AM. ZOOLOGIST, 12:137-149 (1972).
Chalones and the Control of Normal, Regenerative, and Neoplastic
Growth of the Skin
THOMAS S. ARGYRIS
Department of Biology, Syracuse University, Syracuse, New York 13210
SYNOPSIS. Chalones, inhibitors of cell division have been isolated and studied from a
number of mammalian tissues, most notably, the epidermis. The epidermal chalone is
a glycoprotein. It exhibits considerable, but not complete specificity.
The epidermal chalone decreases mitotic activity by inhibiting cells in the G-2 phase
of the cell cycle from entering mitosis, and probably also by inhibiting cells in the
G-l phase of the cell cycle from entering mitosis. To inhibit cells in G-2 from entering
mitosis the chalone requires adrenalin, and for maximal activity, hydrocordsone. It is
not known if adrenalin and hydrocortisone are required for chalone inhibition of cells
in G-l.
In addition to inhibiting cell division in normal epidermal cells, the epidermal
chalone can inhibit cell division in regenerating epidermal cells induced to proliferate
by chemical damage. The phase of the cell cycle in which the chalone inhibits
regenerating epidermal cells from entering mitosis is not known.
Epidermal tumors contain a decreased amount of chalone. Mitosis in epidermal
tumors is inhibited by treatment with epidermal chalone. Tumor cells are inhibited
from entering mitosis from either the G-l or G-2 phases of the cell cycle.
Chalones are said to inhibit mitosis by a negative feedback mechanism. However,
experiments which presumably result in a decrease in chalone concentration do not
result in an increase in mitotic activity. It is suggested that if chalones are physiological controllers of cell division they do not act by a simple negative feedback
mechanism, but require the action of a substance to decrease their concentration.
THE
ISOLATION OF CHALONES FROM
MAMMALIAN TISSUES
One of the unsolved problems of biology
is the mechanism by which growth is controlled in adult mammalian tissues and organs. Basically, investigators have suggested that the release of stimulating substances, loss of inhibitors, or the interplay
of both control growth (Abercrombie,
1957, 1965; Argyris, 1968; Bullough, 1964,
1965; Goss, 1964).
Hard experimental evidence had been
difficult to muster for any of the above
speculations, until Bullough and Laurence
(1960) presented evidence that in the
wounds of mice the local loss of tissue mass
results in the loss of an inhibitor, which
in turns stimulates epidermal mitotic activity. Furthermore, they suggested that the
inhibitor is probably tissue specific, and is
Author's research supported by NSF Grant GB12554.
The author wishes to thank Mr. Steve Mueller
and Mr. Larry DeYoung for critical reading of this
manuscript.
137
also involved in the control of normal epidermal mitotic activity. They called this
inhibitor the epidermal chalone (Bullough, 1962), and predicted that chalones
are critical in the control of mitotic proliferation in all tissues.
If the loss of an inhibitor or chalone is
responsible for the stimulation of epidermal mitotic activity in wound healing, and
if the chalone is involved in the control of
normal levels of epidermal mitotic activity, then the chalone should be extractable
from normal epidermis. Moreover, when
injected into an animal the chalone should
inhibit epidermal mitotic activity. Bullough and Laurence (1964) prepared an
aqueous homogenate of mouse epidermis,
and tested it for its ability to inhibit mitosis in pieces of mouse ears in vitro. Table 1
presents results of a typical experiment.
Clearly, the epidermal extract inhibits epidermal mitotic activity dramatically.
Moreover, the chalone appears to show
considerable specificity, since extracts of
other organs, such as kidney, liver, lung,
and brain, do not inhibit epidermal mitot-
138
THOMAS S. ARGYRIS
TABLE 1. Inhibition of epidermal mitotic activity in vitro by epidermal extracts.
Approx. dry wt macerated epidermis or its equivalent
aqueous extract/4 ml medium
Total macerate
Filtrate
Supernatant
0
0.07 mg
0.14 mg
0.7 mg
1.4 mg
7.0 mg
14.0 mg
5.1 ± 0.40
5.2 ± 0.41
5.4 ± 0.25
4.0 ± 0.45
4.9 ± 0.66
3.6 ± 0.17
3.0 ± 0.24
3.2 ± 0.43
3.2 ± 0.45
2.8 ± 0.25
3.3 ± 0.47
2.4 ± 0.34
2.1 ± 0.40
2.5 ± 0.44
2.1 ± 0.55
1.7 ± 0.17
2.3 ± 0.37
1.2 ± 0.22
1.4 ± 0.29
1.4 ± 0.28
(From Bullough and Laurence, 1964.)
ic activity in vitro (Table 2) . However,
the epidermal chalone inhibits mitosis in
vitro in epidermis, cornea, and esophagus,
but does not, at the dosage used, inhibit
mitotic activity in intestinal epithelium.
Injection of the chalone into a mouse
also results in a strong inhibition of epidermal mitotic activity. Clearly then, Bullough and Laurence have isolated a substance from epidermis which can inhibit
epidermal mitoses in vivo and in vitro. In
both in vivo and in vitro experiments the
effects of a single injection of chalone have
been studied for a period of 4 hours. The
question arises as to how long the mitosis
inhibiting effect of a single injection of
chalone lasts. Bullough and Laurence
(1964) have investigated this question in
vitro. They have found (Table 3) that
after 5 hours in vitro the chalone no longer inhibits epidermal mitotic activity. According to Bullough and Laurence (1964),
these results with the chalone were similar
to those obtained after the injection of
adrenalin, also a potent inhibitor of epidermal mitotic activity. They, therefore,
asked themselves what would happen to
epidermal mitotic activity if chalone and
adrenalin were given together. As Table 3
indicates, the addition of chalone and
adrenalin together in vitro does not significantly enhance the inhibition of epidermal
mitotic activity produced by either alone,
nor does it prevent the recovery of epidermal mitotic activity after the first 5 hours.
