FITC-labeled goat anti-rabbit IgG diluted 1:50.
For tubulin, cells were treated with stabilization buffer consisting of
0.1 M piperazine-A',A'/-bis(2-ethanesulfonic acid) sodium salt adjusted
to pH 6.9 with potassium hydroxide, 1 mM ethyleneglycol bis(2-aminoethyl ether)-jV,yV'-tetraacetic acid, and 4% polyethylene glycol 6000;
and permeabilized using 0.5% Triton X-100 (28); fixed in 4% formal
dehyde; and incubated in undiluted sheep anti-bovine tubulin (Caabco,
Houston, TX), followed by FITC-labeled rabbit anti-sheep IgG (Cappel)
diluted 1:50. For keratin and vimentin, cells were fixed for 5 min in
methanol at —
15°C,incubated in either DAKO rabbit anti-keratin
(Accurate Chemical and Scientific, Westbury, NY), guinea pig antiprekeratin (Miles Scientific, Naperville, IL), mouse monoclonal ami
cytokeratin (Labsystems, Chicago, IL), or mouse monoclonal ami
vimentin (Labsystems) diluted 1:20, followed by incubation with an
appropriate, labeled second antibody.
In order to detect II rav p21, three rat monoclonal antibodies were
used, YA6-172, Y13-238, and Y13-259 (gifts from Dr. T. Shih, Labo
ratory of Molecular Oncology, National Cancer Institute, Frederick,
MD). These antibodies recognize different antigenic determinants of
p21; YA6-172 and Y13-238 are specific for H-ras p21, and Y13-259
reacts with both H-ras p21 and Kristen ras p21 (29). Cells were fixed
for 10 min in methanol at —
15°Cand incubated in antibody (10 Mg/
ml), followed by FITC-labeled anti-rat IgG (Cappel) diluted 1:40.
For controls the specific antibody was replaced with nonimmune
serum at the same dilution as the primary antiserum (for polyclonal
antibodies) or with IgG diluted to the same concentration as primary
antibody (for monoclonal antibodies). As an additional control in the
p21 studies, antibody Y13-238, which had been adsorbed for 24 h with
excess purified H-ras p21 synthesized in Escherichia coli (a gift from
Dr. S. Hattori, Laboratory of Molecular Oncology, National Cancer
Institute, Frederick, MD), was used in the primary antibody step. The
staining protocol for p21 was confirmed using Harvey sarcoma virustransformed NIH 3T3 cells, strain 13A (a gift from Dr. Shih), as a
positive control and using uninfected NIH 3T3 cells as a negative
control.
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AND H-ras P21 IN JB6 CELLS
RESULTS
Short-Term Exposure to TPA
In these experiments, cells of logarithmically growing cul
tures were exposed to TPA (0.10 Mg/ml) for different times
ranging from 2 min to several days.
Morphological Alterations in the Presence of TPA
In subconfluent cultures, the morphological changes resulting
from the addition of TPA were similar in P+ and P~ cells. After
30 min, cells were reduced in size and formed long cytoplasmic
processes. Scanning electron microscopy showed ruffles at the
cell periphery (data not shown). However, after confluence
(about 5 to 10 days after addition of TPA), marked differences
between P+ and P~ cells were evident. P~ cells regained their
original appearance and showed density-inhibited growth (Fig.
\A); however, their saturation density was slightly increased
(«30%)as compared to untreated P~ cells (data not shown). In
contrast, P+ cells were highly refractile (Fig. IB), and numerous
foci of mult ¡layeredround cells developed in these cultures (Fig.
1C).
Actin Distribution. Untreated P+ and P~ cells contained a
prominent system of actin cables in their cytoplasm (Figs. "LA
and 3/<), that began to disappear upon exposure to TPA for
only 5 min. At the same time, peripheral actin staining (in
ruffles) became prominent (Fig. 2B). The process was com
pleted about 15 min after addition of TPA (Fig. 2C). Although
the initial reaction of P+ and P~ cells to TPA was similar (Figs.
2D and 35), maintenance of the cells in TPA-containing me
dium until (and after) confluence resulted in reversal of the
morphological alterations only in P~ cells (Fig. 3C). About 5
t
days after addition of TPA, stress fibers began to reappear in
considerable number in these cells culminating in complete
reconstruction of the stress fiber system. In contrast, P+ cells
.£»»*
did not reorganize their stress fiber system (Fig. 2E).
Vinculin Distribution. In untreated cultures of P* and P~ cells,
the vinculin distribution was comparable and followed a stand
ard pattern; i.e., vinculin was concentrated in small adhesion
plaques at the cell periphery where it demarcated regions of
cell-substrate contacts and indicated the termination points of
actin stress fibers (Figs. 2F and 3D). After addition of TPA for
10 min and shortly after actin cable loss was pronounced,
vinculin was lost from adhesion plaques (Fig. 2, G and //);
however, ruffles at the cell surfaces stained positively (Figs. 21
and 3E). As shown for actin staining, by 10 days vinculin
staining was again detectable at stress fiber termination points
in P~ cells (Fig. 3F) whereas in P+ cells only an occasional spot
of vinculin staining could be detected between adjacent cells
(Fig. 2K).
