ICANCER RESEARCH 36, 2636-2643, July 1976]
Epidermal Growth Control Mechanisms, Hyperplasia, and Tumor
Promotion in the Skin1
Friedrich Marks
Institute of Biochemistry, German Cancer Research Center (Deutsches Krebsforschungszentrum),
Summary
In permanently renewing epithelia such as epidermis, the
steady state between cell loss and cell gain is thought to be
controlled
by tissue-own and tissue-specific
inhibitors of
cell proliferation
(chalones). In epidermis two chalone-like
factors have been found, a G chalone retarding the entry of
cells into the phase of DNA replication and a G2 chalone
inhibiting the transition of cells from G2 into mitosis. Both
factors have been isolated from pigskin; they are probably
glycoproteins.
In contrast to the G2 chalone, the G chalone
is resistant to heat and proteolytic
enzymes and is most
probably produced in keratinizing cells. From the purified
D-69 Heidelberg, Germany
The concept of dedifferentiation
as the driving force of
tumor promotion is supported by investigations of effects of
hyperplasiogenic
and promoting agents on the epidermal
adenosine
cyclic
3' :5'-monophosphate
(cyclic
AMP)
sys
tem. The observations are not consistent with the assump
tion that epidemmal cell proliferation
is triggered by a fall in
the epidemmal level of cyclic AMP but favor the idea that
cyclic AMP is involved in the expression of epidenmal func
tion. Provided that this is correct certain effects of different
phorbol esters on epidemmal cyclic AMP may be directly
correlated
to the “dediffementiating― and promoting
activity
of the agent.
G chalone a dose of 0.05 to 0.1 @tg
injected i.p. into an adult
mouse is sufficient to depress epidermal DNA synthesis to
about 50%.
Studies on the response of mouse epidermis to mitogenic
stimuli lead to the conclusion that the tissue can react in
two
different
ways:
1. If the stimulus does not involve severe injury, only the
flow of cell through the steady state of tissue homeostasis is
accelerated (e.g. , after gentle skin massage on chemical
depilation). In this case hyperplasia is not observed or de
velops only slowly if the stimulus lasts for a longer period of
time (functional hyperplasia, e.g. , at body sites that suffer a
strong wear and tear). Under these conditions
the G
chalone mechanism is not disturbed.
2. The automatic response to any kind of severe injury
Introduction
The great majority of malignant tumors arise from epithe
ha. Despite this and the fact that epidermis is probably the
tissue most widely used in experimental
cancer research,
our
knowledge
of the
molecular
processes
that
underlie
epithelial cell proliferation and differentiation as well as of
the proper control mechanisms is very limited. For obvious
reasons growth control mechanisms at present are mainly
studied with tissue cultures, mostly of mesenchymal or
reticuloendothelial
origin.
There
is, however,
reason
to be
lieve that the mechanisms working in vitro are (in a quanti
tative and qualitative sense) different from those in the
living animal. This may be especially true for highly orga
(e.g. , wounding, removal of the horny layer, treatment with
mitogens such as the tumor-promoting
phorbol esters or
acetic acid) is not only compensatory
growth but also a
nized tissues such as the stratified epithelia, the regulatory
functions of which probably depend rather strictly on their
transient
vestigation
loss
of susceptibility
to G
chalone
which
is fol
lowed by a sudden development of a pronounced and long
lasting hyperplasia (hyperplastic
transformation).
In many
aspects hyperplastically
transformed
epidermis resembles
neonatal tissue.
In analogy to the hemopoietic system, it is proposed that
the proliferative compartment
of epidermis consists of dif
ferent types of stem cells and that only already maturating
stem cells are able to respond to G chalone. During the
hyperplastic transformation
this stem cell pool is depopu
lated due to either hasty keratinization (e.g. , after removal
of the horny laryer) or dedifferentiation
(as is discussed for
the mechanism of action of tumor promoters). The result is
a transient chalone-insensitive
cell proliferation.
morphological
I Presented
at
the
Conference
“
Early
Lesions
and
the
Development
of
2636
Therefore,
we believe that the in
mechanisms
of epithelial
lifenation in the whole animal (although
cell pro
it may be a “dirty―
system for many biochemists)
is an urgent demand,
which
will help lead to a proper
understanding
of neoplastic
dis
eases.