Why is the inhibition of epidermal mitotic activity limited to 4-5 hours in vitro? Ts
it because the chalone becomes inactivated? Or is it because the epidermis becomes refractory? These same two questions can be asked regarding the action of
adienalin. First, let us examine the effects
of using either old or fresh chalone on
epidermal mitotic activity. Table 4 shows
that during the first 5 hours in vitro the
addition of fresh chalone to epidermis
causes, as expected, a decrease in epidermal mitotic activity. Also as expected, during the second 5 hours the epidermal mitotic depressing activity of the chalone is
lost. Addition of fresh chalone does not
restore the mitotic inhibition. However, as
Table 5 shows, if we place old chalone
with fresh epidermis, the inhibition of mitotic activity occurs. Therefore, the old
chalone is equally capable of inhibiting
mitotic activity after 5 hours, but does not
do so because the epidermis becomes refractory. Why does the epidermis become
refractory? According to Bullough and
Laurence (1964), the answer to this question becomes obvious if we look at the
effects of old and new adrenalin. Table 4
shows that if one adds adrenalin during
the first 5 hours, epidermal mitotic activity
in vitro is as predicted, inhibited. Moreover, this mitotic activity inhibition is lost
after the first 5 hours. However, addition
of fresh adrenalin during the second
5-hour period causes a marked inhibition
of mitotic activity. Thus, after the first 5
hours, the reason the epidermis shows no
further inhibition by adrenalin is due to
the fact that the adrenalin has lost its inhibiting effect, and not that the epidermis
has become refractory to its action. This,
of course, is precisely the opposite effect
from that which we see with the chalone.
The inability of 5-hour old adrenalin to
inhibit epidermal mitotic activity is further
shown in Table 5; the addition of old
adrenalin to fresh epidermis does not inhibit epidermal mitotic activity, but the addition of fresh adrenalin does. Bullough
CHALONKS AND GROWTH CONTROL
CO
CO
d
II
O
&
K
CO
CM
d
'& ^
II
o'J
d
i—i
©
d
II
CO
d
—
§
mi
C l CO
CM t ^
d d
II+1
"to
r-i
CX) CO
d d
T-H
IO
tit
d d
II It
co r-:
d d
©
8
a
ious ~t
be
1
d d
mi
00
-*
§
ienat<
8
d d
Liv
CO CO
00
Id
O
mi
o
CO CO
d d
mi
Cl
<M
"fel
ui
.2
s
o
13
'Si
CO CO
d d
Illl
co oi
o ^
'g
s
d d
Illl
in d
5C
O
•i'ssf
139
and Laurence (1964) suggest that perhaps
the real inhibiting mechanism for epidermal mitotic activity is neither the chalone
nor adrenalin by itself, but the action of
the two as a complex. Thus, they envision
a chalone-adrenalin complex whose level
controls epidermal mitotic activity.
More recently, Bullough and Laurence
(1968a) have investigated the action of
hydrocortisone on epidermal mitotic activity, since it has been known for many years
that glucocorticoid hormones depress epidermal mitotic activity (Bullough, 1965).
To determine the effect of hydrocortisone
on epidermal mitotic activity, Bullough
and Laurence (1968a) have taken advantage of the fact that after 4-5 hours in vitro,
epidermal mitotic inhibition by chalone
and adrenalin is lost. In Table 6 we see
that if epidermis is placed in vitro for 5
hours in the presence of either chalone,
adrenalin, hydrocortisone, or various combinations of these three, and if after the
first 5-hour period colcemid is added and
mitoses collected for an additional 4-hour
period, there is no significant inhibition of
epidermal mitotic activity, except when
all three substances are used together. This
suggests that hydrocortisone prolongs the
life of the chalone-adrenalin complex, and
it is because of this that hydrocortisone
exhibits a mitotic activity depressing capability.
We, therefore, may conclude that the
epidermis contains a substance called a
chalone which, when complexed with
adrenalin, inhibits mitosis, and that the
life of this complex is prolonged by hydrocortisone.
Since the classical experiments of Bullough and Laurence (1964) isolating the
epidermal chalone, many other laboratories have confirmed the presence of a
chalone in epidermis and its ability to inhibit epidermal mitotic activity in vitro
and in vivo (Iversen et al., 1965; Elgjo,
1969; Elgjo and Hennings, 1971; Frankfurt, 1971; Hennings et al., 1969; Marrs
and Voorhees, 1971).
What is the chemical nature of the
chalone? To date, the evidence suggests
140
THOMAS S. ARGYRIS
TABLE 3. Inhibition of epidermal mitotic activity by chalone and/or adrenalin in vitro.
Time after establishment of culture
Culture medium
Normal
With 1 unit chalone/4 ml
With 0.01 ixg adrenalin/4 ml
With 1 unit chalone ± 0.01 /ig adrenalin/4 ml
2nd-5th hr
6th-9th hr
6.3 -+• 0.23
3.1 •+- 0.23
3.2 -i- 0.11
7.2 -+- 0.24
7.7 H- 0.28
8.1 -i- 0.21
8.5 -+- 0.37
2.7 •+• 0.52
Average numbers of mitoses arrested by colcemid in 4 hours in unit lengths of 1 em ear epidermis sectioned 7 fi thick. N = 10.
(Data from Bullough and Laurence, 1964.)
that the epidermal chalone is a glycoprotein with a molecular weight of about 30,000 to 40,000 (Bullough et al., 1964; Boldingh and Laurence, 1968; Marrs and Voorhees, 1971).
The questions which next arise are:
How specific is the epidermal chalone, and
do other tissues have chalones, and if so,
how specific are they?
We have already pointed out that Bullough and Laurence (1964) have demonstrated that the crude extracts of epidermal chalone can inhibit mitoses in vitro in
the cornea, esophagus, and sebaceous
gland in addition to the epidermis, but not
mitoses in intestinal epithelium. Why epidermal chalone should effect such a wide
range of epidermal derivatives is not clear.
However, this may be due simply to the
fact that the epidermal extracts are not
pure and possess multiple chalone activities. Supporting this notion is the fact that
recently Bullough and Laurence (1970a)
have been able to obtain a pure epidermal
chalone free of sebaceous gland chalone
activity. Others have also found that the
epidermal chalone has considerable specificity (Elgjo, 1969; Elgjo and Hennings,
1971; Frankfurt, 1971). Thus, the evidence strongly suggests that the chalone
has considerable specificity. The precise degree of specificity of the epidermal chalone
will not be known, however, until its purification has been accomplished.
