Download 12Macromolecular

[CANCER RESEARCH 43, 5552-5559,
November 1983]
12<)-Tetradecanoylphorbol-13-acetate
Actions on Macromolecular
Synthesis, Ornithine Decarboxylase, and Cellular Differentiation
of the Rat Embryonic Visceral Yolk Sac in Culture1
Brian E. Huber2 and Nigel A. Brown3
Department of Pharmacology,
The George Washington University Medical Center, Washington, D. C. 20037
ABSTRACT
spectively, were unchanged by TPA treatment. VYS protein
synthesis, measured by [3H]leucine incorporation, was initially
tude of biological responses including hyperplasia, gene activa
tion, alteration of differentiation, enzyme induction, and cell.sur
face effects (for review, see Refs. 3, 12, and 60). At present, it
is not known which of these responses is/are essential for the
promotional process.
Due to the many embryonic and developmental characteristics
associated with tumor promotion (for review, see Ref. 55), we
are currently investigating the effects of tumor promoters on
mammalian embryogenesis. In the accompanying paper (23), we
described a technique utilizing whole rat embryos in culture as a
model system for the study of the actions of promoters. This
embryo culture technique has the speed, flexibility, and manip
ulative capacity of an in vitro system but maintains intact, nor
mally differentiating mammalian embryonic tissue. Thus, unlike
isolated cells in culture, the tissues of the VYS maintain the
orientations, communications, and interactions characteristic of
normal tissue in vivo. Hence, this whole-embryo culture system
provides a unique method for the investigation of certain hy
potheses concerning the biological effects of tumor promoters
which would be difficult or impossible with either in vivo or cell
culture techniques.
In this system, TPA,4 the most potent phorbol ester epidermal
increased by TPA treatment but returned to control values by
the end of the culture period. This increase in [3H]leucine incor
poration was not due to a TPA-mediated change in the secretory
function of the VYS.
The data suggest that the tumor promoter-induced disruption
promoter (2, 19), disrupted the morphology and function of the
VYS, the outermost embryonic membrane of the cultured rat
embryo (23). The effect was characterized by the abnormal
progressive separation of the 2 cellular layers comprising the
VYS. The structure-activity relationship for a series of promoting
of the VYS is not associated with cellular proliferation, ornithine
decarboxylase induction, or alterations in differentiation. Effects
on the cell surface, altering cell-cell interactions and/or commu
agents to cause this disruption was consistent with other pro
motional actions of the compounds but in addition indicated that
the effect may be related to late-stage promotion. This supported
the hypothesis that embryonic tissue, in general, might need only
late-stage promotional events to produce complete promotional
effects (54).
In the present study, certain general hypotheses concerning
the biological effects of TPA were tested for their validity in the
whole-embryo culture system. We have examined ornithine de
carboxylase (ODC; EC4.1.1.17) activity, because ODC induction
has been hypothesized to be essential and specific for promotion
(5, 42) and has been suggested to be associated with Stage II
promotion (56). In addition, TPA effects on the differentiation of
the VYS were quantitatively assessed by determining hemoglo
bin content and synthesis, since the formation of functional fetal
erythrocytes is a major VYS event over this period of embryoge
nesis. Finally, protein, RNA, and DNA synthesis were determined
in TPA-treated VYS to compare to the in vivo skin effects of TPA
where it has been classified as a gene-activating agent (5, 20).
We have reported previously that 12-O-tetradecanoylphorbol13-acetate (TPA) disrupts the morphology and functional devel
opment of the rat embryonic visceral yolk sac (VYS) maintained
in a whole-embryo culture system. The TPA-mediated disruption
of the VYS is characterized by the abnormal progressive sepa
ration of the cellular layers that comprise the VYS and appears
to be related to late-stage promotion. The present study further
characterizes this effect of TPA on the VYS of rat conceptuses
in vitro.
VYS ornithine decarboxylase levels were not induced but
rather were initially depressed by TPA treatment. There was no
major effect of TPA treatment on VYS hemoglobin content, as
measured by absorbance at 414 nm and polyacrylamide gel
electrophoresis. Changes in VYS hemoglobin synthesis during
the culture period, measured by [14C]leucine incorporation with
subsequent autoradiography, was likewise not a major effect of
TPA treatment. VYS DMA synthesis and VYS RNA synthesis,
measured by [3H]thymidine and [3H]uridine incorporation, re
nication might best explain these actions of TPA.
INTRODUCTION
The 2-stage protocol of initiation and promotion has been an
extensively utilized model system for the study of carcinogenic
mechanisms (for review, see Ref. 52). A unifying concept of
agents that have initiating properties is that those agents are or
can be converted to an electrophilic form which can react with
cellular macromolecules including DNA (38). Promoting agents,
however, are nonelectrophilic compounds that produce a multi1Supported by NIH Grants CA 32306-01 and RR 5359-20 Project 3-82. Pre
sented in part at the Joint Meeting of the American Society for Pharmacology and
Experimental Therapeutics and the Society of Toxicology, 1982 (22). This paper is
the second in a series.
