Download of the hypophysis

HORMONES I N CANCER
T'ITT.
INFLUENCE
OF THE HYPOPHYSIS
FRITZ BISCHOFF, L. C. MAXWELL
AND
H. J. ULLMANN
WII'H A HI8T0rAWIC.4L REPORTBY
RICHARD D. EVANS
(From the D e p w f m e n t of Cancer Rrsearck of t h e 8aqita Barbara Cottage Hospital, H . J .
Ullmniir~,Dirrctor 1)
The present paper is a summation of results obtained leading to the
conclusion that the pituitary gland may be concerned in neoplastic
extension. Preliminary reports (1, 3, 3) of our earlier findings have
been presented, and it was originally inteiided that further publication
should be withheld until the results warranted some degree of finality
as to conclusion. In the meantime others (4,5, 6 ) have entered the
field and new relations ( 7 ) of the anterior lobe hormones to each other
have been established. It appeared timely, therefore, to present our
evidence demonstrating the effect of the pituitary, since it is now clear
that more ground must be covered before the mechanism of the hormonal influence of the pituitary upon neoplastic extension is elucidated.
PLANOF STUDY
I n studying the possible rSle of the pituitary in neoplastic extension,
two methods are feasible: the active glandular principles may be administered, or the gland or fraction o€ the gland elaborating the active
principle may be removed, and the eflects upon tumor growth noted.
We have resorted to roentgen irradiation of the pituitary region in an
attempt to inhibit the normal function of the gland and have studied
the effect of dosage of all the established actire principles of the gland,
Transplantable rat sarcoma R10, mouse sarcoma 180, rat carcinoma
256, and a spontaneous mammary mouse carcinoma were used in demonstrating the influence of the followiiig experimental procedures upon
the growth behavior of the neoplasm :
(1)Pituitary irradi.c l t'1011.
(2) Parenteral administration of standardized growth-promoting
pituitary extracts, of standardized Fonad-stimulating extracts of the
pituitary, or of standardized urine of pregnancy preparations.
1 The completion of these studies was nicide possiblc by a graiit from tlic International
Cancer Research Foundation, Wm. H. Donner, Preu.
2 Presented a t the Anniial Meetings of tllc Federition of Anierirnn Societics for Experimental Biology, Montrc:tl, April 1941, and Philadelphia, April 1933.
329
330
FRITZ BISCHOFF, L. C. MAXWELL AND H. J. ULLMANN
(3) Pituitary irradiation combined with the administration of the
extracts described above.
A pathological study of the pituitaries, of the gonads, and of the
related glands of the experimental animals was made.
Measures of Variability: Because of individual variation in the
growth both of transplantable and spontaneous neoplasms, it is impossible to decide whether or not an experimental procedure affects tumor
growth unless measures of variability are applied. I n all of our experiments a control series of litter mates was inoculated from the same
tumor, and the tumor growth in the treated animals compared with the
controls. This procedure is essential, as tumor growth depends not
only upon the age of the animal, and possibly upon the species factor,
but also upon the material used for the graft. Tumor size was recorded as the mean of the two measurements made at right angles.
The mean was calculated in the usual manner, and the standard deviation of the mean was calculated by the formula
where d is the mean tumor diameter and n the number of animals in
the series. When a comparison of control and treated series was made,
the square root of the sum of the squares of the individual standard
deviations was calculated, and three times this figure was assumed to be
significant beyond reasonable probability. I n most of our experimental groups the number of tumor-bearing animals approximated
twenty, an equal distribution between controls and treated animals
being attempted. An error is introduced by applying the formula to
the relatively small numbers in the series. For this reason three times
instead of twice the difference has been taken as the limit of significance. Whenever a significant result was obtained, the experiment was
repeated. In a colony of rats of the size a t our disposal (500), it is
impractical to study groups of more than thirty, because of the difficulty of grouping animals of the same age.
I n comparing the tumor data of others with our results we have
taken the liberty, in those instances where tumor weight or area is
given, of recalculating such data as mean tumor diameters. The error
involved in such a recalculation is slight. The apparent difference is,
of course, much greater when the data are presented as areas, volumes,
or weights than as diameters. Thus an eightfold difference in tumor
weight represents only a 100 per cent difference in tumor diameter.
EXPERIMENTAL
Preparation
Growth-prom,oting Eztracts: The extracts containing the growthpromoting principle were prepared from beef pituitary anterior lobes
according to the methods of Bugbee et a1 (8), of Van Dyke and Wdlen-
HORMONES IN CANCER
331
Lawrence (9), or of Evans (10). A preparation supplied by Doctor
Kamm of Parke, Davis L Company was also used. The potency of the
extracts was established upon female rats according to the method of
Evans and Simpson (10). This method was not described a t the time
of our early standardizations, which depended upon a significant increase in body weight of groups of female and male rats dosed with the
extract as compared with controls. Standardization of the extracts
was initiated at the time the preparation was administered to the tumorbearing animals, thus eliminating the factor of possible deterioration
in potency. Extracts prepared according to the method of Bugbee et al.
were found toxic to mice, which died following a week of daily injection,
This extract was also toxic to one series of young rats, f o r after a
twenty-day period the dosed rats weighed less than the controls. I n
older rats the toxic effect was not observed, and acceleration of growth
occurred.
Gonadotropic Elr;tracfs: The anterior lobe gonad-stimulating extracts from sheep pituitaries were prepared and standardized according to the methods of Wallen-Lawrence and Van Dyke (11, 12). The
preparation from the urine of pregnancy was made according to
Dickens (13).
