- Email: [email protected]
The epidermal chalone
Experimenfal
192
THE A
W.
PRELIMINARY
S. BULLOUGH,
Birkbeck
EPIDERMAL
College,
AT
and
EDNA
University of London, and Organon Newhouse, Lancasshire, Scotland Received
Ilovember
36, 192-200
(1964)
CHALONE:
ATTEMPT
C. L. HEWETT
Cell Research
ISOLATION
B. LAURENCE Laboratories
Limited,
26, 1963
RECENTLYBullough
and Laurence [4] have described the inhibitory action of epidermal extracts on the mitotic activity of mouse ear epidermis. They have concluded that in normal epidermis mitotic control is exercised through a diffusible inhibitor, for which the name of epidermal chalone has been proposed [ 11. The epidermal chalone is probably produced within the epidermal cells and it is evidently tissue-specific in its action. It is already apparent that at least three distinct chalones may be produced in mouse skin [‘L], and there is also evidence that others may exist for instance in kidney and liver [I, 3]. It now appears possible that each tissue in the body may product its own specific chalone. A second important point that has been established is that in the absence of adrenalin the epidermal chalone loses its full power to inhibit epidermal mitosis [4]. It appears that the actual mitotic inhibitor may be an unstable chalone-adrenalin complex. The experiments described below record the preliminary attempts that have been made to isolate the epidermal chalone and to determine something of its physical properties.
MATERIAL
AND
METHODS
The epidermal chalone was extracted from epidermis taken from the backs bf adult Strong CBA mice. The epidermis was macerated according to the technique described by Bullough and Laurence [4] and the chalone was obtained in a clear aqueous solution after spinning in a refrigerated centrifuge. Except where otherwise stated, all the extracts were freshly made for each experiment. For easein comparing different extracts, the chalone concentrations were estimated in terms of a standard preparation in which 1 unit was arbitrarily considered as the activity contained in an aqueous extract of 1 mg dry weight of mouse epidermis (including the stratum corneum). In the present type of experiment, 1 unit is also the amount which induces a mitotic depression of approximately 50 per cent. Experimental
Cell Research
36
Isolution
of epidermcrl
chrrlone
193
The inhibitory action of the chalone was tested on the ear epidermis of adult male Strong CBA mice 4-6 months old. The epidermis was maintained in vi&o in a phosphate buffered saline medium with 0.02 M glucose and an oxygen gas phase (for details see [3]). In all cases colcemid was added to arrest the developing mitoses in the metaphase. It should be noted that in all the tables N represents the number of animals used; standard errors are given with the means. RESULTS
The preliminary experiments recorded in Table I are included merely to demonstrate that the epidermal chalone is present in the supernatant after centrifugation and that it is therefore soluble in water. The chalone can be extracted either with distilled Lvater or with normal saline, but since it has recently been shown that inorganic salts may account for a large proportion of the total epidermal water extract, the distilled Avater may have behaved in effect as a saline solution. However, from other experiments described belo\\-, it is clear that the chalone is soluble in salt-free water. T’AI
1. Inhibitory
erect
of aqueous marl mitotic
extrcrcts crctioity
of macerated in vitro.
epidermis
on epider-
Average numbers of mitoses arrested by colcemid in 4 hr in unit lengths of 1 cm ear epidermis sectioned 7 p thick. A’ = 10. Culture medium with
0
Total macerate Supernatant
6.0 & 0.33 6.2 + 0.34 ‘I’.~BLF: II. Attempted
Concentrations of chalone/4 ml culture medium 0.1 unit 1.0 unit 10.0 units 4.5 * 0.34 4.6kO.33 dialysis
3.1 + 0.16 3.4iO.18
of’ epidermcxl
2.1 10.29 2.5 1- 0.18
chnlone.