2 Predoctoral student in The Department of Pharmacology, George Washington
University, Washington. D. C. To whom requests for reprints should be addressed.
3 Present address: Medical Research Council Laboratories, Carshalton, Surrey,
SM5 4EF, United Kingdom.
Received February 22, 1983; accepted July 13, 1983.
5552
* The abbreviations used are: TPA, 12-O-tetradecanoylphorbol-13-acetate;
VYS,
visceral yolk saofs); ODC. ornithine decarboxylase; p.c., post coitum; DMSO,
dimethyl sulfoxide; HBSS, Hanks' balanced salt solution; TCA, trichloroacetic acid.
CANCER
RESEARCH
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VOL. 43
TPA Effects on Macromolecular
MATERIALS
(335.0 mCi/mmol), and Enhance autoradiography enhancer were from
New England Nuclear (Boston, Mass.); [5-3H]uridine (20 Ci/mmol) and
[mef/7y/-3H]thymidine (50 Ci/mmol) were from Moravek Biochemicals,
Inc. (Brea, Calif.); L-[2,3,4,5-3H]leucine (144 Ci/mmol) was from ICN, Inc.
(Irvine, Calif.); sodium lauryl sulfate was from BDH Biochemicals (Poole,
United Kingdom); molecular weight markers were from Bio-Rad (Rich
mond, Calif.); Coomassie Brilliant Blue was from Eastman Kodak (Roch
ester, N. Y.).
Animals, Embryo Culture, and Treatment. Breeding and housing of
Sprague-Dawley rats, embryo culture techniques, and TPA treatment
protocols have been described extensively in the accompanying paper
(23). At 10.4 days p.c. (0 is the midpoint of the dark cycle during which
mating occurred), rat conceptuses were explanted from uteri and aseptically dissected free of decidua, Reichert's membrane, and the parietal
yolk sac. Each conceptus at this developmental stage consists of an
early-headfold embryo, amnion, and chorion surrounded by the VYS.
Two conceptuses were placed in a 30-ml serum bottle containing 4 ml
medium, incubated for 2 hr, then treated with TPA or DMSO, and cultured
up to 28 additional hr. The culture medium consisted of 80% rat serum,
which was immediately centrifuged (41), heat inactivated (41), filtered,
and supplemented with Waymouth's medium as described previously
(23). TPA was dissolved in freshly distilled DMSO and added to the
medium in volumes not exceeding 2.5 n\.
ODC Assay. Tissue derived from the VYS was analyzed for protein
content and ODC activity at the start of the culture period (10.4 days
p.c.) and after 8 and 28 hr in culture. The VYS was dissected free of the
remaining conceptus tissue in cold HBSS, washed 3 times in cold HBSS,
and then homogenized by sonication in 150 n\ Tris-HCI buffer (50 HIM
Tris, pH 7.4, containing 4 mM EDTA, 5 HIM dithiothreitol, and 0.625 rriM
pyridoxal phosphate). Each homogenate contained from 2 to 4 VYS. The
homogenate was then centrifuged at 13,000 x g for 30 min at 4°,and
the supernatant was assayed for protein content (7) and ODC activity.
ODC activity was measured by a modification of the method of Bulger
and Kupfer (8) as reported previously (21).
Hemoglobin Determination. Hemoglobin content of the VYS was
determined at the start of the treatment period (2 hr after the start of
culture) and after 26 hr of treatment. Using 35- x 10-mm Petri dishes
containing HBSS at 37°, the amnion, chorion, ectoplacental cone, and
embryo were dissected from the VYS and discarded. The VYS and
HBSS were then transferred to 15-ml centrifuge tubes, the dissecting
dish was washed 3 times with HBSS, and this wash was added to the
centrifuge tube to ensure total collection of yolk sac blood elements. The
tubes were then centrifuged at 2000 x g for 10 min at 23°, the
supernatant was discarded, and the remaining pellet was homogenized
by sonication in 600 n\ Tris-HCI buffer (50 mM Tris, pH 7.0, containing
0.3% Triton X-100, 25 mM KCI, 5 mM MgCI2, and 1 mM 2-mercaptoethanol). This homogenate was then centrifuged at 13,000 x g for 10 min
at 4°, and the supernatant was assayed for protein content (7) and
content.