Do other organs have substances which
show chalone activity? Substances with
chalone activity have been isolated from
esophagus, hamster cheek pouch epithelium, lymphocytes, stomach, and eye lens
(Frankfurt, 1971; Hall, 1969; Houck et al.,
1971; Philpott, 1971; Voaden, 1968). The
degree of purity of the chalone isolated,
and the rigorousness of the tests used to
determine its specificity vary considerably,
but we feel, as does Bullough (1965), that
it is safe to conclude that chalones are
present in tissues other than those of the
skin.
TABLE 4. Effect of adding fresh extract of epidermal chalone to 5-hr old cultures of epidermis.
Time after establishment of cultures
6th-9th hr
2nd-5th hr
First culture medium
Xo. of
mitoses
Final culture medium
No. of
mitoses
Normal
6.2 -4-0.55
Normal
10 units old chalone/4 ml medium
10 units fresh chalone/4 ml medium
7.1 ± 0.35
5.4 ± 0.41
5.5 ± 0.46
10 units chalone/4 ml medium
2.3 ± 0.23
Normal
10 units old chalone/4 ml medium
10 units fresh chalone/4 ml medium
12.0 ± 0.80
8.4 ± 0.66
9.1 ± 0.76
0.1 jig adrenalin/4 ml medium
2.0 ± 0.48
Normal
0.1 jig old adrenalin/4 ml medium
0.1 /ig fresh adrenalin/4 ml medium
7.4 ± 0.16
7.2 ± 0.34
2.6 ± 0.40
Average numbers of mitoses arrested by colcemid in 4 hours in unit lengths of 1 cm ear epidermis sectioned 7 ^ thick. N = 15.
(From Bullough and Laurence, 1964.)
141
CHALONES AND GROWTH CONTROL
TABLE 5. Effect of adding fresh epidermis to culture media containing 5-hr old extracts of epidermal chalone and of adrenalin.
Normal
medium
10 units chalone/
4 ml medium
0.1 ^g adrenalin/
4 ml medium
With culture medium, chalone and adrenalin
freshly prepared
5.6 ± 0.31
1.4 ± 0.14
1.8 ± 0.24
With culture medium, chalone and adrenalin
5 hr old
1.5 ± 0.21
6.1 -i- 0.52
5.8 ± 0.41
Average numbers of mitoses arrested by coleemid in 4 hours in unit lengths of 1 cm ear epidermis sectioned 7 ft thick. N = 15.
(From Bullough and Laurence, 1964.)
THE LOCUS OF ACTION OF THE CHALONE
IN THE CELL CYCLE
Granted that chalones which can inhibit
cell division exist in tissues, the question
arises: In what phase of the cell cycle do
the chalones act to inhibit mitotic activity?
The principle experiments of Bullough
and his colleagues determining the effects
of the chalone on epidermis have been
done using his in vitro system. They place
pieces of mouse ears in flasks, with the
appropriate medium, add colcemid, and
collect mitosis for about a 4-hour period
(Bullough and Laurence, 1964). Decreases
in mitotic activity which occur with
chalone treatment may be due to inhibition at any of the three principle phases of
the cell cycle, G-l, S, or G-2. Experimental evidence has been presented by Gelfant
(1962) using H 3 TdR (tritiated thymidine)
labelling experiments that the inhibition of mitoses in epidermal cells during
the 4-hour in vitro incubation period occurs in G-2. He has clearly demonstrated
that the epidermal mitoses which appear
during the first few hours after the ear skin
is placed in flasks are unlabelled, indicating that the cells must have been in G-2.
He has also shown that the addition of
adrenalin to epidermis in vitro also inhibits cells in G-2 (Gelfant, 1966). Elgjo and
Hennings (1971) and Frankfurt (1971)
also have shown that the injection of epidermal chalone into mice inhibits epidermal or esophageal cells, respectively, in the
G-2 phase of mitosis. Thus, we have ample evidence that chalones inhibit cells in
G-2, and it is this inhibition which results
in a decrease in mitotic activity in vitro
and in vivo. But to show that the chalone
is of the greatest importance to the problem of growth control, we must present
evidence that it is acting on cells in G-l.
This is true because most cells that are
stimulated to enter mitosis by a wide variety of growth promoting stimuli are in G-l
(Baserga, 1965; Gelfant, 1962). This very
important point has been fully realized by
those interested in chalones as controls for
cell division (Bullough, 1965; Iversen,
TABLE 6. The effects of epidermal chalone, adrenalin, and hydrocortisone on mitotic activity in
mouse ear epidermis in the second 4 hours in vitro.
Control epidermis
M±SE
N
-i- 0.19
•+- 0.19
-t- 0.19
± 0.27
20
20
20
10
8.4 ±0.17
25
8.7 ± 0.18
15
8.7 ± 0.18
15
8.3
8.3
8.3
7.9
Epidermis treated with:
The following per
4 ml medium
1 unit chalone
0.01 fig adrenalin
0.01 fig hydrocortisone
1 unit chalone
+ 0.01 fig adrenalin
1 unit ehalone
0.01 fig hydrocortisone
0.01 fig adrenalin
0.01 fig hydrocortisone
1 unit chalone
+ 0.01 fig adrenalin
-j- 0.01 fig hydrocortisone
M± SE
N
Mitotic depression
('.%)
8.3 •+• 0.22
7.4 ± 0.25
7.7 -i- 0.31
7.6 ± 0.26
18
20
20
10
0
11
7
4
5.4 ± 0.14
25
36
4.0 ± 0.16
15
54
4.3 ± 0.16
15
51
Average numbers of mitoses arrested by colcemid in 4 hours in unit lengths of 1 cm of ear
epidermis sectioned 7 n thick. N, number of mice.
(From Bullough and Laurence, 1968a.)