Average numbers of mitoses arrested by colcemid in 4 hr in unit lengths of 1 cm ear epidermis sectioned 7 p thick. N = 20. Culture medium with
0
Fresh chalone Dialysed solution Dialysate
6.2iO.18 6.2rir0.18 S.Z?O.lS
Assumed concentration 0.1 unit 4.1+ 0.46 3.910.37 6.0 IL 0.64
of chalone/4 ml medium 1.0 unit 10.0 units 3.3 * 0.17 3.4 ir 0.22 6.5 & 0.24
1.9io.27 2.4&0.31 5.9 IO.34
Note. In estimating the chalone content of the dialysed solution and of the dialysate it was assumed that the chalone was equally distributed between them. 13 -
641806
Experimental
Cell Keseurch
36
194
U’. S. Bullough,
C. I,. Hewett
and Ednn B. I,rrurence
Experiments were nest undertaken to determine whether the epidermal chalone is dialpsable. A solution of 930 units of epidermal chalone in 400 ml water \vas dialysed against 70 ml water at about 3°C for 2-l hr using a WIlophane membrane. Two separate experiments were performed and the combined results are shown in ‘Table Il. From this it is evident that the epidermal chalone did not pass through the membrane. In Table III the efl’ects of boiling the chalone solution are recorded. The xvater extract was raised to lOO”C, maintained at this temperature for 10 min, and then rapidly cooled and used at once. This treatment caused a complete loss of the antimitotic action. Early in this investigation it was important for practical purposes, and especially for the preparation of a stable standard extract, to discover ho\\ best the epidermal chalone might be preserved. It was found that a water solution loses its activity within several days at about 0°C and within several weeks at - 20°C. However, it was found that the activity can be preserved by freeze-drying. The figures in Table IV refer to a chalone extract that had been freeze-dried and kept for only ‘2 lveeks, but similar results have since TanrAE III. Average
Instability
numbers
of epidernml
of mitoses
chrxlone when 10 min.
arrested by colcemid in 4 hr in unit sectioned 7 p thick. LV - 10. Epidermis
Untreated control epidermis
Fresh
numbers
of mitoses
of
epidermnl
medium
with
0
Fresh chalonc Freeze-dried chalone iVote. Experimental
The
chalone Cell
chalone after
10 units
5.5 -t 0.37
lyophilisation.
lengths
of chalone/d
ml medium
of 1 cm car epidermis
ml medium
0.1 unit
1 .O unit
10.0 units
6.2 F 0.27
4.5 + 0.39
3.4 i- 0.29
2.6 i 0.26
6.2 IL 0.27
3.5 I- 0.28
3.0 i 0.47
2.6i0.37
was lyophilised Research
chalonc/~f
6.3 +0.34
Concentration Culture
for
of 1 cm car epidermis
1 unit
arrested by colcemid in 4 hr in unit sectioned 7 p thick. A’ = 10.
nt 100°C
with ISoiled
2.1-to.17
Stability
I\'.
treated
10 units
3.6 k 0.32
TABLE
lengths
ml medium
1 unit
6.2 20.32
Average
chalone/l
maintained
36
2 vvecks
before
assay.
Isolrrtion
of epiclermrrl chnlone
193
been obtained with lyophilisetl epidermal extracts after 3, 6 and 12 months. It may he noted that, for convenience, the chalone extracts were lgophilisetl with mannitol, which alone was without any effect. The fact that the chalone does not dialyse indicates that it possesses a T.\I~I,kI Average
numbers
\‘.
Alcohol
of mitoses Average
arrested sectioned numbers
fractionation
of epidermrrl
by colcemid in 4 hr in unit 7 ,U thick. LV = 24 throughout. of mitoses
in untreated
chtrlone.
lengths
control
of 1 cm ear epidermis
= 6.1 k 0.22.
Extract
derived
1.0 mg epidermis
4 ml culture Total water Precipitate Prrcipitate Precipitate Prccipitatc Precipitate I
medium
with
extract of from 50 % from 50-60 from 60-70 from 70-80 from SO-90 90 % alcohol
‘I’A~I,I~
Average
numbers
r-h--. No. of mitoses
chalone alcohol 56 alcohol % alcohol 96 alcohol o/: alcohol
3.3 kO.15 5.7 i 0.22 5.5-to.17 4.3 i-o.13 3.4kO.12 4.5 20.18 5.2 l!I 0.27
VI. Dose-response of mitoses
arrested
Units chalone/ 4 ml medium
s
0.0901 0.001 0.01 0.07 0.10 0.14 0.7 1.0 1.4 7.0 10.0 14.0 100.0
30 45 50 65 40 35 75 169 63 2110 80 35 10
relationship
7-h-y No. of mitoses
Percentage depression
Percentage depression
2.2 + 0.15 5.5 ‘- 0.13 5.4 i 0.20 3.2 2 0.21 2.4io.11 4.0 2 0.20 4.8?0.19
46 7 10 30 44 26 15
64 10 11 4x 61 34 22
of epidermnl rhrrlone.
by colcemid in 4 hr in unit sectioned 7 ,u thick.