Hemoglobin
and ODC Levels
MgCI2,1 mM 2-mercaptoethanol, and 240 mM sucrose). This homogenate
was then centrifuged at 13,000 x g for 10 min at 4°,and the supernatant
AND METHODS
Chemicals. TPA Batch 26, was from Consolidated Midland Corp.,
(Brewster, N. Y.); L-[1-'"C]ornithine
(46.0 mCi/mmol), L-[U-'4C]leucine
hemoglobin
Synthesis, Hemoglobin
was determined
spectrophotometri-
cally by absorbance at 414 nm using the method of Kabat ef a/. (28).
Hemoglobin content was quantified by using Am of rat hemoglobin
standards to yield hemoglobin concentration in mg/ml.
Polyacrylamide Gel Electrophoresis. Polyacrylamide slab gel elec-
was assayed for protein content (7) and radioactivity. To determine
radioactivity, a sample of the homogenate was precipitated with 5 ml of
cold 10% TCA and centrifuged at 2000 x g for 5 min at 23°, and the
pellet was washed twice with 5 ml of cold 10% TCA. This pellet was
then collected on Whatman GF/C glass microfiber filters and assayed
for radioactivity. The remaining supernatant from the cellular homogenate
was then made 0.6% in sodium lauryl sulfate and 1% in 2-mercaptoeth
anol, heated at 70°for 15 min, and recentrifuged at 13,000 x g for 10
min at 4°.Samples of this final supernatant were then applied to the gel
with 100 Mg of protein loaded per well. Molecular weight standards
(phosphorylaseb, bovine serum albumin, ovalbumin, carbonic anhydrase,
soybean trypsin inhibitor, and lysozyme) and rat hemoglobin standards
were run as markers. The gels themselves were poured as gradients
ranging from 10% acrylamide-0.8% bisacrylamide to 15% acrylamide1.2% bisacrylamide. Gels were run for 8 hr by the method of Laemmli
(29) and then stained in 0.05% Coomassie Brilliant Blue in 25% isopropyl
alcohol-10% glacial acetic acid for 16 hr. Three batches of destain (10%
¡sopropyl alcohol-10% glacial acetic acid) were then used over a period
of 24 hr. The gels were then photographed, impregnated with Enhance
autoradiography enhancer for 1 hr, placed in 37° water for 1 hr to
precipitate the Enhance, and dried. The gels were then exposed to
photographic film for 10 days to obtain an autoradiographic plate. For
quantitation, both the photograph of the Coomassie-stained gel (positive
plate) and autoradiograph (positive plate) were first scanned on a Joyce
Loebl densitometer (plate from the Coomassie-stained gel was scanned
on reflectance mode, while the autoradiograph was scanned on transmittance mode). The densitometer scans were subsequently analyzed
using an Apple II plus computer with attached graphics tablet digitizer.
Macromolecular Synthesis. We have determined the effect of TPA
on the incorporation of leucine into protein, thymidine into DNA, and
uridine into RNA of the VYS. Rat conceptuses were explanted from uteri,
placed in culture, treated with TPA at 2 hr, and cultured for an additional
24 hr as described previously. In addition, conceptuses were also labeled
for 60 min with [3H]leucine (10 ^Ci; 144 Ci/mmol), [3H]thymidine (12 /jCi;
50 Ci/mmol), or [3H]uridine (10 ^Ci; 20 Ci/mmol) prior to analysis at 2, 6,
and 24 hr after TPA treatment. Incorporation of radiolabeled precursors
into nucleic acids and protein of the VYS was then determined by a
modification of the Schmidt-Thannhauser procedure (50). The VYS and
blood elements were obtained as described for hemoglobin determina
tion, centrifuged at 2,000 x g for 5 min at 23°,washed twice in 10 ml
cold HBSS, and then homogenized by sonication in 50 mM phosphate
buffer, pH 7.4.
For [3H]leucine incorporation into VYS protein, one labeled VYS with
blood
buffer,
nation
added
elements was homogenized by sonication in 200 n\ phosphate
and a sample of the homogenate was taken for protein determi
(7). To the remaining homogenate, 5 ml of cold 10% TCA were
and, after 20 min, centrifuged at 2000 x g for 5 min at 23°. A
sample of the supernatant was taken to determine radioactivity in the
acid-soluble cell fraction, and the remaining supernatant was discarded.
The pelleted precipitate was washed twice with 5 ml cold 10% TCA and
then collected on Whatman GF/C glass microfiber filters. The centrifuge
tubes and filter papers were subsequently washed with an additional 5
ml cold 10% TCA and 5 ml 70% ethanol, and the filters were assayed
for radioactivity.