142
THOMAS S. ARGYRIS
Moreover, the liver extract which supposedly contains the liver chalone has no
effect on liver DNA synthesis. Hennings et
Specific activity of DNA (cpm//*g)
al. (1969) suggest that the fact that the
liver
skin
Hours after
inhibition
of DNA synthesis is not obvious
water
extract
extract
first injection
9-13
hours after initiation of treatuntil
—
10.4
11.6
ment indicates that the chalone does not
—
10.5
11.9
5y3
9.9
8.0 -i- 2.0
11.3 -i- 0.5"
act on cells already in the DNA synthesis
—
12
6.8
10.6
phase
of the cell cycle, but only on cells
b
7
.
3
•+•
0
.
1
13%
10.7
12.9 -+- 1.3"
are about to enter the DNA synthewhich
18
8.3
15.2
—
5.4
30
12.8
sis phase. If this is true, then one should be
48
—
10.6
11.0
able to show autoradiographically a dea
Three
experiments.
crease in the number of H 3 TdR labelled
b
Two experiments.
At zero time and 4, 8, and 12 hours later, groups cells after chalone treatment. Such data for
the epidermis have to date not been
of mice were injected ip with 0.25 ml of distilled
water or of a solution containing 5 mg of skin or
presented. Elgjo and Hennings (1971) also
liver extract. The animals were killed at the times
indicated after
zero time. Mice were injected with have shown that one injection of epider30 /tc of 3H-thymidine 30 minutes before killing
mal extract inhibits DNA synthesis in
and the specific activity of epidermal DNA was de- hamster ear epidermis in vivo, whereas a
termined. Both male and female mice were used,
but each experimental group is compared with a comparable amount of liver extract does
control group of the same sex and age.
not. Again, the claim is made that there is
(From Hennings et al., 1969.)
a decrease in H 3 TdR labelled cells, but no
1970), and a number of laboratories have data are presented.
Direct evidence that chalone can inhibit
initiated investigations into the effect of
chalones on the G-l phase of the cell cy- cells from entering DNA synthesis has
cle. To date, however, evidence that been presented by Frankfurt (1971) for
chalones can inhibit cells in G-l from en- the forestomach epithelium of the mouse.
tering DNA synthesis phase is not une- He shows that the injection of epidermal
chalone decreases the number of H 3 TdR
quivocal.
Baden and Sviokla (1968) have tested labelled cells in the forestomach epithelithe ability of a quantity of chalone capa- um of the mouse (Table 8), whereas liver
ble of inhibiting epidermal mitosis in vitro extract does not. Similarly, Hall (1969)
to inhibit incorporation of tritiated thy- presents evidence that the epidermal
midine (H3TdR) into DNA in vitro. chalone isolated from hamster cheek
They have found that the epidermal pouch epidermal cells can, when added to
chalone does not inhibit DNA synthesis in a culture of hamster cheek pouch epithevitro in ear epidermal cells during a peri- lium, inhibit the number of H:)TdR
od of 2 hours. Similarly, Hennings et al. labelled cells in the cheek pouch
(1969) have shown that a single injection epithelium. Moreover, similarly prepared
of chalone which inhibits epidermal mi- extracts from the cheek pouch connective
toses in vivo, during the first 4 hours after tissue do not inhibit cheek pouch epiderinjection, does not significantly decrease
H 3 TdR incorporation into epidermal TABLE 8. Effect of epidermal chalone on tritiated
thymidine labelling of cells of the epithelium of
DNA. Repeated injections of chalone rethe forestomach.
sult in a decrease in epidermal DNA synIndex labelling in
thesis (Table 7). However, the significance
basal cells (%)
Material injected
of this result, in the opinion of this reControl
8.0 ± 0.5
viewer, is clouded by the lack of sufficient
Liver extract
8.5 ± 1
statistical treatment of the data, and the
Epidermal extract
2.8 ± 1
Adrenalin
13.4-t- 1.6
fact that liver extracts also appear to inhibit epidermal DXA synthesis at 9-i/2 hours.
(From Frankfurt, 1971.)
TABLE 7. Effect of multiple injections of shin or
liver extract on epidermal DNA synthesis.
143
CHALONES AND GROWTH CONTROL
TABLE 9. Inhibition of mitotic activity of regenerating epidermis in vivo after Tween 60 application.
Mitoses in 100 ^m
ear epidermis
Material injected
Control
Epidermal extract
Liver extract
Protein
( m g)
Intensive
hyperplasia
3.2
1.6
0.8
0.4
0.2
1.6*
2.2
3.2 ± 0.19
0.43 ±0.03
0.94 ± 0.11
1.43 ± 0.5
2.3 -i- 0.4
3.2 ±0.76
2.7 ± 0.86
3.2 ±0.7
« Heating at 100°C for 1 min.
(From Frankfurt, 1971.)
mal H 3 TdR labelling. These very interesting results appear to clearly establish the
fact that chalones can inhibit cells in G-l
from entering DNA synthesis. However,
their full acceptance as evidence that
chalones act only as inhibitors of cell division is prevented by the fact that in the
experiments of Hall (1969), low concentrations of chalone can stimulate the number of cells showing H 3 TdR incorporation. Therefore, as these authors clearly
recognize, more work is needed to establish
the role of chalones in mitotic control.
THE CHALONE IN REGENERATION
AND TUMORIGENESIS
The notion of chalones arose from the
interpretation of experiments on wound
healing. Bullough and Laurence (1960)
suggested that the local loss of tissue mass
resulted in the loss of an inhibitor, or
chalone. It was in a later paper (Bullough
and Laurence, 1964) that they expanded
their idea to include the role of adrenalin
in the chalone-adrenalin complex, with
the complex being the controlling mechanism for cell proliferation. The questions
arise as to whether there is a decrease in
the chalone concentration in wounds and
whether the addition of chalone to wounds
inhibits mitotic activity in the regenerating epidermis. There is no direct evidence that in wounds the chalone concentration drops. However, there is evidence
that the administration of a chalone extract to a regenerating epidermis inhibits
its proliferation.