lengths
Untreated control epidermis
Cbalonc treated epidermis
7.5 7.0 7.0 6.3 6.4 6.9 6.4 6..5 6.2 6.5 6.4 7.3 6.5
7.3 -t 0.31 6.520.26 5.8 i- 0.30 4.4 + 0.23 4.4 i 0.22 4.7 & 0.35 3.8 * 0.29 3.2 + 0.01 2.9lO.18 2.4 -t 0.09 2.4 k 0.10 2.3 -t 0.23 0.3 + 0.17
* 0.20 -t 0.19 i 0.20 2 0.23 + 0.26 T 0.40 i 0.21 i 0.01 k 0.24 f0.15 -to.15 2 0.54 i 0.32
from 10.0 mg epidermis
of 1 cm ear epidermis
Experimental
Approximate percentage dcprcssion 3 8 17 30 31 32 41 51 53 63 62 69 95
Cell Research
36
W. S. Bullough,
196
C. L. Hewett
and Edna B. Laurence
relatively large molecule and suggests that it might be precipitated with alcohol. In Table V the results of four combined experiments involving alcohol fractionation indicate that most of the chalone remains soluble in 70 per cent alcohol and that most of it precipitates between 70 and 80 per cent alcohol. Holvever, in order to determine more accurately the distribution of the Tanr,e
1’11. Distribution
The quantities
of chalone
of epidermol
present
chalone
in the various
are deduced from Fig. 1 using shown in Table V.
medium
Total water extract of Precipitate from 50 % Precipitate from 50-60 Precipitate from 60-70 Precipitate from 70-80 Precipitate from 80-90 Residue in 90 % alcohol Total chalone accounted
TAHLE
VIII.
Failure
Average
numbers
with chalone alcohol 9/o alcohol “: alcohol % alcohol % alcohol
depressions
from 10.0 mg epidermis
7-W Pcrcentage mitotic depression (Table V)
0.90 -
Chalone concentration (estimated from Fig. 1) units
61 10 11 48 61 34 22
0.10 0.70 0.05
for
mitotic
derired
1.0 mg epidermis -.---. PerChalonc centage concentration mitotic (estimated depression from Fig. 1) (Table 1’) units 46 7 10 30 44 26 15
fractions.
the percentage
Extract
4 ml culture
alcohol
7.80 1.15 5.60 0.18 0.03 6.96
0.85
of TO-80% crlcohol fraction to inhibit mitosis maintained for more than 5 hr in vitro.
of mitoses
arrested by colcemid in 4 hr in unit sectioned 7 ~1 thick. N = 10. Time
after
lengths
in epidermis
of 1 cm ear epidermis
establishment
of cultures 6-9
4 ml culture Alone With With
1 unit 1 unit
Experimental
of fresh of 70-80
medium
2-5
chalone % alcohol
Cell Research
36
fraction
111
6.9 + 0.21 3.9 i- 0.18 3.9 i: 0.23
11r
With original epidermis
With fresh epidermis
7.2 ir 0.19 7.5 i 0.23 7.2 r 0.29
7.0 i 0.42 4.2 k 0.32 3.7 i 0.42
Isolation
of epidernml
chrrlone
197
chalone between the various fractions it was necessary to relate the percentage mitotic depression obtained in each case to the number of units of chalone that are known to produce this depression. A graph relating chalone concentration to epidermal mitotic depression has already been published [4J, IOO-
90 -
80.
30.
20.
I’ig. l.-The relationship between the concentration of epidermal chalone and the induced epidermal mitotic depression.
0.0001
0.001 0.01 Untts epdermal
0.1 chalonel4
1.0 ml medium
10.0
100
but with further evidence now available a new table has been compiled (Table \‘I). These results are shown as a graph in Fig. 1, and from this, using the percentage mitotic depressions shown in Table V, it was possible to estimate the actual quantities of epidermal chalone that were present in the various alcohol fractions. The results are in Table VII and Fig. 2, and it is immediately obvious that by far the greatest chalone concentration was present in the 70P 80 per cent alcohol fraction. However, before this conclusion could be finally accepted it was necessary to show that the mitotic inhibitor present in the 70-80 per cent alcohol fraction was in fact the epidermal chalone. From previous work it was already known that one of the most remarkable characteristics of this chalone is the fact that it requires the presence of adrenalin as a cofactor [4]. It is known that Experimenfal
Cell
Research
36
W. S. Bullouqh,
198
C. L. Hewett
nnd Edna
B. Laurence
when the epidermal chalone is added to a saline culture medium the epidermal mitotic activity is depressed for not more than about 5 hr; I-)?-this time the loss of the endogenous adrenalin into the medium is almost complete [4]. In Table VIII, which contains the combined results of t\\o CYperiments, this point is illustrated both with an aqueous chalone extract and with the 70-80 per cent alcohol fraction. In both cases it was found that during the (ith-9th hr the epidermal mitotic rate returned to normal, although when after 3 hr fresh epidermis (containing adrenalin) was introduced, the original chalone extracts still possessed the power to induce a mitotic depression. In the experiments recorded in Table IS this point is demonstrated even Tanr,~
IS.
