VOIL. 48, 1962 BIOCHEMISTRY: SZENT-GYORGYI ET AL.

1439

25 Cf. i-o-galactoside mutants in Jacob, F., and J. Monod, J. Mol. Biol., 3, 318 (1961).
26 Snell, E. E., Vitamins and Hormones, 16, 77 (1958).
27 Burns, R. O., and R. D. DeMoss, personal communication.

CONSTITUENTS OF THE THYMUtJS GLAND AND THEIR

RELATION TO GROWTH, FERTILITY, MUSCLE, AND CANCER*
BY ALBERT SZENT-GY6RGYI, ANDREW HEGYELI, AND JANE A. MCLAUGHLIN
INSTITUTE FOR MUSCLE RESEARCH AT THE MARINE BIOLOGICAL LABORATORY, WOODS HOLE,
MASSACHUSETTS

Communicated June 15, 1962

The fact that no low-molecular biologically active substances have unequivocally
been isolated yet from the thymus gland indicates that either there are none present
or else that their demonstration and isolation is connected with major difficulties.
In the absence of characteristic chemical reaction, isolation of such substances de-
pends on a test, based on some biological reaction. Growth involves the interaction
of a great number of reactions, and so we hoped to be able to demonstrate active
material, possibly present, by its influence on growth. The thymus is most active
in young, growing animals.
Normal growth being a slow process, we turned to the faster pathological growth
of cancer. This work led us three years ago1 to the conclusion that "our extracts
contained two active substances, the one promoting, the other inhibiting malignant
growth, and the result depended on their balance." In a crude extract of the
gland, both substances are present and compensate one another, and no activity
is observed. The inhibitor and promotor action can be demonstrated only after
the separation of the two. Since the chemical or physical properties of the two sub-
stances are very similar, this separation is difficult, which may explain why these
activities have hitherto not been unequivocally demonstrated. Another property
which makes isolation of the two substances difficult is that they show no cleancut
water or fat solubility and tend to adhere to any substance or precipitate produced
during the course of preparations. They have no acute drug action either.
For convenience, we will call the substance promoting malignant growth "pro-
mine," the retarding substance "retine."
The Test.-Groups of 5-10 mice were injected subcutaneously with ascites tumor.
Two days later, daily injections of various extracts were begun. The growth of the
tumor was followed by noting its outlines. Final results were obtained on the tenth
day by sacrificing the animals, isolating and weighing the tumors, and comparing
their weight with that of the tumors in the control group. Various factors had to
be considered.
Animal material: To exclude individual variations, inbred Swiss albino mice
were used. The growth of tumors in such animals depends on the age of the ani-
mal, growth being faster in young than in old animals. We found, for instance,
that in four-week-old mice the average weight of the tumor was 4,823, in four-
month-old mice 2,735, and in mice older than one year 542 mg three weeks after
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inoculation. It follows that in any test, mice of the same age have to be used.
We used 6-8 week old ones.
The tumor: In the beginning, we used the spontaneous mammary tumor of C3H
mice of Bar Harbor and could demonstrate both the inhibiting and the promoting
action. Later, we shifted to the Krebs 2 ascites tumor of the National Cancer In-
stitute. The tumor was propagated intraperitoneally as ascites tumor and then
injected subcutaneously to produce a local solid tumor on which our fractions were
tested. To obtain comparable growth, it is essential that, within a series, all ani-
mals should be injected with identical material in an identical way. So a number
of animals were injected with ascites tumor intraperitoneally. Then on the 8th-
10th day, the ascites of the single animals was tapped, united, and stirred gently.
Coagulation was prevented by keeping the temperature at 32 E 20C. 0.25 ml of
this ascites was injected subcutaneously into the shoulder region. In two days,
a palpable tumor was produced. Occasionally, an animal showed no visible tumor
and was rejected. Sarcoma 180 obtained from Merck and Company reacted to
our extracts similarly to the ascites tumor.
Extracts: Neutral aqueous solutions were injected daily in a volume of 0.25
ml subcutaneously on the belly. Control animals received 0.25 ml saline.
Units: A "unit" of retine we called the quantity (free from promine) which in-
hibited the growth of cancer by 50 per cent. This quantity is contained in 15-20
grams of fresh thymus. Four units, injected daily, inhibited growth completely
and caused regression. A typical experimental result was the following:
Weight of the tumor in control animals 1050 :1 150 mg
Weight of the tumor, in animals treated with 4 units of retine, daily 255 30 mg