Frankfurt (1971) induces epidermal
proliferation in the ears of mice by the
application of Tween 60, an irritant. He
then injects epidermal chalone into the
mice and finds a dramatic decrease in mitotic activity in the regenerating epidermis
(Table 9). In addition, he has studied the
effect of the chalone injection on the net
accumulation of epidermal cells in the regenerating epidermis. As Table 10 indicates, the injection of chalone results in a
decrease in the total number of epidermal
cells/unit length of ear epidermis. Thus,
the evidence is clear in epidermis regenerating after chemical damage, that the
chalone can inhibit mitotic activity, and
indeed, prevent the full expression of the
proliferative response. We feel it is reasonable to predict that the proliferative response of the epidermis surrounding a cut
can also be inhibited by the injection of
the chalone. Precisely in what phase of the
cell cycle the inhibition occurs in regenerating cells remains to be investigated.
The fact that the injection of a chalone
can inhibit the proliferation of rapidly dividing cells in regenerating epidermis
raises exciting questions such as: Can
chalones inhibit the proliferation of tumor
cells, and do tumors contain less chalone
than the normal tissue from which they
arise? A number of laboratories have investigated these questions.
Bullough and Laurence (19686), using
a V X 2 epidermal carcinoma of the rabbit,
find than an extract of this tumor can
TABLE 10. The effect of liver or epidermal extracts
on the number of cells in a unit area of mouse ear
epidermis at various hours after treatment with,
Tween 60.
Number of cells in 100 ^m ear
epidermis
injected
Control
Liver extract
Epidermal
extract
Oh
34 h
46 h
25.3 ± 2.3 49.0 ± 6.9 63.0 ± 3.8
—
48.7 ± 2.6 58.8 ± 8.3
34.0 ± 1.8 38.1 ± 4.1
(From Frankfurt, 1971.)
144
THOMAS S. ARGYRIS
TABLE 11. Effect of skin or liver extract on tumor
DNA synthesis.
Specific activity of DXA
( / )
Hours
after
injection
liver extract
2
4
6
8
10
16.6 -(- 3.1"
20.8
27.1 ± 1.6a
17.8 ±
14.7 •+• 5.3"
Per cent
depression
of DNA
skin extract synthesis
17.6 -+- 1.9"
5.3 ±3.8"
5.7
3.1 -f- 1.6"
15.1 ± 3.4*
—
72
70
84
20
Tumor-bearing hamsters were injected ip with
50 mg of liver or skin extract and sacrificed at the
times indicated after injection. The animals received 100 fid °H-thymidine 40 niin before sacrifice.
The depression of DNA synthesis is expressed as
percentage of the average specific activity of DNA
18.9 cpm/^g) after injection of liver extract.
" Two tumors.
b
Four tumors.
(From Elgjo and Hennings, 1971.)
inhibit mitotic activity of mouse ear epidermis in vitro, and that like the epidermal chalone adrenalin is required, as well
as hydrocortisone, for its full inhibiting
activity. These investigators present calculations from which they infer that the concentration of chalone in the tumor cells is
about 1/10 that of normal epidermis. The
V x 2 tumor cells are sensitive to the action
of epidermal chalone and tumor chalone,
since they show a decreased mitotic activity, both in vitro and in vivo, when treated
with either chalone, under the appropriate
circumstances. Bullough and Laurence
have extended their studies to include other tumors, such as the mouse HardingPassey melanoma and the mouse L5178Y
lymphoma
(Bullough and Laurence,
1968c; 1970&). The results are essentially
similar to those obtained with the Vx2
epidermal tumor. The tumor contains a
chalone which can inhibit mitotic activity
in the homologous tissue. The full action
of each chalone in vitro requires the
presence of adrenalin and hydrocortisone.
Others (Elgjo and Henning, 1971; Mohr
et al., 1968; Rytomaa and Kiveniemi,
1968) also have presented evidence that
chalones can inhibit the mitotic proliferation or overall growth of a variety of
tumors.
As in the case of the normal epidermis,
we may ask in what phase of the mitotic
cycle are the tumor cells inhibited. In the
above experiments cited (Bullough and
Laurence, 1968&, 1968c; 19706; Elgjo and
Hennings, 1971), the mitotic activity has
been studied for a 4-hour period after the
addition of colcemid, either in vitro or in
vivo. In all probability, as we have explained earlier for normal epidermal cells,
the inhibition of tumor mitotic activity occurs in the G-2 phase of the cell cycle.
Therefore, the question immediately is
raised: Is there any evidence that chalones
inhibit tumor cells in G-l? Elgjo and Hennings (1971) have clearly demonstrated
that the injection of epidermal chalone significantly decreases DNA synthesis in a keratinizing epithelioma of the hamster (Table 11), as evidenced by a decrease in
H 3 TdR incorporation into tumor cell
DNA. In addition, these investigators
claim, but do not present evidence, that
there is a decrease in the number of
H 3 TdR labelled cells in the tumor after
chalone injection, suggesting that cells in
G-l are inhibited from entering the S
phase. Recently Houck et al. (1971) also
have presented evidence that extracts from
normal lymphoid tissue can specifically
inhibit H 3 TdR incorporation into DNA
in human lymphocytes activated by phytohemagglutinin and in leukocytes from leukemia patients.
Thus, the evidence strongly suggests that
tumors contain chalones, but perhaps at
reduced concentrations. Moreover, tumors
appear to show decreased mitotic activity
or DNA synthesis when treated with the
appropriate chalone. This latter point may
lead some to predict that chalones might
be useful in treating neoplastic conditions
in man. Iversen (1970) presents a thoughtful analysis of this possibility.
THE COMPETENCE OF TISSUE MASS LOSS
TO STIMULATE CELL DIVISION
The hypothesis has evolved that the
chalone complex can control the rate of
proliferation through a negative feedback
mechanism (Bullough, 1962, 1965). This
notion arose primarily from the previously
CHALONES AND GROWTH CONTROL
FIG. 1. Hair growth around a wound, 21 days
after injury. Arrows point to the growing hair
follicles. The surrounding skin has a white appearance because it is clipped. (From Argyris and
Argyris, 1959.)
cited work of Bullough and Laurence
(1960) on wound healing. They have suggested that the local loss of tissue mass
results in a drop in the level of chalone,
which in turn stimulates cell division in
the epidermis and in the other tissues surrounding the wound. Is, in fact, the local
loss of tissue mass sufficient to result in a
decrease in the concentration of chalones?