Fibronectin Distribution. As illustrated in Figs. 2L and 3G,
untreated P+ and P~ cells exhibited a thick matrix of fibronectin.
In the presence of TPA, the density of this matrix decreased
slowly in cultures of P+ and P~ cells until it was markedly
reduced by about 24 h after addition of TPA (Figs 2, M-O and
3//). By 10 days after addition of TPA, there was a further
reduction in fibronectin in cultures of P+ cells (Fig. 2P); in
contrast, P~ cells reacquired a fibronectin matrix similar in
density to that present before treatment (Fig. 37).
Distribution of Intermediate Filaments and Tubulin. Neither
polyclonal antibodies raised against bovine epidermal keratin,
Fig. 1. Phase-contrast microscopy of nonpromotable (P~) and promotable
(P*) JB6 cells grown in the presence of TPA. A, confluent culture of P~ (clone
30) cells, 5 days. Cells are flat and cell borders are obscure. B, confluent culture
of P+ (clone 41) cells, 5 days. Cells are round with distinct borders. C, confluent
culture of P* cells showing multilayered foci (arrows), 10 days. Approximately x
100.
which stain suprabasal as well as some basal cell layers (DAKO,
data sheet information), nor monoclonal antibodies raised
against porcine kidney epithelial cells grown in vitro, which do
not stain epidermal cells, were able to detect keratin in P+ or
P~ cells. However, cells of both lines contained numerous
vimentin filaments, which upon exposure to TPA did not alter
their pattern of distribution. Microtubules were present
throughout the cytoplasm, preferentially radiating from the cell
center towards the periphery. They, too, did not change their
distribution during exposure to TPA.
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AND H-ras P21 IN JB6 CELLS
Fig. 2. Changes in the distribution of actin (A-E), vinculin (F-K), and cellular fibronectin (L-M) with TPA treatment of P* cells. In the presence of TPA these
changes persist. Actin cables disappear rapidly following exposure to TPA (A-E). x 290. A, untreated; B, TPA-treated for 5 min; C, TPA-treated for 10 min; D, TPAtreated for 24 h; E, TPA-treated for 10 days. Loss of vinculin plaques follows loss of actin cables (F-K). F, untreated; G, TPA-treated for 5 min; H, TPA-treated for
10 min; /, TPA-treated for 24 h; K, TPA-treated for 10 days. The density of the fibronectin matrix is gradually reduced (L-P). L, untreated; M, TPA-treated for 20
min; N, TPA-treated for 40 min; O, TPA-treated for 24 h; P, TPA-treated for 10 days.
Distribution of the Harvey ras Gene-encoded Protein p21
In order to detect H-ras p21, we performed indirect immunofluorescence using three monoclonal antibodies. Our controls
showed that (a) all three monoclonal antibodies gave rise to
intense specific fluorescence of the plasma membranes of
Harvey sarcoma virus-transfomed NIH 3T3 cells, line 13A; (b)
untransformed NIH 3T3 cells did not stain with these antibod
ies; and (c) neither normal rat IgG nor Y13-328 antibody
preincubated with p21 gave rise to specific immunofluorescence
in 13A cells.
A specific staining for H-ras p21 was characteristic for all
mitotic cells of untreated as well as subconfluent, TPA-treated
cultures of both P+ and P~ cells (Fig. 4A). After the cultures
became confluent, however, a positive staining was observed
also at membranes of the round interphase cells that accumu
lated in foci of P+ cell cultures in the presence of TPA (Fig.
4B). This is consistent with a previous report in which specific
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CYTOSKELETAL
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AND H-ras P21 IN JB6 CELLS
Fig. 3. Variations in the staining patterns of P cells for actin (A-C), vinculin (D-F), and fibronectin (G-l) with duration of TPA treatment. TPA-induced changes
are initially similar to those seen in P+ cells, but these changes do not persist even in the continued presence of TPA. x 3000. A-C, actin staining. A, untreated; B,
TPA-treated for 24 h; C, TPA-treated for 10 days. D-F, vinculin staining. D, untreated: E, TPA-treated for 24 h; F, TPA-treated for 10 days. G-l, fibronectin staining.
G, untreated; H, TPA-treated for 24 h, /, TPA-treated for 10 days.
Fig. 4. H-ras p21 staining of P* cells using monoclonal antibody Y13-238. A, untreated P* cell. Mitotic cell stains positively, x 300. B, multilayered focus of P*
cells treated with TPA for 10 days shows p21 staining at cell membrane, x 150. C, control. Multilayered focus of P* cells treated with TPA for 10 days stained with
Y13-238 preincubated with excess amount of v-H-ras p21 synthesized in E. coli, x 150. D, untreated P~ cell. Mitotic cell stains positively, x 150. E, P~ cell treated
with TPA for 10 days. Only mitotic cells stain positively, x 150.