Efect of Pituitary Irradiation
A summation of the results of pituitary irradiation is given in Table
L3 Only animals with non-regressing tumors are considered, and data
concerning animals which succumbed to the irradiation are eliminated.
It is apparent from the data in Table 1, that the rate of growth of all
four types of tumors studied may be significantly retarded by pituitary
irradiation, but the effect is observed only when body growth is virtually arrested. Moreover the effect is transient, and massive dosage
of irradiation is required to produce the necessary retardation of body
growth. Protocols of the various series follow.
Prolocols
I n series I1 (See Table I), the roentgen dosage proved fatal f o r over 50 per cent
of the rats, Tumor inoculation (carcinoma 256) was performed the fifth day following
the initial dosage of irradiation. Body growth was stopped for a period of seventeen
days. I n the following period the gain in weight f o r controls and rayed rats was the
same. The rate of tumor growth was marlredly retarded during the period when the body
growth was inhibited.
I n series I radon seeds containing 2 millicuries in 0.3 mm. gold were implanted at
the base of the skull in the region of the pituitary, a small piece of skull at the top of the
3 In the pitnitary irradiation experiments the roentgen factors were F. D. 37 em., K. V. P.
200, M. A. 30, A1 2 mm. In experiments I T , l V , V, VI, and X (rat series) a 0.25 mm. Cu Alter
was used in addition. All nieasurenients of T were made in air with a Victoreen condenser
r-meter. I n experiments I1 and I V (rat series) the head of the animal was irradiated. I n
experiments VIIT, VI, IX, XI, and XITI, the area unshielded by lead over the pituitary approximated 1.42 sq. em. I n d l other rat experiments described in this paper the area exposed
approximated 0.64 sq. cm. For the mome the area irradiated was between 0.4 and 0.5 sq. em.
In all irradiation experinmiits the entire body of the animal with the exception of the area
exposed over the pituitary region was shielded with lead.
tG
w
cc
Control 15
Rayed 6
Control 7
Rayed 5
See footnote on page 331.
1x1
VIII'
180
180
Cont,rol 13
Rayed 10
IV
R-10
Control 8
Rayed 7
Control 10
Rayed 8
IX
R-10
Control 10
Rayed 8
\'I
256
Control 9
Rayed 8
Control 14
Rayed 14
VIII
256
XIX
Control 13
Rayed 8
1
256
256
Control 21
Rayed 9
I1
series
256
Tumor
Number
of
Tumors
25
24
f 1.9
6.0 f 0.7
5.0 f 1.0
f0.7
5.5 f 1.0
8
14
15
18 f 1.5
11 f 1.6
10 f 1.0
10 f 1.1
15
14
f2.3
f2.4
10.0 f 1.0
9.3 f0.9
13 f 1.3
10 f 1.0
32
32
32
25
21
21
11 f0.8 27 f 1.9
7 f 1.0 18 f 2 . 4
12 f0.6
8 f0.9
f 1.7
f 1.6
13th to
17th day
19 f 1.1 41
11 f0.9 35
7th to
12th day
f3.7
f3.0
16.3 f 1.2
15 f 1.6
38
38
39
34
42
37
31
29
36 f 2.1
23 f 3 . 1
18th to
23d day
27 f 2.0
19 f 1.5
40th day
Tumor Diameters in Millimeters
-
2.0
+ 2.2
- 4
+ 2.5
+ 9
+ 5
- 4
+29
+=
+ 9
+ 2.3
+ 0.9
+54
+23
+47
- 4
+31
+53
0
+28
+ 8
+37
- 4
+28
+46
+54
+=
13th to
17th day
+12
+27
+ 6
+33
+ 3
7th to
12th day
+ 3
0
+69
+ 3
+58
+26
+57
+1
18th to
23d day
Body Weight Increments in Grams
TABLEI: Efl& o j Pituiiury Irradiation Upon Rate of Tumor and Body Growth *
r
4000r
4000r
4000to12000r
W
8000to18000r
20000to23000r
3300 r
5OOO to 27000 r
Radon seed implants
3500 r
Remarks
IIORMONWS IN CANCER
333
head having been reniovecl SO that the needle containing the seed could be inserted. Eight
out of ten rats so treated survived the treatment. Seven showed no ill effects except cesHation of growth f o r thirteen days. Tumor inoculation was made five days after radon
implantation, a group of litter mates serving as controls. The results show that the rate
of tumor growth was significantly retarded at the time when the body growth was inhibited. The first measurements (not given in Table I ) differ by 6 times the standard
deviation of the mean. The fourteenth day of tumor life the controls and treated animals
were again growing a t the same rate. A t this date the tumors of the treated animals had
reached the size of the controls.
I n several series of experiments, including V I and I V (Table I ) , irradiation showed
no effect upon tlie rate df tumor growth. I n these experiments the hody weight curve
was retarded as (.ompared with the controls, hiit an appreciable increase in weight did
occur. I n one of these experiments sufficient irradiation was given a t a later period so
that a decrease in body wright occurred during the latter period of tumor life. I n one
experiment the caiidal extrcmities were i*agetlwith the same dosage applied to the heads of
a group of litter m&s.
A n appreciable loss of body weight, followed later by an increase, was observed, but no effect upon tumor growth was notctl.