Epithelia such as epidermis suffer permanent wear and
tear. This results in a continuous loss of cells. Since this is,
at least under normal conditions, precisely matched by the
formation
of new cells, there must be a stringent
the mates of cell proliferation
and maturation
control
which
allows
of
a
perfect feedback between cell loss and cell gain. The con
trol of epidenmal cell proliferation and differentiation is
brought about by a complex pattern of interacting mecha
nisms which is still far from being understood in detail.
Several
Epithelial Cancer,―October 21 to 23, 1975, Bethesda, Md. This work was
supported by the Deutsche Forschungsgemeinschaft.
integrity.
of the control
hormones
such
as estrogens
and androgens,
corti
costemoids, and catecholamines are involved. Furthermore,
the underlying mesenchymal tissue as well as nutritional
factors such as vitamin A may play an important role. How
CANCER
RESEARCH
VOL. 36
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Epidermal Growth Control Mechanisms
even, all of these more or less ‘
‘exogenous'
‘
influences may
have only a modulatory effect on tissue-own control mecha
nisms. According to the theory of Weiss and Kavanau (48), a
tissue is a self-regulatory system that controls the steady
state between cell loss and cell gain by an interplay of
stimulatory and inhibitory factors, which are thought to be
produced by the tissue itself. These might be, for example,
the cyclic nudleotides and the prostaglandins. Both types of
substances are known to be synthesized by epidermis; and
there is some evidence that cyclic AMP2 and prostaglandin
E2 inhibit cell proliferation and promote cell maturation,
whereas cyclic GMP and prostaglandin F21,exert the oppo
site effects. However, the biological activities of cyclic nu
cleotides and prostaglandins are not tissue specific; in
stead, they are thought to be the “2nd
messengers―of
external on local effectors.
In the last 10 years the concept
of tissue-own
and tissue
specific inhibitors of epidermal cell proliferation has gained
increasing interest and support. These “chalones―
(6) are
thought to be intercellular carriers of information within a
feedback circuit between maturating tissue cells (where
they are assumed to be synthesized) and the proliferative
population (where they exert their effect) (22, 23). The chal
one concept still offers by fan the most simple and elegant
explanation of tissue homeostasis. Therefore, its proof or
disproof should be worth the effort. This can be done only
by the demonstration and isolation of chalone-like factors.
concept
was originally
developed
to
explain certain observations on wounded epidermis (8) and
skin treated with carcinogenic hydrocarbons (22, 23, 25,
26),
epidermis
may
be considered
to be the
“classical―
tissue of chalone research.
Ten years ago it was known that aqueous extracts from
skin and epidermis are able to inhibit epidemmal mitosis in
vitro and in vivo (9, 24). In vitro the inhibitory effect was
augmented by adrenalin and cortisol (10). It was probably
caused by a factor (“epidemmal
chalone―)which could be
inactivated by proteases and heat (7). In 1968 Hondius
Boldingh and Laurence (21) reported on the isolation of the
inhibitor
highly purified
in our laboratory.
The starting
material
was
the same pigskin extract used before for the isolation of
epidermal G2chalone (21). As a test of its inhibitory activity,
it was dissolved
in 0.9% NaCI solution
and injected
i.p. into
female NMRI albino mice (7 to 8 weeks old). From 12 to 15
hr later, when the inhibitory effect has reached its maximum
(32), 35
@Ciof Imethyl@:IH1thymidine
were injected
i.p. After
45 mm the animals were killed. Five mm prior to sacrifice.
the hair of the back was removed
by means of a cosmetic
depilator (Pilca Creme; Olivin, Wiesbaden, Germany). Im
mediately after the mice were killed, the depilated skin was
dissected and immersed in ice-cold 0.8 M perchlomic acid.
The epidermis
was scraped off with a scalpel (it could also
be separated from the dermis by the acetic acid procedure).