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AND H-roi P21 IN JB6 CELLS
staining of the 11 »v/.v
gene product adjacent to the membrane
was detected in transformed cells (30). Nondividing flat cells in
the monolayers adjacent to foci were not stained by the anti
body. Likewise, interphase cells of confluent P~ cell cultures
(Fig. 4, D and E) showed no staining for p21 in the presence of
TPA.
Reversal of the Morphological Changes after Removal of TPA
from r Cells
The effects of TPA on the morphology, cytoskeleton, and
extracellular matrix of P+ cells grown in monolayers were
reversible upon removal of the promoter from the culture
medium. Cells in the monolayers and especially the round cells
in the foci became flat and returned to their original appearance
with abundant actin cables, vinculin-containing
adhesion
plaques and dense fibronectin mats. This reversal, which re
quired at least 96 h for cells that had been exposed to TPA (0.1
¿¿g/ml)
for as long as 10 days, resulted in the complete disap
pearance of foci from the cultures.
We isolated foci from confluent, TPA-treated cultures of P+
cells and reseeded the cells in medium lacking TPA in order to
confirm that round focus-forming cells could return to their
original morphology. The reseeded cells established monolay
ers, were density inhibited, and resembled the original P+ cells
in both general morphology and cytoskeletal and matrix ar
rangements. They were negative for H-ras p21. Cells isolated
from five foci were also tested for anchorage-independent
growth in 0.33% agar medium free of TPA. As shown in Table
1, with the exception of focus 3, these cells yielded a very low
number of colonies.
Our findings clearly indicate that the short-term exposure to
TPA (up to 10 days) causes P+ cells to change phenotype and
growth pattern remarkably, both aspects becoming similar to
those of transformed cells; but these changes were reversible
upon removal of TPA from the medium. However, the en
hanced anchorage independence of focus 3 indicates that pro
gression to transformation is under way in a subpopulation of
cells.
Long-Term Experiments
Enhancement of Anchorage-independent
Growth
As described in "Materials and Methods," we established by
repeated selection rounds clonal cell lines, Ci through C5, each
derived from a colony that developed in TPA-containing soft
agar. As shown in Fig. 5, during the passage of Ci to C5, the
capacity for anchorage-independent growth in TPA-free semisolid medium increased in cultures of P+ cells. Fig. 5 also
indicates that, in the presence of TPA, anchorage-independent
Table 1 Colony formation in standard agar medium of clone 41 cells after
isolation from foci obtained in the presence of TPA
No. of colonies/ IO4
Focus no.°
cells
50
1
2
70
3
180
4
50
5
SO
Untreated clone 41
40
(controls)
* Foci were obtained after exposure of confluent monolayers to TPA (100 ng/
ml) for 5 days. Single foci were plucked from monolayers, trypsinized, and seeded
in TPA-free 0.33% agar medium to obtain colonies.
C0
C,
C2
Clonal
C3
C4
C5
Cell Line
Fig. 5. Number of anchorage-independent colonies of C0 to C5 cells/dish in
the absence (O) and in the presence (•)of TPA. Anchorage-independent growth
in the presence of TPA is to a large extent reversible. Anchorage-independent
growth in the absence of TPA increases stepwise in C0 to C5 cells.
Table 2 Presence of H-ras p21 staining in foci of TPA-treated monolayers
Untreated
Exposed to 0.1
jig/ml TPA
24 h
lOdays
Otime
10 days
(P~)Clone
Clone 30
Nofoci+'++++
(P*)C, 41
(P+)Cj
(P*)C3
(P*)RT
101 (T)" Positive staining for H-ras p21, as shown in Fig. 4/f. was present in all foci
of TPA-treated monolayers.
growth of P+ cells in semisolid medium was remarkably
in
creased at higher passage levels. This increase was partially
reversible upon return to standard medium lacking TPA.
Morphological Analysis ofC,-C5 Cell Cultures
When grown under standard culture conditions without TPA,
Ci cells isolated after a single cycle of TPA-induced anchorage
independence resembled untreated P+ cells (C0) in growth pat
tern, configuration, and distribution of actin, \ inculili, and
fibronectin. In contrast, C2-C5 cells, isolated as clones after 2
to 5 cycles, were small and highly refractile (Fig. 6). Fig. 6
indicates that the increase in both size and number of anchor
age-independent colonies in TPA-free semisolid medium is
accompanied by morphological transformation in monolayer
cultures which is clearly seen in Ci-Cj cells. The number of
actin cables in these cells were remarkably decreased (compare
Fig. 1A with Fig. 7Aand Fig. 7Q. The amount of cell-associated
fibronectin in cultures of C2 cells was only slightly decreased,
but that of Cj-Ci cells was significantly reduced (Fig. 7, D to
F). We found no significant changes in the amount and distri
bution of vinculin in cultures Ci through C5. Cells in these
cultures did not express the \\-ms p21 protein nor did they
form foci. However, after confluence and in the presence of
TPA, the cultures proceeded to form foci and simultaneously
the focus-forming cells were again capable of expressing the II
raspll (Table 2).