I n series W I I and IX the weights of tlie rayed rats were kept relatively constant
over a period of twenty-four days, (luring which period the hody weights of the control
rats doubled. I n some of the rayed rats no increaqe in weight occurred even after this
period. One series was inoeulated with sarcoma, the other with carrinoma. I n both
these series irradiation wos given five (lays before tumor inoculation. I n series V I I I twothirds the dose was repeated ten days later, while in Series IX the interval between the
first and second rayings was five days. A third dose was given in series V I I I about the
twentieth day after inoculation. This proved lethal to tive of the animals. I n the rats
inoculated with carcinoma a decrease in rate of tumor growth equalling or exceeding 3
times the standard deviation of the mean was observed the first twenty days of tumor life.
In the rats inoculated with sarcoma a decrease in rate of tumor growth equalling 3 times
the standard deviation of the mean was observed a t the firht measurement. At a later date
four spontaneous regressions occurred in the controls, one in the rayed rats.
The animals in series XTX, approximately thirty (lays of age, were divided into three
groups: 16 controls, 13 subjected to 5000 r, 200 K. V. P., 2 mm. Al, and 12 given 2800 r,
125 K. V. P., unfilteretl. Jn the firbt ten-day period the rats subjected to the longer wavelength dosage gained 23 gm. (mean) in weight and the controls 32 gm. For the corresponding ten-day period the rats subjected to the shorter Wavelength irradiation showed a
significant retardation in body weight. All the rats were subsequently subjected to the
higher voltage irradiation. The retardation of body weight following the dosage of
6000 r was not permanent. About the thirteenth day after tlosage a significant increase
in weight was noted. The i-nying was therefore repeated a t ten-day intervals and was
increased to 8000 r a t the last raying. Six (lays after the last irradiation the rats, with
their litter mate controls, iind a series of 14 thirty-day-old rats were inoculated with carcinoma 256. Unfortunately the percentagc of tumor takes was low, only 43 per cent f o r
the young controls, 19 per cent f o r the old litter mate controlh, and 40 per cent f o r the
irradiated rats. The tumors which developed were rapidly growing, approximating 4 cm.
in diameter the nineteenth (lay of tumor life, there being no significant difference between
the control and irratliatctl heries. The treated rats gained appreciably in body weight.
Simpson Spontaneous Marn wavy Carcinonaa: I n many of tlie ani-
mals studied multiple tumors and metastases developed during the
course of the experiment. The effect of the simultaneous development
of two or more tumors added to the complexity of correlation, so that
data f o r multiple tnmom are not considered. The controls were
divided into two groiips, since it WNH obvious that certain tumors appearing in the cervical and adjacent regions developed more slowly
than tumors appearing in the more usual regions of mammary gland
334
FRITZ BISCBOFF, L. C. MAXWELL AND H. J. ULLMANN
TABLE
I1 ; Effect of Pituitary Irradiation Upon Growth of the Spontaneous Mammary Mouse
Carcinoma
Controls
NO.
57
47
75
92
32
61
69
35
58
76
94
91
89
29
69
37
67
48
77
73
Rate of Tumor Growth Beginning with a Mean Diameter of 10 mm.
Location of Tumor
20 days
30 days
40 days
Right ant. mammary
Right sec. mammary
Left ant. mammary
Right ant. mammary
Left inguinal mammary
Left inguinal mammary
Left inauinal mammary
Right &t. mammary
Left axillary
Right auricular
Right shoulder
Left parotid
Right shoulder
Right parotid
Right parotid
Left shoulder
Cervical
Right shoulder
Left parotid
Left parotid
50days
25
16
17
17
24
20
15
18
27
20
20
20
30
26
17
23
23
26
26
23
37
30
19
25
30
19 f 1.3
23 f 1.5
27 f2.0
24
13
12
17
16
12
21
16
20
19
25
18
29
14
13
18
17
13
16
24
26
33
16
14
18
23
14
28
17
27
28
21
23
19.5 f 1.6
22 f 2.0
18 f 1.3
25
32
36
22
17
18
31
33
20
31
25
Pituitary Rayed
23
68
64
26
88
96
98
78
43
14
93
24
Right pelvic
Left last mammary
Post mammary
Right post. mammary
Left post. mammary
Right last mammary
Left post. mammary
Left post. mammary
Left hip
Ant. mammary
Cervical
Post-cervical
20
17
17
10
11
16
16
16
13
17
22
20
21
10
15
23
17
17
14
19
21
26
13
17
23
34
20
22
21
16
20
28
15 f 1.0
18 f 1.4
19 f 1.5
19
22
20
26
22
28
development. Because of the possible effect of back-scattering, animals with cervical tumors were placed mostly in the control groups.
The rate of tumor growth, measured as the mean tumor diameter beginning from the time when the tumors averaged 10 mm. in diameter, is
given in Table 11. In many instances the tumor was not discovered
until a diameter of 10 mm.had been reached.
HORMONES IN CANCER
335
The rate of tumor growth in mice which had been subjected to
pituitary radiation during the course of the period of observation is
significantly less ( 3 times the standard deviation of the mean) as compared with the rate of growth of unirradiated tumors located in similar
tissues. The difference is not significant when the comparison is made
with the cervical group. See Table 11.
Efect of Grozoth-Promoting Eat racts of the Pituitury
The effect of the growth-promoting principle of the anterior lobe
upon rate of tumor growth was studied in four series of rats inoculated
with carcinoma 256 and one series with sarcoma R10, and in three series
of mice inoculated with sarcoma 180. See Table 111.