Ability
Average
numbers
of 70680 76 alcohol fraction to inhibit mitosis given brief crdrenalin wash after 5 hr in vitro.
of mitoses
arrested
by colcemid in 4 hr in unit sectioned 7 ,u thick. N = 5. Time
after
lengths
in epidermis
of 1 cm ear epidermis
establishment
of cultures 6-9 hr
4 ml culture Alone With With
1 unit 1 unit
5 hr
in
S. Instability
Average
numbers
vitro
fraction epidermis
Experimental
of 70-80
of mitoses
arrested
Fresh Normal 3.6 i 0.32
Cell
7.6*0
8.2 i- 0.86
3.9 + 0.93
3.0 i- 0.32
9.4kO.14
3.1 +0.31
was immersed
Research
36
hr
for
% alcohol fraction for 10 min.
5 min
chalonc
treated
extract
with
in saline
when
by colcemid in 4 hr in unit sectioned 7 ,u thick. N = 5. Epidermis
7.0 IL 0.21
8.4 IO.51
3.8 k 0.42
2-5
chalone % alcohol
TABLE
Untreated control epidermis
7.2 k 0.94
medium
of fresh of 70-80
Note. After adrenalin/ml.
Untreated
After adrenalin wash at 5 hr
with
maintained
lengths
1 unit/4
medium
0.23 pug
at 100°C
of 1 cm ear epidermis
ml medium 70-80
. .56
of
y0 alcohol
fraction
l30iled
Normal
Boiled
6.3 i- 0.31
4.0 -t 0.37
6.8 kO.19
Isolntion
of’ epiderrnrrl
chtrlone
1%
more clearly. After 3 hr in vitro, by which time all the endogenous adrenalin \vas presumed to have been lost, the epidermis \vas washed briefly in a dilute solution of adrenalin and then returned to the original chalone solution. ,A second mitotic depression was then obtained both in the case of the aqueous chalone extract and of the 50-80 per cent alcohol fraction.
Pig. 2.-The concentrations of cpidermal present in the various alcohol fractions.
chalone 50
60
70 Alcohol
80 fmtlon
90
100
These results are in themselves sufficient to sho\v that the mitotic inhibitor present in the 70-80 per cent alcohol precipitate is indeed the epidermal chalone. However, in one final test, recorded in Table S, it was demonstrated that the active agent present in the 70-80 per cent precipitate can be destroyed by boiling for 10 min. SUMMARY
AND
CONCLUSIONS
1. The epidermal chalone, which with adrenalin as cofactor evidently depresses the epidermal mitotic rate in an adult mouse, is shown to be water soluble, non-dialysable, precipitated by alcohol, and destroyed by boiling. It does not survive long in lvater solution, even at - 2O”C, but it has been shown to survive for at least a year when lyophilised. 2. Preliminary attempts have been made to purify the water extract by fractional precipitation with alcohol. It has been found that more than 80 per cent of the chalone recovered was present in the precipitate obtained \vhen the alcohol concentration was raised from 70 to X0 per cent. Experimental
Cell
I
36
200
u’. S. Bullough,
C. I,. Hewett
3. These results are not inconsistent chalone may prove to be a protein.
and Edna B. Laurence
with
the possibility
that the epidermal
We are grateful to Organon Laboratories Limited for their continued this research, to the Central Research Fund of the University of London for equipment, and to Dr. R. P. Edkins who lyophilised the extracts. REFERENCES 1. BULLOUGH,
TV. S., Biol. Reu. 37, W. S. and LAURENCE, 24, 289 (1961).
2. BULLOUGH, 3. __ ibid. 4. __ ibid.
33,
176
5. SAETREN,
H.,
Ezptl
Experimental
Cell
307
(1962).
E. B., Et@
(1964).
Research
Cell Res.
36
11, 229 (1956).
Cell
Res.