After homogenization the specific radioactivity of the DNA
was measured (32). Most of the G chalone activity was
found in the 55 to 72% ethanol precipitate
of aqueous
pigskin extract. It could be destroyed neither by heating the
Epidermal Chalones
Since the chalone
munication)] is due to a delay of the G1-S transition rather
than to an inhibition of DNA synthesis itself or to a decrease
of pool labeling. Although the inhibitor in question is also
not species specific and again entirely restricted to keratin
izing epithelia (37), it has been shown not to be identical
with the antimitotic chalone isolated previously (14, 31).
Thus, we have now 2 epidermal chalones, a G chalone
retarding the entry of cells into the S phase3 and a G2 chal
one preventing the transition from G2 into M phase. Similar
observations have been made in other tissues.
In the past 5 years the epidermal G chalone has been
which was preliminarily
characterized
as a glyco
neutral solution to 100°(for 1 hm)nor by denaturing agents
such as phenol, urea, 1 M formic acid (70°),or ionic deter
gents (100°).It was furthermore completely resistant to tryp
sin , Pronase, collagenase , RNase, DNase, fi-neuramin Idase, and hyalunonidase digestion, whereas complete mac
tivation was achieved with either 0.1 M sodium hydroxide on
1 M hydrochloric
acid at room temperature
(6 hm). In pigskin
extract the factor occurs in a highly aggregated state simu
lating a molecular
weight of several hundred thousand
dal
tons. Due to this complication, the starting material had to
be disaggregated prior to any attempt at purification. This
was achieved by treatment with dithiothreitol and sodium
dodecyl sulfate and afterwards by blocking free sulfhydryl
groups by alkylation with iodoacetamide. The material was
pre
protein with a molecular weight between 2.5 and 4 x 10@ then fractionated by Pronase digestion, ultrafiltration,
daltons. It was exclusively found in and exerted its effect
cipitation with cetyl pymidinium chloride, and phenol extmac
tion. By this procedure, which has been recently described
only on epidermis and closely related kematinizing epithelia
(30, 37). However,
despite this remarkable
success doubts
in more detail (33), an approximately 800,000-fold enrich
skin
were still raised as to the validity of the whole concept. The ment of the G1 chalone over the crude lyophilized
main reason for scepticism was the fact that the ‘
‘epidemmal extract was achieved. A dose level as small as 0.05 to 0.1 @tg
chalone―turned out to be a pure inhibitor of mitosis without
of the purified factor was sufficient to depress DNA labeling
any effect on DNA synthesis (1, 42). However, in 1969 (20) it in mouse epidermis in vivo to 50% and more (33). This
was shown that epidemmalor skin extracts did in fact inhibit corresponds to 2 to 4 j.@g/kg body weight.
the incorporation of labeled thymidine into epidenmal DNA
Such a high biological activity (which is in the mangeof
when injected into mice. The inhibition occurred after a lag that of a hormone), together with tissue specificity and the
phase of several hr (20, 32) and [as demonstrated later by
autoradiogmaphy (Refs. 17 and 37; K. Elgjo, personal com
3 That
2 The
abbreviations
used
are:
cyclic
AMP,
adenosine
cyclic
3'
:5-mono
phosphate; cyclic GMP, guanosmnecyclic 3' :5'-monophosphate; TPA, 12-0tetradecanoyl-phorbol-1 3-acetate; PDB, phorbol-1 2, 13-dibenzoate.
epidermal
G,
chalone
actually
inhibits
cell
proliferation
(and
not
pool labeling. etc.) has been unequivocally demonstrated by the observation
that the depression of thymidine incorporation into epidermal DNA seen
after injection of purified G, chalone is followed by a corresponding depres
sion of mitotic activity (see Footnote 5).
JULY 1976
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2637
F. Marks
observation that embryonic, neonatal, and hyperplastic epi
demmises do not respond to the inhibitor in vivo and in vitro
Epidermal Hyperplasia, Tumor Promotion, and Chalone
Mechanism
(see below) rule out the possibility that the observed inhibi
tion was the result of unspecific
cytotoxic
effects.