Tumorigenicity ofC\-Ci
Cells
To determine the tumorigenicity of Ci-C3 cells, we injected
1 or 5 x IO6 cells of each clonal cell line s.c. into nude mice
(Table 3). Untreated P+ cells and cells of passage Ci did not
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CYTOSKELETAL
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AND H-ras P21 IN JB6 CELLS
Fig. 6. Phase-contrast microscopy of cells of C»
(CI 41) (A), C, («),C2 (C), C3 (D) (X 150) and soft agar colonies of C„
(E), C, (F), C2 (G), and C, (H) cells grown
in TPA-free medium (x 40). Morphological transformation and anchorage-independent growth are increasingly evident in C2-C3 cells.
yield tumors. After 3 months, 4 of 8 animals that had been
given injections of C2 cells and 3 of 8 animals that had been
injected with Cj cells developed tumors that histologically were
undifferentiated carcinomas. All TPA-transformed JB6 clone
41 P+-derived RT 101 cells, the positive controls obtained by
six cycles of TP A exposure in the absence of cell division (31),
gave rise to tumors of similar morphology when injected into
nude mice.
DISCUSSION
Phorbol esters can induce a variety of biological effects that
result over a period of time in conversion of initiated mouse
epidermal cells and their progeny to precancerous and cancer
ous cells. Recently the idea was put forward that, during mul
tistage promotion, tumor promoters might interact with cellular
or activated oncogenes and by elevating their expression might
5929
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CYTOSKELETAL CHANGES AND H-ras P21 IN JB6 CELLS
SJf
'A
Fig. 7. Irreversible changes in actin (A-C), and fibroneetin (D-F), following repeated exposure to TPA and clonal expansion. Actin cables are prominent in C|
cells (A) but are markedly reduced in C2 (B) and C, (C) cells. The fibroneetin matrix is dense in cultures of C¡cells (D), slightly reduced in cultures of C2 cells (£),
and markedly diminished in cultures of C3 (F) cells, x 380.
Table 3 Tumorigenicity of subclones Ca-C3 after injection into athymic NCrNu/
Nu mice
Tumor-bearing mice/
injected mice
Concentration
(cells/animal)
(X ID6)
Time after
Injection
(days)
notypic modulation, and oncogene expression we have made
use of the mouse epidermis-derived JB6 cell line. We have
compared the changing patterns of cell morphology and of Hras oncogene expression of promotable (P+) cells of clone 41
with those of nonpromotable (P~) cells of clone 30 during
Histology
CoC,C2c,RT
exposure to TPA. We demonstrated
that the immediate effects
of the partially trans
P~ cells and are com
SO,148,
of TPA, namely, the reversible induction
18265,
formed cell type, are similar in P+ and
9021UndifferentiatedcarcinomaUndifferentiatedcarcinomaUndifferentiatedcarcinoma
64,
1010/80/84/83/85/5S111546,
yield a population of continually dividing cells (12-14). More
over, it was also shown that tumor promoters may alter the
expression of cooperating genes with consequent progression
to tumor cell phenotype (32).
To examine the interaction between tumor promoters, phe-
parable to those reported previously (2). They consist mainly
of a loss of actin cables, rearrangement of vinculin-containing
adhesion plaques, and decrease in cell-associated fibroneetin.
These changes are thought to be initiated at the plasma mem
brane by the interaction of TPA with its receptor, PKC (33).
Interestingly, Woolen and Wrenn (34) have shown recently
that, during PKC activation by TPA, there is a translocation of
PKC from the soluble cell fraction to the cell membrane. PKC
is well known to have a pivotal role in regulating protein
functions, such as phosphorylation of the adhesion protein
5930
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CYTOSKELETAL
CHANGES
AND H-ras P21 IN JB6 CELLS
vinculin (35). Alterations of vinculin and, possibly, other at
tachment proteins such as a-actinin (36) may bring about
disruption of actin cable anchorage at the cell membrane re
sulting in disorganization of actin bundles (4). The cascade of
events is further enhanced since, as shown here and in previous
reports (37-39), the loss of actin cables is followed by a decrease
in cell-associated fibronectin. Our findings support the sug
gested transmembrane relationship between actin cables and
fibronectin matrix (40-42).