21,
394
(1960).
support of for a grant
192
THE A
W.
PRELIMINARY
S. BULLOUGH,
Birkbeck
EPIDERMAL
College,
AT
and
EDNA
University of London, and Organon Newhouse, Lancasshire, Scotland Received
Ilovember
36, 192-200
(1964)
CHALONE:
ATTEMPT
C. L. HEWETT
Cell Research
ISOLATION
B. LAURENCE Laboratories
Limited,
26, 1963
RECENTLYBullough
and Laurence [4] have described the inhibitory action of epidermal extracts on the mitotic activity of mouse ear epidermis. They have concluded that in normal epidermis mitotic control is exercised through a diffusible inhibitor, for which the name of epidermal chalone has been proposed [ 11. The epidermal chalone is probably produced within the epidermal cells and it is evidently tissue-specific in its action. It is already apparent that at least three distinct chalones may be produced in mouse skin [‘L], and there is also evidence that others may exist for instance in kidney and liver [I, 3]. It now appears possible that each tissue in the body may product its own specific chalone. A second important point that has been established is that in the absence of adrenalin the epidermal chalone loses its full power to inhibit epidermal mitosis [4]. It appears that the actual mitotic inhibitor may be an unstable chalone-adrenalin complex. The experiments described below record the preliminary attempts that have been made to isolate the epidermal chalone and to determine something of its physical properties.
MATERIAL
AND
METHODS
The epidermal chalone was extracted from epidermis taken from the backs bf adult Strong CBA mice. The epidermis was macerated according to the technique described by Bullough and Laurence [4] and the chalone was obtained in a clear aqueous solution after spinning in a refrigerated centrifuge. Except where otherwise stated, all the extracts were freshly made for each experiment. For easein comparing different extracts, the chalone concentrations were estimated in terms of a standard preparation in which 1 unit was arbitrarily considered as the activity contained in an aqueous extract of 1 mg dry weight of mouse epidermis (including the stratum corneum). In the present type of experiment, 1 unit is also the amount which induces a mitotic depression of approximately 50 per cent. Experimental
Cell Research
36
Isolution
of epidermcrl
chrrlone
193
The inhibitory action of the chalone was tested on the ear epidermis of adult male Strong CBA mice 4-6 months old. The epidermis was maintained in vi&o in a phosphate buffered saline medium with 0.02 M glucose and an oxygen gas phase (for details see [3]). In all cases colcemid was added to arrest the developing mitoses in the metaphase. It should be noted that in all the tables N represents the number of animals used; standard errors are given with the means. RESULTS
The preliminary experiments recorded in Table I are included merely to demonstrate that the epidermal chalone is present in the supernatant after centrifugation and that it is therefore soluble in water. The chalone can be extracted either with distilled Lvater or with normal saline, but since it has recently been shown that inorganic salts may account for a large proportion of the total epidermal water extract, the distilled Avater may have behaved in effect as a saline solution. However, from other experiments described belo\\-, it is clear that the chalone is soluble in salt-free water. T’AI
1. Inhibitory
erect
of aqueous marl mitotic
extrcrcts crctioity
of macerated in vitro.
epidermis
on epider-
Average numbers of mitoses arrested by colcemid in 4 hr in unit lengths of 1 cm ear epidermis sectioned 7 p thick. A’ = 10. Culture medium with
0
Total macerate Supernatant
6.0 & 0.33 6.2 + 0.34 ‘I’.~BLF: II. Attempted
Concentrations of chalone/4 ml culture medium 0.1 unit 1.0 unit 10.0 units 4.5 * 0.34 4.6kO.33 dialysis
3.1 + 0.16 3.4iO.18
of’ epidermcxl
2.1 10.29 2.5 1- 0.18
chnlone.