During
the purification procedure, almost the whole inhibitory ad
tivity ofthe crude extract was retained, indicating that there
is only 1 inhibitor of DNA synthesis in skin. Inhibitory effects
measured with crude skin or epidermis extracts may, there
fore, be attributed with a certain degree of confidence to the
epidenmal G chalone.
However, this holds true only when
the effect is measured in vivo and with rather low dose
levels, whereas under in vitro conditions
there is always the
danger of being misled by cytotoxic side effects or the
dilution
of the
labeled
precursor
pool
by unlabeled
thymi
dine and its derivatives, etc.
The purified chalone from pigskin has some peculiar
properties. Although, according to a preliminary analysis, it
is a (glyco-)peptide which can be extracted with phenol, it is
completely resistant to proteolytic digestion. This might be
The successful demonstration of an epidermal chalone
may be taken as a proof for the existence of a local feedback
regulation
of epidermal
cell proliferation.
Provided
this
mechanism controls the steady state between the rates of
cell death and cell renewal, any influence disturbing this
equilibrium
(such as wounding
on treatment
with mitogens)
should have a measurable effect on it. Furthermore, this
feedback system is expected to play an important mole in
the development of preneoplastic and neoplastic diseases.
Local treatment of mouse skin with tumor-promoting
phorbol
esters such as TPA or PDB induces
inflammation
and strong epidermal hyperplasia. Shortly after the appli
cation of the agent and prior to the development
of hyper
plasia, epidermis no longer responds to injections of G1
chalone (28). After a time interval that depends on the dose
level as well as on the type of the promoter (1 to 3 days), the
due to the protective
effect of carbohydrate
residues.
On
chalone mechanism is gradually reestablished (28). The
polyacrylamide gels it cannot be made visible with the usual
lack of response cannot be overcome by raising the chalone
staining
methods;
most probably
it is eluted
during
the
dose level or by altering the time interval between chalone
staining procedure, perhaps again due to its high content of
injection and pulse labeling of DNA. This indicates that the
carbohydrate (18. 33). Finally, it shows heterogeneity with
receptor mechanism for chalone has been transiently
respect
to the molecular
size with
apparent
molecular
switched off. There are many complicated but only 2 simple
weights ranging from less than 10@daltons to more than 1O@ explanations: (a) the mitogen interacts with the receptor
daltons (ultrafiltration,
gel chromatography),
even after ex
molecule, perhaps during an overall attack on the cell mem
tensive proteolytic digestion and in the presence of deter
brane; (b) similar to the hemopoietic system (29), epidermis
gents. This may indicate some kind of oligomemization and
has a heterogeneous stem cell population consisting of
homeopolar
aggregation , probably
via polysacchanide
bridges. In extracts from mouse epidermis most of the
chalone activity was found in a fraction between 10@and 10@
daltons (J. Schweizer and F. Marks, unpublished results).
Therefore, the molecular weight of the “monomer―
may be
in this range.
According
to the feedback hypothesis, the chalone is
expected
to be synthesized
by kematinizing
epidermal
cells.
This is indicated by several lines of evidence: (a) the factor
is found exclusively
keratinizing
and
separately,
in keratinizing
basal
cells
epithelia
were
separated
from
keratinizing
one was restricted
epidermis
of
keratinized,
cells
chick
lacked any inhibitory
extracted
G2 chal
from
is not
However,
yet
when
the tissue was cultivated in vitro in the presence of serum
the appearance
strated
of G, chalone
concomitantly
activity
could
with the development
be demon
of a horny
layer
(S. Bertsch and F. Marks, unpublished results); (d) extracts
from
a nonkeratinizing
inhibitory
epidermal
carcinoma
lacked
any
activity.4
On the whole. the epidermal G inhibitor fulfills all the
requirements
effect
of a chalone.
in minute
amounts,
phase of DNA replication,
cies specific),
Like a hormone
it retards
the entry
it displays
of cells
it is tissue specific
and it is produced
in the course
system only ‘
‘late―
(already maturating
but still proliferating) types of committed stem cells are
controlled by chalones, whereas the plunipotential stem
cells and the stem cells in the early stages of differentiation
communication).
which
activity.
in the hemopoietic
and
(16) whereas
embryos,
induces dedifferentiation of susceptible to unsusceptible
cells.