This is a very difficult question to answer
because the experimental design requires
that we be able to remove a small amount
of tissue without "unpackaging cells," or in
any way changing the intercellular environment that may initiate cell division. We
have devised a technique by which tumor
tissue is inoculated into an organ and the
tumor allowed to invade the organ. Tumor
invasion results in the removal of the tissue surrounding the advancing edge of the
growing tumor, and thus results in the
local loss of organ mass, as occurs after
damage. We will describe the use of this
technique in two experimental situations.
Figure 1 shows that cutting mouse skin
possessing resting hair follicles results in
the stimulation of growth of the resting
hair follicles surrounding the wound (Argyris and Argyris, 1959). Is it the local loss
of skin mass associated with the cut which
results in the stimulation of growth of the
resting hair follicles surrounding the
wound?
We inoculate subcutaneously C57BL/6J
mice possessing resting hair follicles with
the Ehrlich ascites tumor. Within a few
days the tumor is transformed into a solid
nodule and invades the overlying skin.
Shortly, the skin is completely invaded and
replaced by tumor tissue. Since we have
effectively removed skin, just as occurs after damage, we would expect that the resting hair follicles surrounding the wound
should be stimulated to grow, if the local
loss of skin mass is a sufficient cue to trigger hair growth. But the resting hair follicles surrounding the wound do not grow.
Therefore, the loss of skin mass is not a
sufficient cue for the stimulation of hair
growth. However, before we can accept
this conclusion, a number of objections
have to be met.
One may object because of the possibility that the tumor debilitates the mice so
that the resting hair follicles cannot grow,
even though they have received cues for
growth. A large series of experiments (Argyris 1968; Argyris and Trimble, 1964a)
indicate clearly that the tumor does not
debilitate the mice. Secondly, we have met
the objection that the tumor may be in
some way preventing the resting hair follicles from responding to the growth promoting effect of the loss of mass, even though
the mice are not debilitated. We have inoculated groups of mice with the Ehrlich
ascites tumor. After 21 days, when the tumor has clearly invaded the overlying skin
and there is ulceration, the resting follicles
surrounding the wound are plucked.
Plucking is a stimulus for hair growth. If
the tumor affects the competence of the
resting hair follicles to respond to legitimate cues for growth, then the plucked
hair follicles should not grow. But, they in
146
THOMAS S. ARGYRIS
fact do grow. Similarly, wounding skin adjacent to an area of skin completely invaded by the tumor stimulates hair growth
(Argyris and Trimble, 1964a). Finally, we
have shown that, occasionally, spontaneous waves of hair growth can occur in mice
with resting hair follicles bearing tumors,
and the waves of hair growth move up
right to the edge of the ulcerated tumor.
Thus, the reason resting hair follicles surrounding a tumor do not grow is not because they are incapable, but because they
have not received a legitimate cue to grow.
Another objection that may be raised is
that the monitoring devices for mass are
not organ-specific, and they monitor only
mass. Since the tumor has replaced the
skin, no loss of mass is detected, and therefore no hair growth occurs. This is unlikely. Loss of mass which results in growth, as
in compensatory hypertrophy, is largely
organ specific (Abercrombie, 1957; Argyris, 1968). In the case of the skin, if the
monitoring devices for mass are not organspecific, we should be able to induce hair
growth by cutting out the the tumor after
it has replaced the skin. However, cutting
out the tumor does not induce hair growth
(Argyris and Trimble, 1964a).
Thus, the evidence presented, as well as
other evidence not detailed here (Argyris,
1968; Argyris and Trimble, 1964a), demonstrates that the use of the Ehrlich ascites
tumor invasion to remove skin mass is
a legitimate method. Moreover, the local loss of skin mass produced by the invading tumor is not a sufficient cue for the
stimulation of growth of resting hair follicles. Thus, it is unlikely that the loss of
mass per se in wound healing is sufficient
to initiate growth of the resting hair follicles. If the decrease in hair follicle
chalone is responsible for initiating hair
growth (Bullough, 1965), the loss of mass
observed in wounds is not sufficient to lower the level of the chalone. Some additional mechanism must be involved. We have
speculated that the stimulation of hair
growth in wound healing is initiated by
the release of a stimulating substance (s)
(Argyris and Trimble, 1964a). If so, and if
the chalone is involved in controlling hair
growth, perhaps the stimulator (s) results
in the inactivation of the hair follicle
chalone. Further experiments are necessary
to decide this issue.
Is there any evidence in other organs
that the local loss of mass accompanying
damage is not a sufficient cue for the initiation of growth of the tissues surrounding
the wound? We have investigated this
question in the mouse kidney, where we
have demonstrated that damage to the kidney cortex results in the stimulation of
mitotic activity of the kidney cortical tissue
surrounding the damaged area (Argyris
and Trimble, 1964&). Using the Ehrlich
ascites tumor, we have shown that the local loss of kidney mass approximating that
lost after damage is not a sufficient cue to
stimulate mitotic activity in the kidney
cells surrounding the tumor, as occurs in
kidney cells surrounding a damaged area.
We have also demonstrated that the lack
of a proliferative response on the part of
the kidney cells is not due to their being
made incompetent to divide by the invading tumor (Agryris and Trimble, 1964b;
Argyris et al., 1969). They do not proliferate because the local loss of kidney mass is
not a sufficient cue for their growth.
We, thus, conclude that the loss of mass
associated with kidney damage is not a
sufficient cue to initiate an increase in cell
proliferation. We again speculate that kidney damage results in the release of a stimulating substance(s) presumably, which
may directly or indirectly stimulate growth.
If chalones are involved in controlling kidney growth, these stimulators could be inactivating the kidney chalones, decreasing
their concentration, and thus stimulating
proliferation (Argyris, 1968; Argyris and
Trimble, 19646).