Our investigation shows that, in the presence of TPA, the
morphological changes are only transient in P~ cells, thus
indicating that these cells are capable of overriding the TPAinitiated signals. The transient nature of various morphological
changes induced by TPA, such as a change in cell shape and
cell adhesion (21, 43, 44), loss of actin cables (18), and a
decrease in the number of gap junctions (22), has been reported
for various types of cells. However, our results also clearly
indicate that there exist fundamental differences between P+
and P~ cells. In contrast to P~ cells, P+ cells apparently lack
the ability to acquire tolerance to TPA. P+ cells retain the
altered phenotype and proceed to form foci during the extended
presence of TPA. Although experiments to date have not dem
onstrated any differences between P* and P~ cells in PKC
activity or substrates (45), considering the central role of PKC
(and the cascade of events that follows PKC activation) in
effecting the TPA signals, the hypothesis remains attractive
that P+ cells may lack capability to reduce the activation of the
enzyme at the cell membrane to normal levels during prolonged
exposure to TPA, whereas P~ cells may have such a mechanism
available. This is in agreement with recent results showing a
loss of intercellular communication between BALB/c 3T3 cells
in the presence of TPA during growth phase; however, after
confluence, these cells establish normal communications. In
contrast, the transformation-sensitive variant of these cells was
not capable of doing so (46). It was suggested that the under
lying mechanisms are mediated by the action of PKC and its
intermediates (47). If an inhibitory effect of TPA on intercel
lular communication and gap junction formation could be
detected in the JB6 cell system, it would be of great interest to
determine whether the inhibitory effect is persistent in P+ cells
and transient in P~ cells and whether it is regulated by the
action of PKC. In evaluating our findings in relation to pub
lished data, it is tempting to speculate that TPA acts on initiated
or postinitiated cells by a sustained activation of PKC and that
the cascade of events which follows selectively provides preneoplastic cells with proper intracellular, cell-cell, and cell-sub
strate conditions to escape normal growth control and progress
to a tumor cell phenotype.
In this respect it is especially interesting that we found
enhanced expression of the H-ras oncogene product p21 only
in those interphase P+ cells that formed foci in the presence of
TPA and after confluence, thus identifying the oncogene expres
sion as a relatively late event. p21 has been detected previously
in cells escaping from G0-Gi, the resting phase of growth, and
in mitotic cells implying an important role of the H-ras gene in
cell division and growth. It has been shown that microinjection
of either transforming p21 or normal p21 into quiescent cells
results in rapid alteration of cell morphology and stimulation
of DNA synthesis and cell division, although the activity of
normal p21 is much lower than that of transforming p21 (8, 9).
At present, it seems unlikely that P+ cells contain the oncogenic
form of the H-ras gene, because DNA extracted from P+ cells
does not transform NIH 3T3 cells in transfection assays (25),
and p21 is not expressed in permanently transformed P* cells
(this study). Our findings imply that the transient stimulation
of the (cellular) H-ras gene may be necessary to trigger, in the
preneoplastic and morphologically altered cells, enhanced di
vision and proliferation thus enabling these cells to overcome
the growth-inhibiting effects of the surrounding normal cells
(13). Because normal p21 has a low activity with respect to
mitogenesis and morphological transformation, we consider in
agreement with Weinstein et al. (48) that the combined effects
of TPA on the cytoskeleton (actin cables), adhesion plaques,
extracellular matrix (fibronectin), H-ras gene expression and,
possibly, cell communication are necessary for preneoplastic
P+ cells to yield foci or accomplish clonal expansion. Our results
also find support in recently published data by Yuspa et al. (49)
indicating that in keratinocytes an activated ras gene provides
the proliferative signal for tumor formation, yet exposure to
tumor promoters is necessary to selectively reverse the quiescent
growth state of the cells that contain the activated ras gene. An
alternative possibility not excluded by our data is that p21
expression is not causally related to but rather is indicative of
TPA-induced focus formation.
Our present study showed that the initial morphological
changes, focus formation, and induction of p21 expression in
monolayers are reversible upon removal of TPA from P+ cells.
Previous results (24, 50) had shown acquisition of tumorigenicity after one cycle of TPA-induced anchorage-independent
transformation, harvesting the largest clones for further culti
vation. However, we found it necessary to subject these cells to
two or more cycles in order to achieve neoplastic transformation
as shown by tumor production. Interestingly, neoplastic behav
ior as shown by morphological transformation, anchorageindependent growth, and tumorigenicity increases stepwise in
these subclones. For example, the ability to assemble a network
of actin cables and an extracellular fibronectin matrix and to
establish adhesion plaques was progressively diminished, actin
cables having been the first to disappear permanently. Such
progressive and stepwise acquisition of the transformed phe
notype of P+ cells during the repeated treatment with TPA is
consistent with skin tumor promotion in vivo. The production
of papillomas and carcinomas requires long-term and frequent
application of the promoter.