The 2nd assumption is supported by strong evidence that
do not respond to the inhibitor
to basal cells (15); (c) extracts
9-day-old
and chalone-unsusceptible
cells. The
preferentially the unsusceptible cells or
(37); (b) when
G chalone activity was found almost exclusively
in the extract
chalone-susceptible
mitogen stimulates
its
into the
(but not spe
(L. G. Lajtha, personal
In order to decide between the 2 possibilities mentioned
above, epidemmal cell proliferation was induced by pure
mechanical means.―For this purpose the horny layer of
depilated mouse back skin was removed by gentle rubbing
with cosmetic sandpaper (3), a method that resembles the
dermatologist's
techique of “stripping―
with adhesive tape.
After 15 hr, this treatment results in a sharp peak of DNA
labeling (3).5 This is followed by a mitotic wave and, subse
quently, by the development of a pronounced epidermal
hypemplasia which lasts for at least 5 days. At 9 hr after
stimulation, the treated epidermis no longer responded to
injections of G chalone; only after 48 hr was the respon
siveness reestablished. Again the lack of response could
not be overcome by increasing the chalone dose level or by
altering the time interval between chalone injection and
of epidermal
cell maturation.
5 Bertsch,
4 S.
2638
Bertsch
and
F.
Marks,
manuscript
in
preparation.
K.
Csontos,
J.
Schweizer,
and
F.
Marks.
Effect
of
Mechanical
Stimulation on Cell Proliferation in Mouse Epidermis and on Growth Regu
lation by Endogenous Factors (Chalones). Cell Tissue Kinet. , in press.
CANCERRESEARCHVOL. 36
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Epidermal Growth Control Mechanisms
DNA labeling.
These results cleanly demonstrate that the loss of chalone
responsiveness during the hypemplastic reaction is not nec
essarily due to an interaction of a chemical mitogen with a
receptor site. Furthermore, it is known that the primary
response of epidermis to the removal of the horny layer is a
hasty keratinization of cells; this occurs prior to the onset of
DNA synthesis
and leads to a depopulation
of the basal cell
layer due to an increased vertical cell migration (38). Those
cells are certainly the late stem cells, which under normal
conditions are still committed to proliferation and under
chalone control. Due to the stripping, the pool of these cells
nuns empty so that the regeneration of the tissue must now
transiently been taken over by ‘
‘early'
‘
stem cells that do not
respond to the inhibitor (Chart 1). This course of events is
quite similar to that assumed to take place in the hemo
poietic system, for example, after severe bleeding.
The cell proliferation induced by stripping still seems to
be responsiveto the epidemmalG2chalone (3).5 Thus. a 2nd
feedback circuit, perhaps between late and early committed
stem cells, may exist (Chart 2). This assumption would offer
an explanation for the abundance of G2 chalones in all
tissues thus far examined as well as for the localization of
@
(Th
@!@)-@@—?—-
B
NON-COMMITTED
STEMCELL
tion without
subsequent
hypemplasia is investigated.
Such
stimulation can be achieved, for example, by skin massage
on by chemical depilation. In both cases the mitotic rate
rises nearly 20-fold. and after skin massage the time course
and extent of the proliferative events (DNA synthesis, mito
early
‘late'
C0MMITrEDSTEMCELLS
MATURECELLS
(postmitotic)
Chart 1. A proposed scheme of epidermal cell differentiation and the
effect of G chalone. In analogy to the hemopoietic system only maturating
(late) stem cells are thought to be controlled by G chalone. The pool of these
chalone-sensitive stem cells may be depopulated by either hasty keratiniza
tion (Pathway A) or dedifferentiation (Pathway B). In both cases the conse
quences are the same, i.e. , induction of epidermal cell proliferation (of early
stem cells) which transiently is not under chalone control, and subsequent
development of hyperplasia (hyperplastic transformation). Whereas Pathway
A offers a simple explanation of the proliferative response seen, for example.
after removal of the horny layer. Pathway B may be involved in the process of
tumor promotion and epidermal metaplasia.