Finally, the question arises as to whether
the spontaneous loss of chalone is sufficient to initiate increases in cell proliferation normally. If the decrease in the concentration of a chalone is responsible for
initiating normal hair growth in mice
(Bullough, 1965), we might expect that
hair follicles of skin grafts transplanted
*
CHALONES AND GROWTH CONTROL
147
cle chalone, since wounding the hairless
skin adjacent to the graft does not lead to
growth of the hair follicles in the graft.
Nor are the graft hair follicles incompetent to grow, since plucking them, initiates
their growth (Argyris and Argyris, 1970).
Thus, we conclude that a spontaneous
drop in the level of a hair follicle chalone
does not occur. If the loss of a chalone is
required for stimulating hair growth, some
substance is necessary to initiate the drop
in the level of the chalone. Experiments to
determine if similar arguments apply to
other tissues are needed.
FIG. 2. Haired skin grafts from female mice placed
on female littermate hairless mice, 5 months
after grafting.
onto hairless mice will remain in the
growth phase of the hair cycle, because the
chalone produced by the hair follicles
would diffuse out of the graft into the
surrounding hairless skin of the host. Since
there are no hair follicles in the host hairless skin, presumably no hair follicle inhibitor is produced, and therefore no accumulation of hair follicle chalone will occur to
halt hair growth in the graft. Growth promoting effects of damage can travel across
the scar line separating graft from host
skin (Argyris and Argyris, 1970). To determine if hair follicles of grafts will remain in the growth phase permanently
when the grafts are placed on hairless
mice, haired skin from female mice has
been transplanted onto littermate female
hairless mice of the strain HRS/J (Argyris
and Argyris, 1970). The grafts are accepted and survive well for over a year.
Figure 2 shows two such grafts on the
backs of hairless mice. The haired grafts
undergo normal hair growth cycles. The
hair follicles may remain in the resting
phase for months. These results argue
against the possibility that a drop in the
level of a hair follicle chalone is the primary event for initiating normal hair
growth. If it were so, then the graft hair
follicles should remain in the growth
phase. Nor does the hairless skin, with its
hair follicle remnants, produce a hair folli-
GENERAL SUMMARY AND CONCLUSIONS
The evidence that chalones exist is unequivocal. The fact that they can inhibit
cells in G-2 from entering mitosis is also
clear. Their ability to inhibit cells in G-l
from entering mitosis is also strongly suggested, but more work is needed before we
can be certain.
It is clear that for chalones to inhibit
mitoses in the G-2 phase of the cell cycle
adrenalin is required and, probably, hydrocortisone. It is not known if these substances are necessary for the chalone to
inhibit cells from entering mitosis from G1.
The fact that chalones can be extracted
from tissues does not automatically guarantee that their physiological function is to
control cell proliferation. The history of
the study of growth is replete with
examples of stimulators and inhibitors
which were thought to be involved in the
control of growth (Abercrombie, 1964; Argyris, 1968; Bardos et al., 1968). Similarly,
the study of embryonic development
abounds with examples of inducers which
were thought to control differentiation
(Needham, 1950; Saxon and Toivonen,
1962). To establish that chalones are the
physiological controllers of growth will require more work. One of the criticisms
which must be met is that of Argyris
(1968) that in wound healing the loss of
mass per se does not result in the stimulation of growth. If correct, the results of
148
THOMAS S. ARGYRIS
Argyris (1968) suggest that, assuming
chalones are involved in growth control,
they cannot control growth by a straightforward negative feedback control mechanism (Bullough, 1965). Something must
inactivate the chalone to initiate a decrease in its concentration. A search for
such a substance released from wounds is
of considerable importance. But again, history indicates that the search for wound
hormones has been a frustrating and unrewarding one (Abercombie, 1957; Argyris,
1968). We offer one speculation for consideration. Recently, a considerable amount
of evidence suggests that prostaglandins
are released during trauma (Piper and
Vane, 1971; Ramwell and Shaw, 1970;
Willis, 1969), and that they stimulate cell
proliferation (Franks et al., 1971). Perhaps prostaglandins are wound hormones
(Franks et al., 1971). They may cause a
decrease in the concentration or activity of
the chalone adrenalin complex and thereby initiate growth. Moreover, since merely
very small changes in the cellular environment can release prostaglandins (Piper
and Vane, 1971), these substances may be
involved in the inactivation of chaloneadrenalin complex under "physiological"
conditions thus helping to control cell division.
REFERENCES
Abercrombie, M. 1957. Localized formation of new
tissue in an adult mammal. Symp. Soc. Exp. Biol.
11:235-254.
Abercrombie, M. 1964. Behavior of cells toward one
another. Advan. Biol. Skin 5:95-112.
Argyris, T. S. 1968. Growth induced by damage.
Advan. Morphogenesis 7:1-43.
Argyris, T. S., and M. E. Trimble. 1964a. On the
mechanism of hair growth stimulation in wound
healing. Develop. Biol. 9:230-254.
Argyris, T. S., and M. E. Trimble. 19646. The
growth promoting effects of damage in the damaged and contralateral kidneys of the mouse.
Anat. Rec. 150:1-10.
Argyris, T. S., and B. F. Argyris. 1959. Stimulation
of hair growth during skin regeneration. Develop. Biol. 1:269-280.
Argyris, T. S., and B. F. Argyris. 1970. Hair growth
in skin grafts placed on hairless mice. Anat.
Rec. 168:457-462.
Argyris, T. S., M. E. Trimble, and R. Janicki. 1969.
Control of induced kidney growth, p. 45-60.
In W. W. Nowinski and R. Goss [ed.], Compensatory renal hypertrophy. Academic Press Inc.,
New York.
Baden, H. P., and S. Sviokla. 1968. The effect of
chalone on epidermal DNA synthesis. Exp. Cell
Res. 50:644-646.
Bardos, T. J., H. L. Gordon, Z. F. Chmielewicz, R.
L. Kutz, and M. V. Nadkarni. 1968. A systematic
investigation of the presence of growth-inhibitory
substances in animal tissues. Cancer Res.
28:1620-1630.
Baserga, R. 1965. The relationship of the cell cycle
to tumor growth and the control of cell division:
a review. Cancer Res. 25:581-595.