The results are most convincing in the series bearing the rat carcinoma. No effect was observed when the experiment was initiated
on 75 gm. rats. I n one series of 125 gm. rats and in one series of 250
gm. rats, the accelerating effect of early injection of the preparation
on tumor growth was marked (the difference between tumor growth of
dosed and control animals equalling 3 times the joint standard deviation
of the mean). It should be noted that the administration of the extract
had no appreciable body growth-accelerating effect upon the 75 and 125
gm. rats, one toxic preparation finally producing a retardation of body
growth. Jn the series of old rats, body weight increased 24 gm. in
twenty days, the controls losing 2 gm. during the same period.
The effect of the growth-promoting principle was less marked in the
series of sarcoma-bearing rats and mice. The individual variation in
rate of tumor growth for these particular series is large. While the
mean f o r each series of dosed animals exceeds the mean for the controls,
the difference never equals 3 times the joint standard deviation of the
mean. The results, therefore, would not be significant if the variation
were not in the same direction in each case. It should be noted that the
extracts had no appreciable effect on body growth in the mice series,
even in the series of old mice (30 gm.). A growth-promoting extract
was tested upon five mice bearing spontaneous mammary tumors, another five serving as controls. N o effect was observed.
The cessation of dosage with the growth hormone in rats is followed
by an abrupt loss in body weight. While the studies of Richter and
Wislocki (14) show rather conclusively that water balance is not the
factor in body weight increase, we have nevertheless taken the precaution to eliminate this factor in our tumor growth studies. The solid
content of the tumors of dosed and control rats of a series showing a
significant effect of hormone injection upon tumor growth was determined (see Table 1V for examples). Hydration obviously cannot account for the difference in tumor size.
If the retardation of body and tumor growth following irradiation
of the pituitary body is due to lack of production of growth hormone,
the administration of growth hormone to animals so treated should
result in an acceleration of body and tumor growth.
6.5 f 0.6
8.7 f 1.6
8.3 f 1.0
8.0 f 1.2
4.0 f 1.0 9.0 f 2.1
6.0 f 1.1 9.6 f 1.4
8
8
10
8
8
Controls
V.D., Evans
Controls
Evans, P.D.
Mouse
180
Old Mice
180
19 f4.4
22 f 2 . 6
21 f 3 . 0
23.7 f3.8
18.7 f 4.4
26.5 f4.5
f2.2
f3.8
f3.1
19
22
24
18
-1oto 1
-1oto 1
10 to 20
10 to 20
2.3
2.4
10 to 25 1.9 f0.3
10 to 25 2.4 f0.5
14 to 22 3 f 2.6
14 to 22 10 f3.0
- 2
+24
23
23
5 to 15
5to15
10 to 30
10 to 30
AG
Period
18
1 to 12
1 to 12
10 to 35
10 to 35
20 to 30
20 to 30
39
48
5 to 25
5t025
5.0 f 0.6
6.6 f0.9
11
11
7
AG
Period
Body Weight Gain in Grams
V. D. for Van Dyke, P. D. for Parke, Davis and Company.
13 f 3 . 0
17 f1.8
17.5 f2.0
20.4 f3.0
Abbreviations:
12 f2.0
13 f 1.9
The period expressed in days begins with the day of inoculation.
7
9.9 f 1.5
15.1 f 2.6
f4.3
Controls
Bugbee, V.D.
33 f4.0
40 f 5.0
40
45
Mouse
180
R-10
f 1.6
f 0.6
f3.0
f4.5
8
6
Controls
Evans, P.D.
Rat
20
35
17 f 1.6
23 f 3 . 0
7
6
8
10
Controls
Evans, P.D.
256
Rat (250 gm)
37
29
f2.7
f 4.3
40 f3.0
f 2.6
f 1.5
Controls
Evans, P.D.
Ftat (75 gm)
256
29
28
12
11
Controls
Bugbee, V.D.
Rat (125 gm)
256
16.0 f0.6
11 f 0 . 9
13 f 1.7
32
46
0-35th day
8
7
10
10
Controls
Bugbee
Rat (125 gm)
256
23
24
f3.0
f 6.5
f2.3
f4.4
32
41
25th day
20th day
18 f 2.3
f 1.6
15th day
7 f 1.2
12 f0.9
10th day
23
33
Dosed
Tumor
Tumor Diameter in Millimeters
13.5 f 1.0
Preparation
Animal and
TABLE
111: Effect of Pituitary Growth-Stimulating Extract Upon Tumm and Body Growth *
337
HORMONES IhT CANCER
Two series, X I and XTII, of young rats, thirty-four to thirty-eight
days old, were subjected t o the massivc dosage of irradiation which had
been shown effectivc in retardin! tumor growth. The dose of rociitg.cn
irradiation given the rats in 8cries XTII was three-fourths thc amount
given in Series XI (YO00 r ) , and was not lethal t o any of the animals
(see Table V). Inoculation with carcinoma 256 was made the day following irradiation. One-half of the rats of each group were dosed
daily with a preparation of the growth-promoting principle. I n each
case a significant increase in body weight and in tumor growth was
noted for the rats which had received the growth-promoting principle.
TABLE
IV: Solid Content of Tumors of Rats Dosed with Growth-Promoting Principle
Weight of
Tumor
(Grams)
Per cent
Solids
1.85
0.70
18.5
19.0
Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Dosed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
16.0
2.5
18.5
17.7
TABLE
V : Combined E f l d of Pituitary Irradiation and Pituitary Growth-Promoting Principle Upon
the Rate of Tumor and Body Growth
Increment In
Body Weight
(Grams)
11th Day
Tumor
Diameter
(Millimeters)
11th Day
Series X I
Non-dosed rats 9 , . . . . . . . . . . . . . . . - 7 i2.0
Dosed rats 0 ....................