Average numbers of mitoses arrested by colcemid in 4 hr in unit lengths of 1 cm ear epidermis sectioned 7 p thick. N = 20. Culture medium with
0
Fresh chalone Dialysed solution Dialysate
6.2iO.18 6.2rir0.18 S.Z?O.lS
Assumed concentration 0.1 unit 4.1+ 0.46 3.910.37 6.0 IL 0.64
of chalone/4 ml medium 1.0 unit 10.0 units 3.3 * 0.17 3.4 ir 0.22 6.5 & 0.24
1.9io.27 2.4&0.31 5.9 IO.34
Note. In estimating the chalone content of the dialysed solution and of the dialysate it was assumed that the chalone was equally distributed between them. 13 -
641806
Experimental
Cell Keseurch
36
194
U’. S. Bullough,
C. I,. Hewett
and Ednn B. I,rrurence
Experiments were nest undertaken to determine whether the epidermal chalone is dialpsable. A solution of 930 units of epidermal chalone in 400 ml water \vas dialysed against 70 ml water at about 3°C for 2-l hr using a WIlophane membrane. Two separate experiments were performed and the combined results are shown in ‘Table Il. From this it is evident that the epidermal chalone did not pass through the membrane. In Table III the efl’ects of boiling the chalone solution are recorded. The xvater extract was raised to lOO”C, maintained at this temperature for 10 min, and then rapidly cooled and used at once. This treatment caused a complete loss of the antimitotic action. Early in this investigation it was important for practical purposes, and especially for the preparation of a stable standard extract, to discover ho\\ best the epidermal chalone might be preserved. It was found that a water solution loses its activity within several days at about 0°C and within several weeks at - 20°C. However, it was found that the activity can be preserved by freeze-drying. The figures in Table IV refer to a chalone extract that had been freeze-dried and kept for only ‘2 lveeks, but similar results have since TanrAE III. Average
Instability
numbers
of epidernml
of mitoses
chrxlone when 10 min.
arrested by colcemid in 4 hr in unit sectioned 7 p thick. LV - 10. Epidermis
Untreated control epidermis
Fresh
numbers
of mitoses
of
epidermnl
medium
with
0
Fresh chalonc Freeze-dried chalone iVote. Experimental
The
chalone Cell
chalone after
10 units
5.5 -t 0.37
lyophilisation.
lengths
of chalone/d
ml medium
of 1 cm car epidermis
ml medium
0.1 unit
1 .O unit
10.0 units
6.2 F 0.27
4.5 + 0.39
3.4 i- 0.29
2.6 i 0.26
6.2 IL 0.27
3.5 I- 0.28
3.0 i 0.47
2.6i0.37
was lyophilised Research
chalonc/~f
6.3 +0.34
Concentration Culture
for
of 1 cm car epidermis
1 unit
arrested by colcemid in 4 hr in unit sectioned 7 p thick. A’ = 10.
nt 100°C
with ISoiled
2.1-to.17
Stability
I\'.
treated
10 units
3.6 k 0.32
TABLE
lengths
ml medium
1 unit
6.2 20.32
Average
chalone/l
maintained
36
2 vvecks
before
assay.
Isolrrtion
of epiclermrrl chnlone
193
been obtained with lyophilisetl epidermal extracts after 3, 6 and 12 months. It may he noted that, for convenience, the chalone extracts were lgophilisetl with mannitol, which alone was without any effect. The fact that the chalone does not dialyse indicates that it possesses a T.\I~I,kI Average
numbers
\‘.
Alcohol
of mitoses Average
arrested sectioned numbers
fractionation
of epidermrrl
by colcemid in 4 hr in unit 7 ,U thick. LV = 24 throughout. of mitoses
in untreated
chtrlone.
lengths
control
of 1 cm ear epidermis
= 6.1 k 0.22.
Extract
derived
1.0 mg epidermis
4 ml culture Total water Precipitate Prrcipitate Precipitate Prccipitatc Precipitate I
medium
with
extract of from 50 % from 50-60 from 60-70 from 70-80 from SO-90 90 % alcohol
‘I’A~I,I~
Average
numbers
r-h--. No. of mitoses
chalone alcohol 56 alcohol % alcohol 96 alcohol o/: alcohol
3.3 kO.15 5.7 i 0.22 5.5-to.17 4.3 i-o.13 3.4kO.12 4.5 20.18 5.2 l!I 0.27
VI. Dose-response of mitoses
arrested
Units chalone/ 4 ml medium
s
0.0901 0.001 0.01 0.07 0.10 0.14 0.7 1.0 1.4 7.0 10.0 14.0 100.0
30 45 50 65 40 35 75 169 63 2110 80 35 10
relationship
7-h-y No. of mitoses
Percentage depression
Percentage depression
2.2 + 0.15 5.5 ‘- 0.13 5.4 i 0.20 3.2 2 0.21 2.4io.11 4.0 2 0.20 4.8?0.19
46 7 10 30 44 26 15
64 10 11 4x 61 34 22
of epidermnl rhrrlone.
by colcemid in 4 hr in unit sectioned 7 ,u thick.