For [3H]uridine incorporation into VYS RNA, one labeled VYS with
trophoresis analysis of VYS proteins was performed by a modification of
the method of Laemmli (29). Rat conceptuses were explanted, cultured,
and treated with TPA as described above. After 2 hr in culture, when
TPA (50 nw) or DMSO (2.5 ^) was added to the culture medium, L-[U14C]leucine (5 /*Ci; 335.0 mCi/mmol) was also added. After 28 total hr in
blood elements was homogenized
and a sample of the homogenate
To the remaining homogenate, 5
after 20 min, centrifuged at 2000
culture, the VYS with blood elements were obtained as described for
hemoglobin determination and centrifuged at 2000 x g for 10 min at
23°;the supernatant was discarded. The remaining pellet was washed
fraction, and the remaining supernatant was discarded. The pelleted
precipitate was washed twice with 5 ml cold 10% TCA and then hydrolyzed in 5 ml of 0.5 N KOH for. 1 hr at 37°.The hydrolysate was chilled
twice in cold HBSS and then homogenized by sonication in 400 ^l TrisHCI buffer (50 mM Tris, pH 6.8, containing 0.3% Triton X-100, 5 mM
on ice for 20 min, neutralized with 1.2 ml of 50% TCA and, after 20 min,
centrifuged at 2000 x g for 5 min at 23°.A sample of supernatant was
NOVEMBER
by sonication in 1 ml phosphate buffer,
was taken for RNA determination (51 ).
ml of cold 10% TCA were added and,
x g for 5 min at 23°.A sample of the
supernatant was taken to determine radioactivity in the acid-soluble cell
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5553
8. E Huber and N. A. Brown
taken to determine
radioactivity
in the acid-precipitable,
alkaline-labile
fraction (RNA fraction), and the rest was filtered using Whatman GF/C
glass microfiber filters (as described for [3H]leucine incorporation) to
determine
radioactivity
in the acid-precipitable,
alkaline-stable
fraction
(DNA, protein fraction).
For [3H]thymidine incorporation into VYS DNA, all procedures were
routinely performed as described for [3H]leucine incorporation into pro
tein. However, additional experiments were performed as described for
[3H]uridine incorporation into RNA to determine the percentage of radio
activity incorporated into the acid-precipitable, alkaline-labile cell fraction
(RNA). DNA was determined by the method of Burton (9).
Secretion from the VYS. Rat conceptuses were explanted from uteri
and placed in culture as described previously. After 30 min of equilibration
in culture, [3H]leucine (50 MCi; 144 Ci/mmol) was added to the culture
medium. At 3 hr, each conceptus was washed 4 times in 37° Waymouth's medium, replaced into fresh culture medium (4 ml) which was
treated with TPA (50 nw) or DMSO (2.5 ¿»I),
and cultured as described
previously. At various times, a 500-^1 sample of the culture medium was
obtained to assay for secreted labeled proteins. This sample was precip
itated with 5 ml of cold 10% TCA and centrifugea at 2000 x g for 5 min
at 23°.The precipitate was washed twice with 5 ml cold 10% TCA and
then hydrolyzed with 1 ml 1.0 N NaOH overnight at 37°.This hydrolysate
was neutralized with 500 ^l 50% TCA, and radioactivity was determined.
Measurement of Radioactivity. All samples were counted in 10.0 ml
of Liquiscint counting solution (National Diagnostic; Somerville, N. J.)
using a Beckman LS-255 liquid scintillation counter. Correction for
quenching was made by use of an automatic
results were calculated in dpm.
external standard, and
RESULTS
ODC Activity. The potentialof TPA to affect ODC activityin
the VYS of rat conceptuses was examined. VYS soluble protein
content and ODC activity in the presence of TPA or DMSO
(control) are shown in Chart 1. While protein content of control
VYS increased dramatically over the culture period, ODC activity
remained unchanged. These control observations on growth and
ODC activity have been reported by us previously in a similar rat
conceptus culture technique (21).
After 6 hr exposure to 50 nw TPA (8 total hr in culture), which
is the earliest time at which any morphological effects of TPA on
the VYS can be observed (23), there was an increase in protein
content above controls, but this did not exhibit a dose-response
relationship. After 26 hr of treatment (28 total hr in culture), VYS
protein content was not significantly different from controls at all
dose levels of TPA. In contrast to protein content, VYS ODC
levels were significantly depressed at all doses of TPA after 6 hr
of treatment. After a 26-hr treatment period, only levels in VYS
dosed with 100 nM TPA remained significantly lower than those
in controls.
HemoglobinContent. The VYS differentiatesoverthe culture
period from a developmental stage at 10.4 days which is char
acterized by few blood elements to a stage that has a functioning
circulatory system by the end of the culture period. As a measure
of the effects of TPA on VYS cellular differentiation, we have
determined VYS hemoglobin content by absorbance at 414 nm.
Hemoglobin in the VYS had the typical visible absorption spec
trum of oxyhemoglobin. Table 1 shows the soluble protein and
hemoglobin content in VYS tissue at the start of treatment and
after a 26-hr treatment period to TPA or DMSO (control).