+21 f 5.0
10 f 1.2
17 f2.0
series X I I I
Non-dosed rats 14 . . . . . . . . . . . . . . . - 3 f 2.0
Dosed rats 10 . . . . . . . . . . . . . . . . . . . 8 f 3.2
+
9 f 1.7
17 f 1.9
E f e c t of Gopaad-Stiniulatirhg Extracts
Previous to our studies on pituitary irradiation and tumor growth
we had tried (15, 16) the effect of practically all the pituitary, sex, and
supposedly related physiologically standardized preparations upon the
rate of growth of sarcoma R10. I n all but two cases the effect was
negative. In these two instances (placental and anterior lobe pituitary
preparations) the effect was doubtfully accelerating. The results are
given in Table VI. Since these results were essentially negative and
those with the pituitary growth-stimulating extract positive, it was
assumed that pituitary irradiation affected cancerous extension by
inhibition of the growth factor alone. I n the meantime Zondek, Zondek
and Hartoch (5) have shown that tremendous doses of the urine of
pregnancy gonad-Rtimulating preparation decrease the rate of tumor
growth in mice, and Evans and his co-workers (17) have shown that
mixtures of the gro\l.tli-accelcrating pituitary extract and the urine of
338
FRITZ BISCHOFF, L. C. MAXWELL AND H. J. ULLMANN
pregnancy extract function as the pituitary gonad-stimulating extract.
We have therefore tried the combined effect of pituitary irradiation
and dosage with the gonad-stimulating urine of pregnancy preparation
in order to ascertain whether amounts of the urine of pregnancy preparation which have no effect upon the tumor growth of normal animals,
affect tumor growth in pituitary irradiated animals. See Table VII.
TABLEVI : Effect of VaTious Gonad-Stimulating Preparations Upon Rate of Tumor Growth
Number
of
Tumors
Maximum Deviation of
Mean Tumor Diameter
from Control
R-10 Ovarian
Control 11
Dosed 9
1 x Std. dev. mean
6 Allan-Doisy units daily
R-10 Corpus luteum
Control 15
Dosed 18
1 x Std. dev. mean
0.1 cc. Hynaon, Westcott,
Dunning preparation daily
R-10 Anterior lobe
pituitary
Control 19
Dosed 18
1 X Std. dev. mean
0.1 cc. Parke Davis antuitrin
Tumor
Preparation
Source
Doaage and
Potency of
Preparation
daily
Placental
Control 9
Dosed 7
+2
x Std. dev. mean
0.2 cc. crude emmenin (Collip) equivalent to 8 gm.
placenta daily
R.10 Placental
Control 15
Dosed 9
1
x Std. dev. mean
0.2 cc. crude emmenin (Collip) equivalent to 8 gm.
placenta daily
R-10 Urine of pregnancy
Control 15
Dosed 9
1 X Std. dev. mean
Daily dose of Dickens’ preparation causing opening of
vagina in 6 days
Control 6
Dosed 8
1 X Std. dev. mean
Daily dose of Casimer Funk
preparation causing hypertrophy of seminal vesiclea in
6 days
R-10 Anterior lobe
pituitary
Control 10
Dosed 10
4-2 X Std. dev. mean
5 rat unite Van Dyke preparation in 2 weeks
R-10 Anterior lobe
pituitary
Control 10
Dosed 9
1 x Std. dev. mean
10 rat unitR Van Dyke preparation in 3 weeks
256
Anterior lobe
pituitary
Control 12
Dosed 12
1 x Std. dev. mean
0.1 cc. Parke Davis antuitrin
daily
256
Placental
Control 9
Dosed 8
1 x Std. dev. mean
0.2 cc. crude emmenin (Collip) equivalent to 8 gm.
placenta daily
R-10
R-10
Male urine
.
Protocole
Mice, Series I : Young mice approximately 20 gm. in weight were divided into three
groups after inoculation with sarcoma 180, one group serving as controls, one group receiving 1.0 C.C. urine of pregnancy per day in two doses beginning the sixth day after
inoculation, and a third group receiving the same dosage of urine of pregnancy, in addition to radiation over the pituitary region (4000 r) the fourth day after inoculation. The
HORMONES IN CANCER
339
twentieth day after inoculation the animals were sacrificed, the ovaries and seminal
vesicles weighed, the pituitaries sectioned.
The results of this experiment are signiflcant. The difference in tumor diameters
between controls and the series which was both rayed and dosed exceeds three times the
joint standard deviation of the mean. On a weight basis, the control tumors average over
three times the weight of the rayed and dosed series. The effect of urine of pregnancy
alone on tumor growth is of doubtful significance, the difference being over twice the
standard deviation of the mean. The pronounced physiological effect of the dosage is
amply proved by the great difference in the size of ovaries and seminal vesicles, the ovaries
of the controls averaging 6 mg. in weight, those of the dosed mice 14 and 15 mg. respectively. The seminal vesicles of the controls averaged 47 mg. I n the rayed series the
seminal vesicles averaged 136 mg. I n the dosed series which were not irradiated the
seminal vesicles averaged 185 mg.
Mice, Series ZV: Twenty mice ranging in weight from 15 to 20 gm. were inoculated
with sarcoma 180. Two clays later the mice were given 4000 r pituitary irradiation and
daily doses of 1 C.C. urine of pregnancy. The dosage proved toxic to several of the animals. The percentage of tumor takes was 100 per cent. A t the end of twenty days the
treated mice had lost a n average of 1.4 gm. in weight. The controls gained 2.9 gm. The
results are even more striking than those in series I, the difference between the control
and dosed series approximating six times the joint standard deviation of the mean.