lengths
Untreated control epidermis
Cbalonc treated epidermis
7.5 7.0 7.0 6.3 6.4 6.9 6.4 6..5 6.2 6.5 6.4 7.3 6.5
7.3 -t 0.31 6.520.26 5.8 i- 0.30 4.4 + 0.23 4.4 i 0.22 4.7 & 0.35 3.8 * 0.29 3.2 + 0.01 2.9lO.18 2.4 -t 0.09 2.4 k 0.10 2.3 -t 0.23 0.3 + 0.17
* 0.20 -t 0.19 i 0.20 2 0.23 + 0.26 T 0.40 i 0.21 i 0.01 k 0.24 f0.15 -to.15 2 0.54 i 0.32
from 10.0 mg epidermis
of 1 cm ear epidermis
Experimental
Approximate percentage dcprcssion 3 8 17 30 31 32 41 51 53 63 62 69 95
Cell Research
36
W. S. Bullough,
196
C. L. Hewett
and Edna B. Laurence
relatively large molecule and suggests that it might be precipitated with alcohol. In Table V the results of four combined experiments involving alcohol fractionation indicate that most of the chalone remains soluble in 70 per cent alcohol and that most of it precipitates between 70 and 80 per cent alcohol. Holvever, in order to determine more accurately the distribution of the Tanr,e
1’11. Distribution
The quantities
of chalone
of epidermol
present
chalone
in the various
are deduced from Fig. 1 using shown in Table V.
medium
Total water extract of Precipitate from 50 % Precipitate from 50-60 Precipitate from 60-70 Precipitate from 70-80 Precipitate from 80-90 Residue in 90 % alcohol Total chalone accounted
TAHLE
VIII.
Failure
Average
numbers
with chalone alcohol 9/o alcohol “: alcohol % alcohol % alcohol
depressions
from 10.0 mg epidermis
7-W Pcrcentage mitotic depression (Table V)
0.90 -
Chalone concentration (estimated from Fig. 1) units
61 10 11 48 61 34 22
0.10 0.70 0.05
for
mitotic
derired
1.0 mg epidermis -.---. PerChalonc centage concentration mitotic (estimated depression from Fig. 1) (Table 1’) units 46 7 10 30 44 26 15
fractions.
the percentage
Extract
4 ml culture
alcohol
7.80 1.15 5.60 0.18 0.03 6.96
0.85
of TO-80% crlcohol fraction to inhibit mitosis maintained for more than 5 hr in vitro.
of mitoses
arrested by colcemid in 4 hr in unit sectioned 7 ~1 thick. N = 10. Time
after
lengths
in epidermis
of 1 cm ear epidermis
establishment
of cultures 6-9
4 ml culture Alone With With
1 unit 1 unit
Experimental
of fresh of 70-80
medium
2-5
chalone % alcohol
Cell Research
36
fraction
111
6.9 + 0.21 3.9 i- 0.18 3.9 i: 0.23
11r
With original epidermis
With fresh epidermis
7.2 ir 0.19 7.5 i 0.23 7.2 r 0.29
7.0 i 0.42 4.2 k 0.32 3.7 i 0.42
Isolation
of epidernml
chrrlone
197
chalone between the various fractions it was necessary to relate the percentage mitotic depression obtained in each case to the number of units of chalone that are known to produce this depression. A graph relating chalone concentration to epidermal mitotic depression has already been published [4J, IOO-
90 -
80.
30.
20.
I’ig. l.-The relationship between the concentration of epidermal chalone and the induced epidermal mitotic depression.
0.0001
0.001 0.01 Untts epdermal
0.1 chalonel4
1.0 ml medium
10.0
100
but with further evidence now available a new table has been compiled (Table \‘I). These results are shown as a graph in Fig. 1, and from this, using the percentage mitotic depressions shown in Table V, it was possible to estimate the actual quantities of epidermal chalone that were present in the various alcohol fractions. The results are in Table VII and Fig. 2, and it is immediately obvious that by far the greatest chalone concentration was present in the 70P 80 per cent alcohol fraction. However, before this conclusion could be finally accepted it was necessary to show that the mitotic inhibitor present in the 70-80 per cent alcohol fraction was in fact the epidermal chalone. From previous work it was already known that one of the most remarkable characteristics of this chalone is the fact that it requires the presence of adrenalin as a cofactor [4]. It is known that Experimenfal
Cell
Research
36
W. S. Bullouqh,
198
C. L. Hewett
nnd Edna
B. Laurence
when the epidermal chalone is added to a saline culture medium the epidermal mitotic activity is depressed for not more than about 5 hr; I-)?-this time the loss of the endogenous adrenalin into the medium is almost complete [4]. In Table VIII, which contains the combined results of t\\o CYperiments, this point is illustrated both with an aqueous chalone extract and with the 70-80 per cent alcohol fraction. In both cases it was found that during the (ith-9th hr the epidermal mitotic rate returned to normal, although when after 3 hr fresh epidermis (containing adrenalin) was introduced, the original chalone extracts still possessed the power to induce a mitotic depression. In the experiments recorded in Table IS this point is demonstrated even Tanr,~
IS.