The VYS protein content at both time periods and in all
treatment groups was higher than in corresponding groups seen
in Chart 1. This increase is due to methodological differences in
tissue preparation and reflects VYS blood elements. After a 26hr TPA exposure period, there were no significant changes in
VYS protein content compared to those in DMSO controls (p >
0.05; Newman-Keul's test; Table 1).
The absolute (ng hemoglobin per yolk sac) and relative (per
centage of total soluble protein) hemoglobin content increased
dramatically over the 26-hr treatment period in control VYS
(Table 1). At the time of treatment, hemoglobin represented 7.6%
of total soluble VYS protein; but after 26 hr treatment with
DMSO, hemoglobin represented 21.1% of total soluble VYS
protein (Table 1). The absolute hemoglobin amount increased
150 -
125
126
Chart 1. Soluble protein content (d) and
ODC activity (8) in TPA- or DMSO-treated VYS
tissue were determined at the start of culture
(O) and after 8 and 28 hr in culture. The follow
ing were added 2 hr after the start of culture:
•,2.5 ,¿DMSO; A, 10 nw TPA; •50 nw TPA;
Ü,100 nm TPA. Points, mean of 8 to 13 deter
minations (from 3 to 5 separate experiments);
bars, S.E.; ", significantly different from DMSO
controls (0.01 <p<0.05,
Newman-Keul's test);
", significantly different from DMSO controls (p
< 0.01, Newman-Keul's test).
100
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75
76
50
60
25
26
28
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28
HOURS IN CULTURE
5554
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VOL. 43
TPA Effects on Macromolecular
Synthesis, Hemoglobin
and ODC Levels
Table1
Effects of TPA on soluble protein and hemoglobin content
VYS soluble protein and hemoglobin content were determined at the start of treatment and after 26 hr in
culture in the presence of DMSO (control) or TPA (50 and 100 nw).
TreatmentDMSO
after
treatment
(hr)0
contentProtein
content
sac)25.1
(Mg/yolk
±1.6" (4)6
of total soluble
protein7.6
hemoglobin/yolk
sac1.9
±0.3 (4)
137.5 ±5.8 (12)
(2.5 Ml/4 ml)
21.1 ±1.4(12)
2626
15.1 ±1.3(13)c
TPA (50 nw)
170.9 ±13.2 (13)
14.3 ±0.8 (6)cng
TPA(100nM)Time
156.2 ±4.8 (6)%
26Hemoglobin
a Mean ±S.E. from 2 to 5 separate experiments.
6 Numbers in parentheses, number of determinations.
c p < 0.01 ; Newman-Keul's test, compared to DMSO controls.
d 0.01 < p < 0.05; Newman-Keul's test, compared to DMSO controls.
±0.06 (4)
27.9±1.2 (11)
24.9 ±1.1 (12)
22.4 ±0.6 (6)"
B
DMSO
r
I IK ti
111
Ü
T
l
*
i.
TPA---
Hb
m
ce
O
co
co
14.4 21.5
31
45
67 93
14.4 21.5
31
45
6793
MOLECULAR WEIGHT (x 103)
Chart
(positive
graphics
hr in the
2. Densitometer scans of VYS soluble proteins on polyacrylamide slab gels in the presence of 0.6% sodium lauryl sulfate. Presented are the photographs
plates) of the Coomassie Brilliant Blue-stained gel above the densitometer scan. These scans were analyzed using an Apple II plus computer with attached
tablet digitizer. A, profile of VYS soluble proteins at the start of the treatment period (2 hr after the start of culture); B, profiles of VYS soluble proteins after 26
presence of DMSO (2.5 0I/4 ml culture media) or TPA (50 nM). Ho, hemoglobin.
from 1.9 nQ hemoglobin per VYS at the time of treatment to 27.9
/¿ghemoglobin per VYS after a 26-hr culture period in the
presence of DMSO.
In contrast to VYS protein content, TPA caused a dosedependent decline in hemoglobin content of the VYS (Table 1).
When expressed as percentage of total soluble protein, TPA
significantly depressed hemoglobin content to 71.6 and 67.8%
of DMSO control values at 50 and 100 nM, respectively. How
ever, when expressed as /¿ghemoglobin per VYS, only the 100
NOVEMBER
nM dose of TPA caused a significant decrease in hemoglobin
content. This discrepancy is due to opposite trends of TPA on
protein content and hemoglobin content of the VYS.
Polyacrylamide Gel Electrophoresis.