Rats, Series ZIZ, Carcinoma 256: Rats approximately forty days of age were divided
into two groups, one serving as controls, the other receiving 5200 r pituitary irradiation.
The following day both series were inoculated with carcinoma 256. The irradiated rats
received daily injections of urine of pregnancy or purified preparations containing the
active principle. A t the end of the experiment the ovaries of the controls ranged in
weight from 21 to 35 mg., the ovaries of the dosed rats from 57 to 123 mg. The seminal
vesicles of the dosed rats averaged four times the normal weight. A significant retardation in the rate of tumor growth was observed in the dosed rats. See Table VII.
Rats, Series F Z I : Rats approximately thirty days of age were divided into two groups
and inoculated with carcinoma 256. One group served as a control series, the other group
received pituitary irradiation (4700 r ) the second day after tumor inoculation and two
daily injections of urine of pregnancy (1.35 C.C. each) the first seven days of tumor life,
followed by a daily injection of a more potent purified urine of pregnancy preparation
(8 rat units per day). There were no fatalities due to radiation.
The percentage of tumor takes was low, being 31 per cent for the controls and 38 per
cent f o r the treated animals. Only 5 tumors developed in each group, but these were of a
very uniform size. A significant retardation in the rate of tumor growth was observed
in the dosed rats.
Rats, Series IS:Forty-day-old rats were given 4700 T pituitary irradiation three days
after inoculation with carcinoma 256, a group of litter mates serving as controls. The
irradiated rats received daily doses of 8 rat units of a purified urine of pregnancy preparation. The retardation in rate of tumor growth was not as marked as that in the series
in which urine of pregnancy was given.
Rats, Series IZ, Sarcoma R10: Young rats approximately thirty-five days of age were
inoculated with the R10 sarcoma and divided into two groups, one serving as controls, one
receiving 2 C.C. of pregnancy urine per day, in two doses, beginning the day after inoculation, and irradiation over the pituitary region (5200 r ) the second day after inoculation.
The dosage proved toxic, scvernl of the animals dying. Dosage was continued f o r ten
days, and the experiment was under observation for fifteen days. I n spite of retardation
of body growth, and under the influence of the pregnancy hormone as gauged by the increase in weight of the seminal vesicles (77 nig. over the controls), no marked effect on
tumor growth was noted.
Ruts, Series VZ: Thirty rats, approximately thirty days of age, were inoculated with
R10 sarcoma and divided into two groups, one serving as controls, the other receiving
4700 r pituitary radiation and daily dosage of 2 C.C. urine of pregnancy. The tenth day
of tumor life the control tumor diameters averaged 33 -+ 2.0 mm., the dosed 17 f 2.4.
Since the difference was not markedly significant, the expcrirnent was discontinued. The
body weight increase for the controls was 36 gm., that f o r the treated rats 10 gm.
0
ip
w
Control
AZ
X-ray
Control
A2
X-ray
Control
AZ
X-ray
Control
-1Z
X-ray
1'11
IS
I1
1-1
I
IV
256
256
R-10
R-10
180
180
+
+ AZ
Control
X-ray
X-ray
hZ
Control
+
+
+
+
('on t rol
X-ray
-%Z
+
11.0 f 0.9
9.0 f 0.7
9.4 f 1.0
3.9 f 0.5
11
8
13.0 + 1.0
14
15
16
10
23.0 f 2.9
17.0 f 2.4
13.9 f 1.1
6.5 f 0.6
17.0 f 1.1
13.0 f 0.9
11.5 f 1.2
17.0 f 1.6
10.3 f 1.2
+36
+10
+39
0
44.0 f 6.0
30.0 f 3.8
21.0 f 5.2
17.0
7
9
s
+30
- 5
31.0 f 2.0
25.0 f 1.1
+25
+ 5
38.0 f 1.2
31.0 f 1.4
21.0 f 1.4
15.5 f 0.7
+a
10th day
+ l
21.0 f 1.5
17.0 f 0.9
14.5 f 1.5
20th day
35.0 f 2.1
27.0 f 2.0
15th day
,
f2.5
20th day
Body Weight Increment in Grams
24.0 f 1.7
15.5 f 1.7
10th day
Tumor Diameter in Millimeters
4000 r
4000 r
4700 r
5200 r
4700 r
4700 r
5200 r
Remarks
the (-Tine qf P T F ~ T U (A.Z.)
I I L C ~itpon Rate of Ttrnwr and Body@owth
15.0 f 1.6
11.0 f 1.3
1
oj
9
12
5
7
10
I
-
Sumher of Tumors
111
-
Series
256
Tumor
TABLE
VII: Combined Eflert qf Pitititmy Irradiation a i d Gonad-Stmtilntzrig Fraction
HORMONES IN CANCER
34 1
I n the case of mouse sarcoma 180 (see Table I, series V111,l IX,’ and
Table VII, series I and TV) a greater effect is noted when pituitary
irradiation (4000 P ) and urine of pregnancy dosage are combined (4 to
5 times the standard deviation of the mean) than when either agent is
used by itself (twice the standard deviation of the mean). In the case
of rat carcinoma 256, the combined effect of urine of pregnancy dosage
and pituitary irradiation (see Tables VII, series 111,VII, IX, and compare with Table I, series 11, VIII) is no greater upon tumor growth
than that obtained by pituitary irradiation alone, but relatively less
irradiation was required. In the case of rat sarcoma R10 (see Table
VI) no effect upon tumor growth was observed with urine of pregnancy
preparation alone, and only a doubtfully significant result (see Table I,
series IX and IV, and Table VII, series I1 and VI) when this was combined with pituitary irradiation.