Ability
Average
numbers
of 70680 76 alcohol fraction to inhibit mitosis given brief crdrenalin wash after 5 hr in vitro.
of mitoses
arrested
by colcemid in 4 hr in unit sectioned 7 ,u thick. N = 5. Time
after
lengths
in epidermis
of 1 cm ear epidermis
establishment
of cultures 6-9 hr
4 ml culture Alone With With
1 unit 1 unit
5 hr
in
S. Instability
Average
numbers
vitro
fraction epidermis
Experimental
of 70-80
of mitoses
arrested
Fresh Normal 3.6 i 0.32
Cell
7.6*0
8.2 i- 0.86
3.9 + 0.93
3.0 i- 0.32
9.4kO.14
3.1 +0.31
was immersed
Research
36
hr
for
% alcohol fraction for 10 min.
5 min
chalonc
treated
extract
with
in saline
when
by colcemid in 4 hr in unit sectioned 7 ,u thick. N = 5. Epidermis
7.0 IL 0.21
8.4 IO.51
3.8 k 0.42
2-5
chalone % alcohol
TABLE
Untreated control epidermis
7.2 k 0.94
medium
of fresh of 70-80
Note. After adrenalin/ml.
Untreated
After adrenalin wash at 5 hr
with
maintained
lengths
1 unit/4
medium
0.23 pug
at 100°C
of 1 cm ear epidermis
ml medium 70-80
. .56
of
y0 alcohol
fraction
l30iled
Normal
Boiled
6.3 i- 0.31
4.0 -t 0.37
6.8 kO.19
Isolntion
of’ epiderrnrrl
chtrlone
1%
more clearly. After 3 hr in vitro, by which time all the endogenous adrenalin \vas presumed to have been lost, the epidermis \vas washed briefly in a dilute solution of adrenalin and then returned to the original chalone solution. ,A second mitotic depression was then obtained both in the case of the aqueous chalone extract and of the 50-80 per cent alcohol fraction.
Pig. 2.-The concentrations of cpidermal present in the various alcohol fractions.
chalone 50
60
70 Alcohol
80 fmtlon
90
100
These results are in themselves sufficient to sho\v that the mitotic inhibitor present in the 70-80 per cent alcohol precipitate is indeed the epidermal chalone. However, in one final test, recorded in Table S, it was demonstrated that the active agent present in the 70-80 per cent precipitate can be destroyed by boiling for 10 min. SUMMARY
AND
CONCLUSIONS
1. The epidermal chalone, which with adrenalin as cofactor evidently depresses the epidermal mitotic rate in an adult mouse, is shown to be water soluble, non-dialysable, precipitated by alcohol, and destroyed by boiling. It does not survive long in lvater solution, even at - 2O”C, but it has been shown to survive for at least a year when lyophilised. 2. Preliminary attempts have been made to purify the water extract by fractional precipitation with alcohol. It has been found that more than 80 per cent of the chalone recovered was present in the precipitate obtained \vhen the alcohol concentration was raised from 70 to X0 per cent. Experimental
Cell
I
36
200
u’. S. Bullough,
C. I,. Hewett
3. These results are not inconsistent chalone may prove to be a protein.
and Edna B. Laurence
with
the possibility
that the epidermal
We are grateful to Organon Laboratories Limited for their continued this research, to the Central Research Fund of the University of London for equipment, and to Dr. R. P. Edkins who lyophilised the extracts. REFERENCES 1. BULLOUGH,
TV. S., Biol. Reu. 37, W. S. and LAURENCE, 24, 289 (1961).
2. BULLOUGH, 3. __ ibid. 4. __ ibid.
33,
176
5. SAETREN,
H.,
Ezptl
Experimental
Cell
307
(1962).
E. B., Et@
(1964).
Research
Cell Res.
36
11, 229 (1956).
Cell
Res.
21,
394
(1960).
support of for a grant