IGO'IALONEI
2
IGi
CHALb@J
®
epidermal G2 chalone in the basal cell layer (15).
It may be supposed that it is the ‘
‘
breakdown' ‘
of the G
chalone control that ultimately leads to hyperplasia. This is
supported when the stimulation of epidemmal cell prolifema
____
,@
“ear@j
NON-COMMITtED
@Iate@
COMMITTED
STEMCELLS
STEMCELL
MATURECELLS
( Postmitotic)
Chart 1. A hypothetical scheme explaining the different localization and
function of epidermal G, and epidermal G7chalones.
motion? First it must be stated that a hypemplastic transfon
mation with concomitant loss of chalone responsiveness is
sis) is quite similar to that seen after stripping . However, the
induced also by stimuli that apparently do not have a pro
responsiveness
moting effect, for example,
motion is a highly complex
of the tissue
to G chalone
is not lost nor is
any hyperplasia is developed.5
Finally, epidermis
which from its morphological
appear
different experimental parameters and is still fan from being
ance seems to be hypemplastic under normal conditions,
such as mouse tail epidermis. is nevertheless under chalone
control .@
From these results we may draw the conclusion that
epidermis responds to an external stimulus in 2 different
ways.
1 . If the stimulus
flow
of cells
through
is accelerated.
does not involve severe injury, only the
the steady
state
of tissue
In this case hyperplasia
homeostasis
is not observed
response
to any kind of severe
injury
is
not only compensatory growth but also a transient loss of
chalone susceptibility which is followed by a sudden devel
opment of hyperplasia. This is certainly not due to a simple
overshooting of tissue repair but is the consequence of a
true ‘
‘transformation'‘
(induced hypemplasia) probably in
dluding the activation of a more undifferentiated stem cell
population.
Are there any relationships between the hypemplastic
transformation of epidermis and the process of tumor pro
JULY 1976
entirely
understood.
It is quite
possible
that the induction
of
epidermal hyperplasia always has promoting consequences
if it is permanently repeated; however, in many cases the
promoting effect cannot be seen due, for example, to the
wrong timing of the experiment or because the “dormant―
tumor cells are killed by the hyperplasiogenic
treatment
before they can be promoted. On the other hand, it has
been postulated that tumor promotion is the consequence
or
develops only very slowly if the stimulus lasts for a longer
period of time (functional hyperplasia, for example, at body
sites that suffer a strong wear and tear such as the mouse
tail). Under these conditions the chalone mechanism is not
suspended.
2. The automatic
acetic acid (28). However, pro
process that depends on many
of a dedifferentiation
of epidenmal cells (27, 39), a concept
that is especially appealing. In this case one would assume
that late (chalone-susceptible) stem cells are transformed
into early (chalone-unsusceptible)
types. The result would
be the same as in the case of an accelerated maturation of
late stem cells, namely, a changeover of epidemmal cell
proliferation
from a chalone-susceptible
to a chalone-un
susceptible population and, consequently, the develop
ment of hyperplasia due to an emptying of the pool of late
chalone-regulated stem cells (Chart 1).
As long as we do not have proper biochemical parameters
of epidermal cell differentiation it is, of course, difficult to
decide between the 2 possibilities. However, observations
made with newborn mice may show a way to resolve the
problem (3, 5). Embryonic and neonatal mouse epidermis
show striking similarities to adult epidermis that has been
2639
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F. Marks
hyperplastically transformed. These analogies are not me
stnicted to morphological
features but concern also negula
tory functions.
Thus, the DNA synthesis in epidermis of
newborn animals in vivo on of embryonic mouse or chick
epidermis in vitro cannot be inhibited by epidermal G chal
one, but the chalone responsiveness is gradually developed
within
the 1st weeks postnatum
(3, 5). Furthermore,
we
never succeeded in inducing a wave of cell proliferation and
hyperplasia in neonatal mouse epidermis, e.g. , by stripping
on treatment with phombol esters or acetic acid (3, 27). From
these results one may conclude that neonatal epidermis is
in the state of hyperplasia
and insensitivity
to the action of
At present
the favored
concept
is that
the
relative
tissue
levels of cyclic AMP and cyclic GMP control the equilibrium
between proliferation and differentiation (46, 47). On the
whole, theme is no question that the cyclic nucleotides are
deeply involved in the regulation of tissue homeostasis in
epidermis.