We have also directly
measured VYS hemoglobin content at the start of treatment and
after 26 hr in the presence of TPA (50 nM) or DMSO (2.5 ¿il)by
polyacrylamide slab gels with 0.6% sodium lauryl sulfate. The
globin band represented 4.0% of the total VYS soluble protein
area at the start of treatment (Chart 2A). After 26 hr DMSO
1983
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5555
S. E. Huber and N. A. Brown
treatment, the globin band increased to 14.7% of the total soluble
protein area (Chart 28), which is approximately the same as the
increase seen in Table 1. This globin band was depressed to
10% of total VYS soluble protein area (Chart 26) when the
conceptuses were treated for 26 hr with 50 nM TPA. This
represents a 32% decrease from 26-hr DMSO control levels and
is consistent with the decrease seen in Table 1. Importantly,
except for this depression in the hemoglobin band, the banding
pattern of the soluble proteins in TPA-treated VYS was qualita
tively and quantitatively identical to that of DMSO controls. Both
treatments produced identical changes in the protein banding
pattern compared to the pattern seen at the start of the treatment
period. For example, there is the appearance of a new protein
with a molecular weight of approximately 30,000 in TPA- and
DMSO-treated VYS that is not found to any great extent before
treatment (Chart 26; see starred arrow).
Chart 3 shows the autoradiograph of the Coomassie-stained
polyacrylamide gel described in Chart 2. Whereas Chart 2 iden
tifies total VYS soluble proteins, Chart 3 identifies only those
proteins synthesized over the 26-hr treatment period by [14C]leucine incorporation. Interestingly, TPA treatment (50 nw)
caused a 1.5 times greater incorporation of leucine into acidinsoluble cellular pools than did DMSO. This same approximate
increase is observed in the total exposed area on the autoradi
ograph being 1.3 times greater in TPA-treated VYS than in
corresponding DMSO-treated samples (Chart 3). The hemoglo
bin band represents 17.9 and 15.3% of the total exposed area
in DMSO- and TPA-treated VYS, respectively (Chart 3). The
absolute amount of hemoglobin synthesized, however, was iden
tical in both treatment groups, as measured by the area under
—DMSO
l
••TPA
l
LJJ
Ü
m
oc
O
Hb
l
15
l
l DMSO
^•1 TPA SOnM
Â¥<
¡i
TPA 100nM
«2
6
ÕS
3
2 HF
24 HR
Chart 4. Effects of DMSO (2.5 nl/4 ml culture media) and TPA on DNA, RNA,
and protein synthesis in the VYS at 2. 6. and 24 hr after treatment. Columns, mean
of 6 to 14 determinations (from 2 to 3 separate experiments); bars, S.E.; ',
significantly different from DMSO controls (0.01 < p < 0.05, Newman-Keul's test);
", significantly different from DMSO controls (p < 0.01, Newman-Keul's test).
the hemoglobin peak. This slight decrease in the relative amount
of hemoglobin synthesis but equal absolute amounts of hemo
globin is consistent with the data in Table 1 and Chart 2. Equally
important is the fact that, except for the slight relative depression
in hemoglobin synthesis, the autoradiographs of TPA- and
DMSO-treated VYS tissues are superimposable.
DNA Synthesis. We have measured DNA synthesis in TPAand DMSO-treated VYS tissue by determining [3H]thymidine
incorporation into acid-insoluble cellular pools. Preliminary ex
periments indicated that: (a) [3H]thymidine was linearly incorpo
rated into VYS acid-insoluble pools up to 4 hr. A 1-hr pulse was
routinely used in all other experiments; (b) 2.9 ±0.39% of the
acid-insoluble dpm were incorporated into the acid-insoluble,
alkaline-labile fraction (RNA fraction); (c) in all experiments, dpm
in the acid-soluble cell fraction confirmed that TPA had no
statistically significant effect on cellular uptake of [3H]thymidine.
[3H]Thymidine (12 ¿¿Ci;
specific activity of 50.0 Ci/mmol) was
routinely used in all experiments. To confirm that TPA did not
affect intracellular pool sizes of thymidine, experiments were
performed using excess cold thymidine as a carrier (12 ^Ci; 50.0
Ci/100mmol).
Treatment with TPA had no significant effect on the incorpo
ration of [3H]thymidine into VYS acid-insoluble pools at 6 and 24
hr after treatment compared with DMSO controls (p > 0.05;
Newman-Keul's test; Chart 4).
INCREASING
MOLECULAR
WEIGHT-»
Chart 3. Densitometer scans of autoradiographic plates of VYS soluble proteins.
Presented are the photographs (positive plates) and densitometer scans of the
autoradiographs obtained from the polyacrylamide gels shown in Chart 2. These
scans were analyzed using an Apple II plus computer with attached graphics tablet
digitizer. Ho. hemoglobin.