In the experiments of Zondek, Zondek and Hartoch 50 to 100 units
of Prolan (urine of pregnancy preparation) daily were found necessary
to produce a retardation in the growth of a mouse tumor, which was the
same as that produced in our mouse series by only 2 rat units of urine of
pregnancy in connection with pituitary irradiation.
It will, of course, be necessary to repeat our experiments using
purified preparations. Since a lesser effect is observed for the rat
tumors, it will be necessary to give larger amounts of a purified preparation, in an attempt to produce results in the rat as striking as those
found in the mouse. The effect may be due to some substance other
than prolan.
Pathological E f c c t s of Pituitary Irradiatiorz
Attempts to abolish permanently the function of the anterior lobe of
rats or of mice through pituitary irradiation were unsuccessful. Even
repeated doses of irradiation totaling 23,000 P (Series XIX) failed to
arrest the weight growth curve permanently except in a few instances.
Histologically the effect is quantitative. Sometimes only fragments of
the anterior lobe remain in the sections. There are regions of debris.
When the anterior lobe is present, normal appearing cells are always
seen. Sex impairment appears to be profound in some cases. In
young females the opening of the vagina is delayed. In males the
testicles are smaller, and damage to the testicular cells is noted. The
results with mice are essentially the same. That pituitary irradiation
does not destroy the sex function is shown by the following observations. In three instances animals went through a normal pregnancy.
The urine of pregnancy hormone had its normal gonadotropic effect
in both irradiated mice and rats.
Pratocols
X-Ray Series XV: Thirty-two rats, thirty to thirty-five days of age, were divided into
two groups, one serving as controls, the other receiving radiation over the pituitary body
in six doses, covering a period of two months. The maximum dose given at any one time
18
342
FRITZ BISCHOFF, L. C. MAXWELL AND H. J. ULLMANN
was 5000 r. Death from overdosage resulted in ten instances. All the animals were
sacriflced ninety days after the flrst dosage. Sections of the adrenals, thyroids, liver,
kidney, testes, and ovaries were prepared and stained with eosin hematoxylin. Sections
of the pituitary were prepared and stained with orange 0 or with safranine acid violet.
No histological changes were observed in the adrenals, thyroids, liver, or kidney. Disappearance of spermatogenic cells lining the tubules of! the testes was observed in three
out of five of the rayed surviving rats and absence of corpora lutea was noted in the
ovaries of three rayed females. I n three of the rats which did not survive the treatment,
no anterior lobe was found on section. I n a fourth there was disappearance of cells
leaving granular d6bris. All types of cells were seen when anterior lobe tissue was
found. I n the animals which survived the treatment, complete damage to the pituitary
body was reported questionable or absent, since normal cells were always found. The
testes of three of the surviving rayed rats did not exceed 0.3 gm. in weight. The mean
for the controls was 1.7 gm. See Table VIII.
TABLE
VIII: Eflect of Repeated Maeaive Pituitary Zmadialion on Body and Organ Weight
Initial
Body
A Weight
A Weight
Laat
Body
Weight
Weight 2 Mo. Later 2 Months 20 Days
Weight
Testes
Weight
Liver
Weight
Kidney
2.6
1.2
9.8
6.6
1.7
1.1
Weight
Testes
Weight
Liver
Weight
Kidney
1.7
0.77
8.3
4.7
1.8
1.2
serif34XVZ
Control
Rayed
63
62
173
110
110
48
27
23
Initial
Body
A Weight
A Weight
Last
Body
Weight
Weight 3 Mo. Later 3 Months 20 Days
Seriea X V
Control
Rayed
61
68
196
119
136
61
17
18
X-Ray Series XVZ: The procedure was the same as that in Series XV, except that
irradiation was performed three instead of six times. Half of the animals succumbed.
No histological deviations from the normal were observed for the tissues examined
(same aa series XV).
X-Ray Series XZX: I n this series of rats (see Table I ) the chief difference in the
sections of the pituitary glands of control and rayed animals consists in the size of thc
section obtained; the cells of the irradiated glands stain well. The ovaries of the irradiated animals frequently have large corpora lutea. The adrenals are unchanged.
Nice, Series I: In this series the essential difference between the pituitary glands of
the irradiated mice and the normal controls is a quantitative one. I n the irradiated series
the pituitary glands either could not be removed intact a t autopsy o r only small fragments of the anterior lobe are present in the sections, together, in some instances, with
granular d6bris. Where such fragments are found, normal anterior lobe cells can be
identified. I n one irradiated mouse, however, an exception is noted, in that a large
anterior lobe is seen. The largest ovaries are those from animals injected with pregnancy
urine, in which large corpora lutea are present. I n two irradiated mice corpora lutea are
present in greater number than in the controls. The results may be interpreted as follows: A single sub-lethal dose of pituitary irradiation to 20 gin. mice leads to a quantihtive pituitary effect. The cells which remain are normal appearing, and the pregnancy
urine hormone is able to excrt its usual gonad stimulation.