In order to investigate possible relationships between
G chalone which in adult epidermis can be induced only by
cyclic AMP, hyperplastic
injury. Although insensitive to G1 chalone, neonatal epider
mis responds to G2 chalone (27). This again supports the
tion alterations of the cyclic AMP level in dorsal mouse
epidermis caused by a single local application of phonbol
hypothesis illustrated by Chart 2.
In light of the concept developed above, this would mean
that everyday tissue regeneration in neonatal epidermis is
brought about mainly by stem cells that do not respond to
chalone. Perhaps the whole tissue, which is cleanly not
fully developed, is still in a state of “noncommitment―
so
that any kind of cell maturation must be forced by exoge
nous factors such as corticosteroids (41) on epidenmal
esters were measured
growth
factor
matically
(4) instead
of
running
more
on less
as is the case of the mature tissue.
conditions the pool of chalone-contnolled
may be very small if it exists at all.
The results obtained
with newborn
auto
Under these
late stem cells
animals
transformation
and tumor promo
in vivo (19). For this purpose
the
animals were killed at different time intervals after the treat
ment. The shaved back skin was dissected without delay
and wetted thoroughly with tnichloroacetic acid. Then the
epidermis was immediately scraped off and homogenized in
5% tnichlomoacetic acid. The cyclic AMP content of the
homogenate was measured by means of the protein binding
method (34). The results of these experiments are shown in
Chart 3.
Immediately after application of the strong promoter TPA,
the cyclic AMP level begins to rise reaching the 1st (“early―)
peak after 1 to 2 hr. Then it declines to a deep depression
with a minimum of 10% of the control level at 18 hr (after 20
clearly mdi
date that chalones are not absolutely necessary for main
nmoles of TPA). At about 20 hr the level begins to increase
taming
again, rapidly reaching a broad peak (700% of the control
after 20 nmoles TPA) between 28 and 36 hr. Only after about
60 hr is the control level reached again.
Both the early and the late elevation can be reduced by
the injection of an c@-adrenergicblocker. After application of
the weak promoter PDB, neither the early peak nor the
depression is observed. This indicates that these alterations
growth
conditio
control
in epidermis
but
that
sine qua non for the maintenance
they
are
a
of the adult
state. The breakdown of this state and its transition into a
“pseudoembryonic'
‘
state as induced by promoting agents
plays certainly a central role in the process of tumor promo
tion and in the development of preneoplastic alterations,
since
it may be the only
way in which
the new
program
of
tissue differentiation called “cancer―
can be realized. It may
be recalled in this connection that, in the adult organism,
epidemmal metaplasia, which is a change of the commitment
of the tissue (for example the formation of interfollicular
epidermis from hair roots), is observed only after injury.
There is certainly no reason to deny that neoplastic develop
ment is a special type of a metaplastic reaction.
Epidermal Hyperplasia, Tumor Promotion, and Cyclic AMP
@
ment with phombol esters (2) have led to the idea that the
nucleotide plays an important mole in epidermal growth
regulation. Meanwhile evidence indicates that cyclic AMP
enhances epidermal cell maturation (11), with growth inhi
bition being a more indirect consequence of this effect (46).
Catecholamines such as adrenalin or isoproterenol are
strong inhibitors of epidermal cell proliferation. Originally,
adrenalin was regarded as a cofactom of epidermal G2chal
one (9), but this could not be confirmed. There is, however,
evidence that the antimitotic effect of the chalone depends
on a certain level of cyclic AMP in the target cell and that
this level can be adjusted by catecholamines (13). It has
been shown (12, 36) that adenyl cyclase of normal adult
epidermis
is coupled
to a /3-adnenengic receptor
cyclic AMP in higher concentrations
inhibitor
of the G2-M transition
and that
is by itself a potent
in vitro (35, 45).