5556
RNA Synthesis. We have measured RNA synthesis in TPAand DMSO-treated VYS tissue by determining [3H]uridine incor
poration into acid-insoluble cellular pools. Preliminary experi
ments indicated that: (a) [3H]uridine was linearly incorporated
into VYS acid-insoluble pools up to 3.5 hr. A 1-hr pulse was
routinely used in all other experiments; (b) only 3.6 ±0.8% of
the acid-insoluble dpm were incorporated into the acid-insoluble,
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VOL. 43
TRA Effects on Macromolecular
alkaline-stable fraction (DNA and protein fraction); (c) in all ex
periments, dpm in the acid-soluble cell fraction confirmed that
TPA had no statistically significant effect on cellular uptake of
[3H]uridine.
The effect of DMSO and TPA on the incorporation of [3H]uridine into VYS acid-insoluble cellular pools is shown in Chart
4. At 2 and 6 hr after TPA treatment, there was no change in
uridine incorporation compared with DMSO controls (p > 0.05;
Newman-Keul's test). At 24 hr, incorporation was depressed in
conceptuses treated with 100 nw TPA.
Protein Synthesis. Protein synthesis was determined in TPAand DMSO-treated VYS by measuring the incorporation of [3H]leucine into acid-insoluble cellular pools. Preliminary experiments
indicated that: (a) [3H]leucine was linearly incorporated into VYS
acid-insoluble pools for at least 4 hr. A 1-hr pulse was routinely
used in all other experiments; (o) dpm in the acid-soluble cell
fraction confirmed that TPA had no statistically significant effect
on cellular uptake of [3H]leucine.
After a 2-hr treatment period with 100 nw TPA, there was a
significant increase in [3H]leucine incorporation into acid-insolu
ble pools compared to DMSO controls (Chart 4). By 6 hr, both
50 and 100 nw TPA caused an increase in incorporation, which
returned to control values by 24 hr (Chart 4).
Since a major biological function of the VYS at this time in
development is the secretion of proteins, we investigated
whether the increase in dpm into acid-insoluble pools after [3H]leucine treatment could be explained by an inhibition of secretion
from the VYS. In addition, we have indicated in the accompanying
paper (23) that the effects of TPA on the VYS suggest an
alteration on the cell surface. Any TPA-mediated change in the
secretory function of the VYS might support this suggestion.
The time course for secretion of labeled proteins from TPA (50
nw)- and DMSO (2.5 ¿<l/4
ml culture medium)-treated VYS is seen
in Chart 5. The data indicate that TPA does not affect the
secretory function of the VYS during the 18-hr experimental
period.
TIME (MIN)
Chart 5. Effects of TPA (50 MM)(•)and DMSO (2.5 ^1/4 ml culture media) (•)
on the secretion of proteins from the VYS. Points, mean of 3 to 5 determinations
(from 2 separate experiments); oars, S.E. There was no statistical difference
between corresponding DMSO and TPA points (p > 0.1. f test).
NOVEMBER
Synthesis, Hemoglobin and ODC Levels
DISCUSSION
The in vivo and cell culture effects produced by TPA have
been divided into 3 major categories: mimicry of neoplastic cells;
modulation of differentiation; and finally, cell membrane and cell
surface alterations (58).
When applied to mouse epidermis or cultured cells, TPA
induces many phenotypic changes which are associated with
the transformed state (for review, see Refs. 10 and 58). Included
in this series of changes is the induction of ODC with the
subsequent increase in polyamine levels and cellular proliferation
(42, 44). Slaga et al. (56) have suggested that TPA-mediated
increases in ODC activity and cellular hyperplasia are related to
Stage II of promotion, whereas others suggest that hyperplasia
may be necessary at only a very early substage of promotion
and, presumably, late stage promotion lacks this requirement
(10). Reports have indicated, however, that promoter-mediated
cellular hyperplasia and ODC induction could be independent
rather than related events (6,10, 43). Other investigations ques
tion the hypothesized essential role of either hyperplasia (11, 33,
36) or ODC induction (25, 63) in tumor promotion entirely.
Glucocorticoids, which inhibit tumor promotion, fail to inhibit the
TPA-mediated induction of ODC (63). These phenomena can be
explained by a nonessential role of ODC in tumor promotion or,
alternatively, by glucocorticoids altering an essential promotional
event which is unrelated to and independent of ODC induction
(10). Huberman ef al. (25) have also indicated that promoterinduced changes in polyamine levels can occur without altera
tions in ODC activity, suggesting the existence of an alternative
polyamine-biosynthetic pathway(s).
The data in accompanying paper (23) suggested that the
effects of TPA on the VYS were not the result of general cellular
toxicity or an overall proliferative response. The data presented
in this paper further support this hypothesis and indicate that the
effects of TPA on the VYS are not the result of cellular hyperpla
sia or ODC induction. Contrary to reported TPA-mediated induc
tion of ODC activity in other systems (5, 6, 42, 43), VYS ODC
levels were initially depressed by TPA treatment. In addition,
TPA had no effect on [3H]thymidine incorporation in the VYS.