Recently, it has been proposed that tumor promoters produce
clonal expansion of preneoplastic cells, while additional muta
tion appears to be required for malignant conversion (51). Our
results indicate that clonal expansion during TPA treatment of
P+ cells may play a role in their escape from normal growth
restriction. However, it is unlikely that P+ cells have to undergo
additional mutation for the /'/; vitro equivalent of malignant
conversion because (a) of the high frequency of TPA-induced
neoplastic transformation (24) and (b) of the fact that P+ cells
remained untransformed despite numerous passages. The latter
implies that the incidence of such spontaneous mutations is
low.
Thus, our data suggest that TPA plays a direct role in
preneoplastic progression. In this aspect, it needs to be empha
sized that tumor promoters have been thought for many years
to act as gene regulators (1, 45, 48, 52). Recently a number of
genes have been shown to confer sensitivity to TPA induced
transformation, among them T24, ras (13) myc, PyLT, and
EIA (16). Of special interest are the pro genes that have been
isolated from promotion-sensitive (P+) JB6 cells and have been
found to be capable of conferring promotion sensitivity to
otherwise promotion insensitive (P~) cells (17, 53, 54). The
mechanism by which the expression of pro genes in cooperation
with TPA might contribute to the expression of H-ras gene
5931
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CYTOSKELETAL
CHANGES
AND H-ras P21 IN JB6 CELLS
product p21, described in this report, and to neoplastic trans
formation in general needs further exploration.
ACKNOWLEDGMENTS
We thank Drs. T. D. Gindhart (deceased), A. B. Roberts, and S.
Sukumar for stimulating discussions. We thank Dr. T. Shih for the
generous gift of rat monoclonal antibodies to detect H-ras p21 and Dr.
S. Hattori for purified H-ras p21 synthesized in E. coli. We appreciate
the excellent technical assistance of S. Kenney.
REFERENCES
1. Boutwell, R. K The function and mechanism of promoters of carcinogenesis.
CRC Crit. Rev. Toxicol., 2:419-443, 1974.
2. Slaga, T. .1.. and Andres, P. B. Cellular and biochemical changes during
multistage skin tumor promotion. In: H. Fujiki et al. (eds), Cellular Interac
tions by Environmental Tumor Promoters, pp. 291-301. Utrecht: VNU
Science Press, 1984.
3. Geiger, B., Tokuyasu, K. T., Dutton, A. H., and Singer, S. J. Vinculin, an
intercellular protein localized at specialized sites where microfilament bun
dles terminate at cell membranes. Proc. Nati. Acad. Sci. USA, 77: 41274131, 1980.
4. Schliwa, M., Nakamura, T., Porter, K. R., and Euteneuer, U. A tumor
promoter induces rapid and coordinate reorganization of actin and vinculin
in cultured cells. J. Cell Biol., 99: 1045-1059, 1984.
5. Hynes, R. O., Destree, A. T., and Wagner, D. C. Relationship between
microfilaments, cell-substratum adhesion and fibronectin. Cold Spring Har
bor Symp. Quant. Biol., 46: 459-470, 1981.
6. Rohrschneider, L. R., Eiseman, R. N., and I filch, C. R. Identification of a
Rous sarcoma virus transformation-related
protein in normal avian and
mammalian cells. Proc. Nati. Acad. Sci. USA, 76: 4479-4483, 1979.
7. Rohrschneider, L. R., and Gentry, L. E. Subcellular localizations of retroviral
transforming proteins define multiple mechanisms of transformation. Adv.
Oncol., 4: 269-305, 1984.
8. Feramisco, J. R., Gross, M., Kamata, T., Rosenberg, M., and Sweet, R. W.
Microinjection of the oncogene form of the human H-ras (T-24) protein
results in rapid proliferation of quiescent cells. Cell, 38: 109-117,1984.
9. Stacey, D. W., and Kung, H.-F. Tranformation of NIH 3T3 cells by microin
jection of Ha-ros p21 protein. Nature (Lond.), 5/0: 508-511, 1984.
10. Balmain. A., and Pragnell, I. B. Mouse skin carcinomas induced in vivo by
chemical carcinogens have a transforming Harvey-raj oncogene. Nature
(Lond.), 303: 72-74, 1984.
11. Balmain, A., Ramsden, M., Bowden, G. T., and Smith, J. Activation of the
mouse cellular Harvey-nu gene in chemically induced benign papillomas.
Nature (Lond.), 307: 658-660, 1984.
12. Hsiao, W. L. W., Gattoni-Celli, C., and Weinstein, I. B. Oncogene-induced
transformation of C3H 10T'/2 cells is enhanced by tumor promoters. Science
(Wash. DC), 226: 552-555, 1984.
13. Dotto, G. P., Parada, L. F., and Weinberg, R. A. Specific growth response
of ras-transformed embryo fibroblasts to tumour promoters. Nature (Lond.),
318:472-475, 1985.
14. Greenberg, M. E., and /ill. E. B. Stimulation of 3T3 cells induces transcrip
tion of the c-fos proto-oncogene. Nature (Lond.), 311: 433-438, 1984.