These results and observations that the cyclic AMP level
seems to be decreased in rapidly proliferating epidermis
such as in psoniatic skin (44, 46) or in epidermis after treat
2640
of the cyclic AMP level have nothing
to do with the induc
tion of cell proliferation, since in the dose applied PDB is a
strong hyperplà siogen. The late elevation after 480 nmoles
PDB was 350% of the control (as high as after 2 nmoles
TPA). These results demonstrate the extraordinarily high
activity of the strong
promoter
TPA in this experiment.
Injection of catecholamines such as isopmoterenol cause
an approximately 10-fold increase of epidenmal cyclic AMP,
and this stimulation can be prevented by f.@-adrenergic
blockers (34). From 1 to 2 hr after application of TPA, this
effect can no longer be observed; however, between 2 and 7
days after treatment it is gradually reestablished (19). The
abolition of the f3-adrenergic elevation of cyclic AMP de
pends not only on the TPA concentration but also on the
type of the phorbol ester showing a direct correlation with
the promoting activity (TPA > phonbol didecanoate >
phorbol dibenzoate
4-0-methyl phorbol didecanoate).
Interestingly, the effect is also seen after treatment of
mouse skin with 3,4-benzpyrene (43). Thus, the promoting
effect of a phonbol ester seems to be related to its ability
(a) to depress
the responsiveness
of epidermal
adenyl
cyclase to /3-adnenergic agonists and (b) to depress after
an initial elevation the basal level of cyclic AMP.
Provided that cyclic AMP is involved in epidenmal cell
CANCER RESEARCHVOL. 36
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1976 American Association for Cancer Research.
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2641
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1976 American Association for Cancer Research.
F. Marks
differentiation,
the observations
fit very
well
with
the
promotion. One may assume that the early cyclic AMP peak
is an expression of the transformation
of cells into a less
differentiated
state, which is afterwards reflected by the
subsequent depression of the cyclic AMP level, whereas the
late elevation is correlated to the medifferentiation of the
tissue. This assumption is supported by several lines of
evidence: (a) both peaks of cyclic AMP obviously coincide
with 2 peaks of histone phosphorylation
(40). According
current concepts this may be regarded as an expression
to
of
gene activation (involving cyclic AMP-dependent protein
kinases), i.e. during dediffementiation and mediffementiation;
(b) tumor promotion can be inhibited by manipulations that
keep the level of cyclic AMP in epidermis high, i.e., manipu
lations that prevent the TPA-induced depression (2); (c) the
late cyclic AMP peak coincides with the onset of reactions
related to epidermal differentiation such as the synthesis of
histidine-mich
protein
and the reestablishment
receptor
AMP (from
mechanism)
a /3-adnenengic
sion of dediffenentiation.
Experiments to prove whether or
not the a-adrenemgic mechanism
is related to the neonatal
12.
13.
14.
The effect on the (membrane-bound)
interpreted
as resulting
/3 receptor
from
an attack
may be
of the
promoter on the cell membrane, during which the receptor
lipid-adenyl cyclase complex is destroyed, perhaps due to
the surface activity of the phorbol ester. However, this can
explain neither the lack of an effect of 4-0-methyl phorbol
didecanoate (which is expected to be surface active) non the
(19) or benzo(a)pyrene
(43), which have
been shown to be as powerful in inhibiting the fJ-adrenemgic
mechanism as is TPA. Furthermore, it is difficult to under
stand the relatively long lag phase of 1 to 2 hr between the
application
of the promoter and the breakdown of the f3
receptor mechanism.
In summary, themeis indeed a body of admittedly indirect
evidence supporting the concept of dediffementiation as the
driving force for tumor promotion; however, we must col
lect considerably
more experimental
data before we can
draw final conclusions.
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2643
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1976 American Association for Cancer Research.
Epidermal Growth Control Mechanisms, Hyperplasia, and
Tumor Promotion in the Skin
Friedrich Marks
Cancer Res 1976;36:2636-2643.
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