The VYS, however, is a rapidly dividing tissue with high basal
ODC levels; therefore, TPA treatment may not increase further
ODC activity or proliferation. We do not at this time have an
explanation for the initial decrease in ODC levels with TPA
treatment.
The second category of TPA mechanisms involves alterations
in normal cell differentiation. TPA has been shown to stimulate
(23, 37, 49), inhibit (13, 15, 26, 35, 47) or alter (48) terminal
differentiation of certain cell lines in culture. As an example, TPA
inhibited the spontaneous differentiation of Friend erythroleukemia cells as measured by benzidine-positive cells and hemoglobin
content (47). How this disruption of differentiation in cultured
cells relates to differentiation and promotion in an intact organism
is not known.
Our preliminary observations (23) suggested that TPA did not
qualitatively inhibit the cellular differentiation of erythroblastic or
endothelial cells of the VYS. The current observations show that
TPA had a slight inhibitory effect on hemoglobin content of the
VYS. This effect is best described as a relative effect on cellular
hemoglobin concentration rather than an effect on the absolute
amount of hemoglobin synthesized. This effect, however, was
1983
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5557
S. E. Huber and N. A. Brown
not as pronounced or substantial as seen in other systems (14,
47). Furthermore, total soluble VYS proteins and soluble proteins
being synthesized in TPA-treated VYS are qualitatively identical
to DMSO controls. Except for the slight decrease in the relative
amount of hemoglobin, we conclude that an alteration in normal
cellular differentiation is not a major effect of TPA on the VYS. It
should be pointed out, however, that the 28-hr treatment period
in our culture technique is less than certain cell culture experi
ments that report a positive TPA effect on altered cellular differ
entiation (47). In addition, DMSO has been reported to stimulate
the differentiation of Friend erythroleukemia cells by the induction
of hemoglobin synthesis (17, 28). The concentrations necessary
for such an effect, however, are 16 to 32 times greater than are
DMSO vehicle concentrations in our experiments; therefore, we
do not believe that this alters our interpretations and conclusions.
The third category of TPA effects in cell culture and in vivo
suggests that the cell membrane may be the initial and major
target of TPA action. Alterations in cell-cell orientation (53),
adhesion (61), membrane (3, 11, 24) and extracellular matrix
components (27,59,60); increased permeability of tight junctions
(45) and membrane fluidity (16); and interactions with growth
factor binding (30, 31) and other surface receptors (18) are
several of the cell surface effects of TPA. Most importantly,
intercellular communication, whereby normal cells have the ca
pacity to exchange chemical or electrical signals, is inhibited by
TPA exposure (40, 57, 62).
The TPA-mediated separation of the VYS layers, described in
the accompanying paper (23), is consistent with a membrane- or
surface-mediated alteration in cell communication and/or adhe
sion. This effect may be similar to the apparent opening of
intercellular spaces in TPA-treated mouse skin (46). However,
one measure of VYS cell surface function, protein secretion, was
not affected by TPA treatment.
It is interesting to note that changes in the cell surface,
mediated via regulatory proteases, may alter the exchange of
information between cells (4). Blood vessel endothelial cells
produce 2 proteases, collagenase and plasminogen activator
(39). During neovascularization, these proteases are capable of
splitting the basement membrane in the area of the developing
vessel (1, 39). TPA has been shown to stimulate this protease
production in rabbit, bovine, and human endothelial cells (32, 34,
39). Thus, overproduction of proteases may cause splitting of
the cell layers of the yolk sac, preventing the controlled growth
of vessels between them. Consistent with this hypothesis are
the data indicating a TPA-mediated increase in protein synthesis.
Contrary to what one would predict, however, RNA synthesis
did not increase prior or during the observed effect on protein
synthesis. This suggests either a posttranscriptional effect of
TPA on protein synthesis or that the RNA synthesis assay was
too insensitive or variable to detect an increase in mRNA synthe
sis over total cellular RNA pools.
In conclusion, it appears that the tumor promoter-induced
disruption of morphology and function of the rat embyronic
visceral yolk sac was due neither to a cellular proliferative re
sponse nor to ODC induction. Alterations in cellular differentia
tion, quantitatively assessed by hemoglobin content and synthe
sis, were also not a major consequence of TPA treatment. A
TPA-mediated effect(s) on the yolk sac cell surface altering cellcell adhesion and/or communication may best explain the actions
of TPA. This hypothesis is currently under investigation.
5558
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5559
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12-O-Tetradecanoylphorbol-13-acetate Actions on
Macromolecular Synthesis, Ornithine Decarboxylase, and
Cellular Differentiation of the Rat Embryonic Visceral Yolk Sac
in Culture
Brian E. Huber and Nigel A. Brown
Cancer Res 1983;43:5552-5559.
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