15. Müller,R., Bravo, R., Burckhardt, J., and Curran, T. Induction of c-fos gene
and protein by growth factors precedes activation of c-myc. Nature (Lond.),
5/2:716-720,1984.
16. Connan, G., Rassoulzadegan, M., and Cuzin, I. Focus formation in rat
fibroblasts exposed to a tumour promoter after transfer of polyoma pit and
myc oncogenes. Nature (Lond.), 314: 277-279, 1985.
17. Lerman, M. I., Hegamyer, G. A., and Colburn, N. H. Cloning and charac
terization of putative genes that specify sensitivity to neoplastic transforma
tion by tumor promoters. Int. J. Cancer, 37: 293-302, 1976.
18. Rifkin, D. B., and Crowe, R. M. Tumor promoters induce changes in the
chick embryo fibroblast. Cell, 18: 361-368, 1979.
19. Blumberg, P. M., Driedger, P. E., and Rossow, P. W. Effect of a phorbol
ester on a transformation-sensitive surface protein of chick fibroblasts. Na
ture (Lond.), 264:446-447, 1976.
20. Zerlauth, G., and Wolf, G. Release of fibronectin is linked to tumor promo
tion: response of promotable and nonpromotable clones of a mouse epidermal
cell line. Carcinogenesis (Lond.), 6: 73-78, 1985.
21. Dion, L. D., Bear, J., Bateman, J., DeLuca, L. M., and Colburn, N. Inhibition
by tumor-promoting phorbol estes of procollagen synthesis in promotable
JB6 mouse epidermal cells. J. Nati. Cancer Inst., 69: 1147-1154, 1982.
22. Kalimi, G. H., and Sirsat, S. M. Phorbol ester tumor promoter affects the
mouse epidermal gap junction. Cancer Lett., 22: 343-350, 1984.
23. Colburn, N. H., Vorder Bruegge, W. F., Bates, J. R., Gray, R. H., Rossen,
J. D., Kelsey, W. H., and Shimade, T. Correlation of anchorage-independent
growth with tumorigenicity of chemically transformed mouse epidermal cells.
Cancer Res., 38:624-634, 1978.
24. Colburn, N., Former, B. F., Nelson, K. A., and Yuspa, S. H. Tumor promoter
induced anchorage independence irreversibly. Nature (Lond.), 281:589-591,
1979.
25. Colburn, N. H., Lerman, M. I., Hegamyer, G. A., and Gindhart, T. D. A
transforming activity not detected by DNA transfection of NIH 3T3 cells is
detected by JB6 mouse epidermal cells. Mol. Cell. Biol., 5:890-893, 1985.
26. Diwan, B. A., Ward, J. M., Rice, J. M., Colburn, N. H., and Spangler, E. F.
Tumor-promoting effects of di(2-ethylhexyl)phthalate in JB6 mouse epider
mal cells and mouse skin. Carcinogenesis (Lond.), 6: 343-347, 1985.
27. Heimer, G. V., and Taylor, C. E. Improved mountant for immunofluorescence preparations. J. Clin. Pathol., 27: 254-256, 1974.
28. Osborn, M., Webster, R. E., and Weber, K. Individual microtubules viewed
by immunofluorescence and electron microscopy in the same PCK 2 cell. J.
Cell Biol., 77: R27-R34, 1978.
29. Furth, M. E., Davis, L. J., Fleurdelys, B., and Scolnick, E. M. Monoclonal
antibodies to the p21 products on the transforming gene of Harvey murine
sarcoma virus and of the cellular ras gene family. J. Virol., 43: 294-304,
1982.
30. Willingham, M. C., Pastan, I., Shih, T. Y., and Scolnick, E. M. Localization
of the src gene product of the Harvey strain of MS V to plasma membrane of
transformed cells by electron microscopic immunocytochemistry. Cell, 19:
1005-1014, 1980.
31. Colburn, N. H., Wendel, E., and Aborizzo, G. Dissociation of mutagenesis
and late-stage promotion of tumor cell phenotype by phorbol esters: mitogen
resistant variants are sensitive to promotion. Proc. Nati. Acad. Sci. USA, 78:
6912-6916,1981.
32. Colburn, N. H. The genetics of tumor promotion. In: }. Carl Barrett (ed.).
Mechanisms of Environmental Carcinogenesis, Vol. 1. Boca Raton, FL:
CRC Press, Inc., in press, 1986.
33. Nishizuka, Y. The role of protein kinase C in cell surface signal transduction
and tumor promotion. Nature (Lond.), 308:693-698, 1984.
34. Wooten, M. W., and Wrenn, R. W. Redistribution of phospholipid/calciumdependent protein kinase and altered phosphorylation of its soluble and
paniculate substrate protein in phorbol ester-treated rat pancreatic acini.
Cancer Res., 45: 3912-3917, 1985.