DISCUSSION
Following our preliminary reports (1, 2) on the effect of pituitary
radiation on tumor growth, Ball, Samuels and Simpson (4) and
McEuen (6) reported on the effect of hypophysectomy, using the same
transplanted tumor, carcinoma 256, as was used in our studies. Unf or-
HORMONUS IN UANUPJB
343
tunately, however, the data of these investigators are so presented that
measures of variability can not be applied. In a series of 9 hypophysectomized rats studied by Ball, Samuels and Simpson the mean tumor
diameter was 20 mm. the third week of tumor life as compared with 28.5
for a series of 16 controls and 35 mm. for a series in which the pituitary
was exposed without removal. At five weeks the respective diameters
were 33,54, and 61. In another series of Samuels and in the series of
McEuen hypophysectomy was performed after the tumors had appeared. In the former the diameters eighteen days after operation
were 52 for the controls against 39 for the hypophysectomiaed animals,
in the latter 44 against 36. Assuming that the standard deviation of
the mean of the tumor diameter is the same in these series as found for
our tumor (the two control series of Ball indicate that it is at least as
large), the difference in rate of growth between tumors in control and
hypophysectomized rats is about 3 to 4 times the standard deviation of
the mean, the effect being no greater than that produced by pituitary
irradiation.
It appears striking that while pituitary irradiation produces no
permanent cessation of growth, the effect during the period of maximum body weight retardation is as great as that produced by complete
hypophysectomy. Moreover, less radiation is required when the area
of radiation is increased. This would indicate that the injury to the
area surrounding the pituitary, possibly interfering with the release of
the active principles from the gland, or with the nutrition of the gland,
is directly responsible for the effect. The experiments with pituitary
irradiation would in themselves offer no final proof that the pituitary
influences tumor growth. However, the experiments in which dosage
of pituitary extracts with known growth-promoting powers counteracted the effect of irradiation, demonstrate rather clearly that the irradiation in some way affected the available supply of the active principle of the gland, especially since the animals were at the age where
they normally have an excess of growth hormone and dosage with
growth hormone is without effect upon body weight.
It has been generally stated that (‘any condition that decreases the
nutrition of the body as a whole, or of the tissue in which a tumor is
located, decreases the rate of growth of the tumor ” (18). Rous (19)
observed this phenomenon with some transplanted tumors, but not with
all nor with spontaneous tumors. Since in our experiments retardation
of tumor growth through pituitary irradiation is manifested only with
concomitant arrest or slight loss in body weight, the question arises as
to whether the action is due specifically to a hormonal effect through the
pituitary, or whether it is merely a general systemic, possibly nutritional effect, brought about by x-ray sickness. In earlier experiments
data have been accumulated for the tumors in question which throw
some light on the subject. In rats poisoned with colloidal cuprous
oxide (unpublished data), so that they lost 6 gm. (mean) in body weight
during a period in which controls gained 26 gm., and in rats poisoned
with synthalin (16), which lost 10 gm. (mean) in body weight while
344
FRITZ BISOHOFF, L. 0. MAXWELL AND H. J. ULLMANN
the controls gained 15 gm.,the difference in the size of tumor 256 as
compared to control animals was less than once the standard deviation
of the mean. With sarcoma R10, a loss in body weight of 43 gm.
(mean) through synthalin poisoning as compared with a 4 gm. loss in
weight of the controls produced no effect upon tumor growth. I n mice
bearing sarcoma 180, sublethal doses of pituitrin (20) which brought
about a 1.3 loss in body weight produced no effect upon tumor growth,
as compared with controls, which gained 1.7 gm. The relative weight
changes in these experiments are of the same order of magnitude as
those produced by pituitary irradiation, and the evidence is therefore
against any explanation based upon a systemic effect. This is further
corroborated by the experiments dealing with replacement therapy, in
which the administration of pituitary extracts counteracted the effect
of pituitary irradiation both in regard to body and to tumor growth.
The question of greatest importance arising from these studies is
whether the so-called growth hormone of the pituitary alone functions
in neoplastic extension through acceleration of growth or whether there
is another quite distinct pituitary mechanism functioning through a
retardation of growth. From the evidence at hand it is impossible to
decide whether the urine of pregnancy substance functions in the absence of the pituitary. To answer this question it will be necessary to
find out whether hypophysectomy in the mouse produces the profound
effect demonstrated by dosage with urine of pregnancy preparations
and whether proper administration of these preparations in rats will
produce greater effects than hypophysectomy or pituitary irradiation.
It is obvious that there is a large species variation in the effect of the
growth hormone in the mouse and the rat. It is also obvious that, whatever the mechanism, a practical application of these pituitary studies
to human neoplastic extension is possible only if the maximum effect in
man is comparable to the maximum effect demonstrated in the mouse,
and of theoretical interest only if the maximum effect in man is comparable to that thus far produced in rats.
SUMMARY
1. Sublethal doses of pituitary irradiation, which bring about a
temporary cessation of body growth, significantly retard the growth
rate of rat sarcoma R10, rat carcinoma 256, and a mouse carcinoma
(spontaneous mammary) if the tumor appears at the period of maximum body weight retardation.
2. In the case of rat carcinoma 256, the above effects were abolished
by simultaneous dosage of pituitary extracts with standardized growthpromoting powers.
3. A cessation of body weight growth produced by various poisons,
equivalent to that following pituitary irradiation, failed to retard tumor
growth significantly.
4. In older rats dosage with standardized growth-promoting preparations of the pituitary significantly accelerate the rate of growth of
HORMONE8 IN U A N O E R '
345
carthoma 256. In younger animals, and with mouse sarcoma 180 and
rat sarcoma 10, the effect was less significant.
5. Dosage of urine of pregnancy augmented the effect of pituitary
irradiation upon the rate of growth of mouse sarcoma 180. The effect
was less significant for rat carcinoma 256 and rat sarcoma 10.
6. Attempts to abolish permanently the function of the anterior lobe
of rats or of mice through pituitary irradiation were unsuccessful.
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