Boldingh, W. H., and E. B. Laurence. 1968. Extraction, purification and preliminary characterisation of the epidermal chalone: a tissue specific
mitotic inhibitor obtained from vertebrate skin.
Eur. J. Biochem. 5:191-198.
Bullough, W. S. 1962. The control of mitotic activity in adult mammalian tissues. Biol. Rev. (Cambridge) 37:307-342.
Bullough, W. S. 1964. Growth regulation by tissuespecific factors, or chalones, p. 124-145. In P.
Emmelot and O. Muhlbock [ed.], Cellular control mechanisms and cancer. Elsevier Pub. Co.,
Amsterdam.
Bullough, W. S. 1965. Mitotic and functional homeostasis: a speculative review. Cancer Res.
25:1683-1727.
Bullough, W. S., and E. B. Laurence. 1960. The
control of epidermal mitotic activity in the
mouse. Proc. Roy. Soc. London B Biol. Sci.
151:517-536.
Bullough, W. S., and E. B. Laurence. 1964. Mitotic
control by internal secretion: the role of the
chalone-adrenalin complex. Exp. Cell. Res.
33:176-194.
Bullough, W. S., and E. B. Laurence. 1968a. The
role of glucocorticoid hormones in the control of
epidermal mitosis. Cell Tissue Kinet. 1:5-10.
Bullough, W. S., and E. B. Laurence. 19686. Control of mitosis in rabbit VX2 epidermal tumors
by means of the epidermal chalone. Eur. J.
Cancer 4:587-594.
Bullough, W. S., and E. B. Laurence. 1968c. Control of mitosis in mouse and hamster melanomata by means of the melanocytic chalone. Eur. J.
Cancer 4:607-615.
Bullough, W. S., and E. B. Laurence. 1970a.
Chalone control of mitotic activity in sebaceous
glands. Cell Tissue Kinet. 3:291-300.
Bullough, W. S., and E. B. Laurence. 19706. The
lymphocytic chalone and its antimitotic action on
a mouse lymphoma in vitro. Eur. J. Cancer
6:525-531.
Bullough, W. S., C. L. Hewett, and E. B. Laurence.
1961. The epidermal chalone: a preliminary
attempt at isolation. Exp. Cell Res. 36:191-200.
F.l^jo, K. 19fi9. Epidermal it'll proliferation during
the first 24 hr. after injection of an aqueous skin
CHALONES AND GROWTH CONTROL
extract (chalone). Virchows Arch. Abt. B
Zellpathol. 4:119-125.
Elgjo, K., and H. Hennings. 1971. Epidermal
chalone and cell proliferation in a transplantable
squamous cell carcinoma in hamsters. 1. In
vivo results. Virchows Arch. Abt. B Zellpathol.
7:1-7.
Frankfurt, O. S. 1971. Epidermal chalone. Effect on
cell cycle and on development of hyperplasia.
Exp. Cell Res. 64:140-144.
Franks, D. J., J. P. MacManus, J. F. Whitfield.
1971. The effect of prostaglandins on cyclic AmP
production and cell proliferation in thymic lymphocytes. Biochem. Biophys. Res. Commun.
44:1177-1183.
Gelfant, S. 1962. Initiation of mitosis in relation to
the cell division cycle. Exp. Cell Res. 26:395-403.
Gelfant, S. 1966. Patterns of cell division: the
demonstration of discrete cell populations, p.
359-395. In D. M. Prescott [ed.], Me'thods in cell
physiology. Vol. 2. Academic Press, N. Y.
Goss, R. 1964. Adaptive growth. Academic Press
Inc., New York.
Hall, R. G. 1969. DNA synthesis in organ cultures
of the hamster cheek pouch. Exp. Cell Res.
58:429-431.
Hennings, H., K. Elgjo, and O. H. Iversen. 1969.
Delayed inhibition of epidermal DNA synthesis
after injection of an aqueous skin extract
(chalone). Virchows Arch. Abt. B. Zellpathol.
4:45-53.
Houck, J. C, H. Irajsquin, and S. Leikin. 1971.
Lymphocyte DNA synthesis inhibition. Science
173:1139-1141.
Iversen, O. H. 1970. Some theoretical considerations
on chalones and the treatment of cancer: A
149
review. Cancer Res. 30:1481-1484.
Iversen, O. H., E. Aandahl, and K. Elgjo. 1965. The
effect of an epidermis-specific mitotic inhibitor (chalone) extracted from epidermal cells.
Acta Pathol. Microbiol. Scand. 64:506-510.
Marrs, J. M., and J. J. Voorhees. 1971. Preliminary
characterization of an epidermal chalone-like inhibitor. J. Invest. Dermatol. 56:353-358.
Mohr, V., J. Althoff, V. Kinzel, R. Suss, and M.
Volm. 1968. Melanoma regression induced by
"chalone": a new tumour inhibiting principle
acting in vivo. Nature (London) 220:138-139.
Needham, J. 1950. Biochemistry and morphogenesis. Cambridge Univ. Press, London.
Philpott, G. W. 1971. Tissue-specific inhibition of
cell proliferation in embryonic stomach epithelium in vitro. Gastroenterology 61:25-34.
Piper, P., and J. Vane. 1971. The release of
prostaglandins from lung and other tissues. Ann.
N. Y. Acad. Sci. 180:363-385.
Ramwell, P. W., and J. E. Shaw. 1970. Biological
significance of the prostaglandins. Recent
Progr. Hormone Res. 26:139-187.
Rytomaa, T., and K. Kiviniemi. 1968. Control of
DNA duplication in rat chloroleukaemia by
means of the granulocytic chalone. Eur. J. Cancer
4:595-606.
Saxen, L., and S. Toivonen. 1962. Primary embryonic induction. Logos Press Ltd., London.
Voaden, M. J. 1968. A chalone in the rabbit lens?
Exp. Eye Res. 7:326-331.
Willis, A. L. 1969. Parallel assays of prostaglandinlike activity in rat inflammatory exudale by
means of cascade superfusion. J. Pharm. Pharmacol 21:126-128.