35. Werth, D. K., Niedel, J. E., and Pastan, I. Vinculin, a cytoskeletal substrate
of protein kinase C. J. Biol. Chem., 25«:11423-11426, 1983.
36. Meigs, J. B., and Wang, Y. L. Reorganization of a-actinin and vinculin
induced by a phorbol ester in living cells. J. Cell Biol., 102: 1430-1438,
1986.
37. Mautner, V., and Hynes, R. O. Surface distribution of LETS protein in
relation to the cytoskeleton of normal and transformed cells. J. Cell. Biol.,
75:743-758, 1977.
38. Ali, I. V., and Hynes, R. O. Effect of cytochalasin B and colchicine on
attachment of major surface protein of fibroblasts. Biochim. Biophys. Acta,
471: 16-24, 1977.
39. Kurkinen, M., Wartiovaara, J., and Vaheri, A. Cytochalasin B releases a
major surface-associated glycoprotein from cultured fibroblasts. Exp. Cell
Res., ///: 127-137, 1978.
40. Singer, I. I. The fibronexus: a transmembrane association of fibronectincontaining fibers and bundles of 5nm microfilaments in hamster and human
fibroblasts. Cell, 16: 675-685, 1979.
41. Hynes, R. O., and Yamada, K. M. Fibronectins: multifunctional modular
glycoproteins. J. Cell Biol., 95:369-377, 1982.
42. Chen, W. T., and Singer, S. J. Immunoelectron microscopic studies of the
site of cell-substratum and cell-cell contacts in cultured fibroblasts. J. Cell
Biol., 95: 205-222, 1982.
43. Boreiko, C., Mondai, Narayan, K. S., and Heidelberger, C. Effect of 12-Otetradecanoylphorbol-13-acetate
on the morphology and growth of C3H/
10T'/2 mouse embryo cells. Cancer Res., 40:4709-4716, 1982.
44. Nagel, D. S., and Blumberg, P. M. Activity of phorbol ester tumor promoters
on enucleated Swiss 3T3 cells. Cancer Res., 40: 1066-1072, 1980.
45. Gindhart, T. D.. Nakamura, Y., Stevens, L. A., Hegameyer, G. A., West, M.
W., Smith, D. M., and Colburn, N. H. Genes and signal transduction in
tumor promotion: conclusions from studies with promoter resistant variants
of JB-6 mouse epidermal cells. Carcinog. Compr. Surv., 8: 341-367, 1985.
46. Yamasaki, H., Enomoto, T., Shiba, Y., Kamm. Y., and Kakunaga, T. Inter
cellular communication capacity as a possible determinant of transformation
sensitivity of BALB/c 3T3 clonal cells. Cancer Res., 45:637-641, 1985.
47. Enomoto, T., and Yamasaki, H. Rapid inhibition of intercellular communi
cation between BALB/c 3T3 cells by diacylglycerol, a possible endogenous
functional analogue of phorbol ester. Cancer Res., 45: 3706-3710, 1985.
48. Weinstein, I. B., Gattoni-Celli, S., Kirschmeier, P., Lambert, M., Hsiao, W.,
Backer, J., and Jeffrey, A. Multistage carcinogenesis involves multiple genes
and multiple mechanisms. In: Cancer Cells I/The Transformed Phenotype,
pp. 229-237. Cold Spring Harbor Laboratory, New York: 1984.
49. Yuspa, S. H., Kilkenny, A. E., Stanley, J., and Lichti, U. Keratinocytes
blocked in phorbol ester-responsive early stage of terminal differentiation by
sarcoma viruses. Nature (Lond.), 314:459-62, 1985.
50. Colburn, N. H. Tumor promoter produces anchorage independence in mouse
epidermal cells by an induction mechanism. Carcinogenesis (Lond.), /: 951954, 1980.
51. Hennings, H., and Yuspa, S. H. Two-stage tumor promotion in mouse skin;
an alternative interpretation. J. Nati. Cancer Inst., 74:735-740, 1985.
52. Hoffman-Liebermann, B., Liebermann, D., and Sachs, L. Regulation of gene
expression by tumor promoters. III. Complementation of the developmental
program in myeloid leukemic cells by regulating mRNA production and in
RNA translation. Int. J. Cancer, 28:615-620, 1981.
53. Colburn, N. H., Lerman, M. I., Hegamyer, G. A., Wendel, E., and Gindhart,
T. D. Genetic determinants of tumor promotion: studies with promoter
resistant variants of JB6 cells. In: Genes and Cancer, pp. 137-155. New
York: Alan R. Liss, Inc., 1984.
54. Colburn, N. H., et al., in press, 1986.
5932
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Role of Cytoskeleton Changes and Expression of the H-ras
Oncogene during Promotion of Neoplastic Transformation in
Mouse Epidermal JB6 Cells
Kiyoshi Takahashi, Ursula I. Heine, James L. Junker, et al.
Cancer Res 1986;46:5923-5932.
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