Judged by the outlines, the tumor showed no growth in the latter group. Since
retardation begins after the second injection, this result actually means regression.
Microscopic examination showed mainly connective tissue.
It was found that promine induced sterility both in males and females.; To pre-
vent conception in females, about twice as big doses of promine were needed in the
females as in the males. The minimum quantity needed to produce sterility in
females we called a "unit." This was contained in about 50 grams of fresh -thymus.
Two units, administered daily, increases the weight of cancer 2-3 times. Fertility
was regained after the treatment was discontinued.
Chemical Procedures.-As material we used the thymus of calves, carefully cleaned
and frozen as soon as possible after death. The glands were stored. transported,
and reduced to a snow in frozen condition. The snow was made to drop directly into
methanol, 100 liters being used for every 80 lb of the gland. The suspension was
repeatedly stirred and stored for 24 hr, after which time solution and residue were
separated by centrifugation. In part of the experiments (Variation 1), a hot con-
centrated barium acetate solution was added till further addition produced no pre-
cipitate. The barium precipitate was separated on a centrifuge. The supernate,
whether treated with Ba or not, was reduced to four liters in vacuo, about room
temperature. The concentrated fluid was acidified with HCl to pH 4 and then
shaken out twice with 350 ml chloroform under addition of 175 ml of ethanol.
Then the gland was extracted a second time with hot methanol, 25 liters being used
for every 80 lb of the starting thymus material. The extract was reduced to two
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liters and shaken out with chloroform as above. The retine content of the various
fractions per lb of thymus used is given in units in Table 1. As will be seen, the
active material is more or less uniformly distributed over the various fractions.

TABLE 1
Ba-treated material
1. Aqueous fraction 2,5-3,5
2. Chloroform extract 4-5
3. Acid aqueous extract of Ba precipitate 3-5
4. Chloroform extract of Ba precipitate 6-8
Not treated with Ba
1. Aqueous fraction 6-8
2. Chloroform fraction 12-15
3. Second hot extract 6-10

Purification: The watery residue, after chloroform extraction, can be liberated

from a great deal of inorganic salts by the addition of four volumes of methanol
with subsequent filtration.
Two main methods were used for further purification. The aqueous extracts
could be purified without considerable loss of activity by shaking them out twice
with 10 per cent 2,4-dichlorophenol. The latter separates readily in a separation
funnel, taking down the active material which can be separated from the dichloro-
phenol (see later). This way, the dry weight can be reduced to about 1/10.
Both promine and retine can be precipitated by reagents precipitating weak
bases, like phosphotungstic acid or Reinecke salt. We preferred Reinecke salt,
which takes down the active substances in presence of 2-3 N HCL. It was used in
the form of a saturated aqueous solution. Five per cent of methanol prevents for-
mation of Reineckate, so alcohol must be evaporated prior to precipitation.
The chloroform extracts were distilled off to dryness in vacuo and the residue
extracted with acetone. In presence of the lipins, the active substances dissolve
in acetone leaving a great mass of inactive resinous material behind. The acetone
was then distilled off, the residue dissolved in chloroform and then shaken with an
excess of Reinecke solution containing 2-3 N HCl. Intense shaking was continued
till no more Reinecke was used up, as indicated by the color of a small centrifuged
sample.
The dichlorophenol can be extracted likewise after being diluted with an equal
volume of chloroform.
The Reinecke precipitates were separated on a Bichner funnel, washed with 2
N HC1 and hexane, and then dissolved in acetone. After addition of HC1 and
water, the Reinecke reagent was shaken out with ethyl ether. The aqueous phase
was then neutralized with NaGH, evaporated to dryness and extracted with
methanol, which left the NaCl undissolved.2
Separation of retine and promine: The two active substances can be separated
by paper chromatography. The concentrated methanol solution was brought
on paper and dried. The paper (Whatman 3) was exposed for ten min to saturated
water vapor and ascending chromatography made. As solvent we used butanol,
saturated with 1 N HCL. The promine, under these conditions, did not move and
could be extracted with methanol. The retine showed an RF of 0.18-0.25 and could
be extracted with methanol containing 10 per cent of 1 N HCL.
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For injections, the extracts were evaporated in vacuo to dryness. Retine, under
these conditions, was not readily soluble in water and had to be dissolved in a small
amount of propylene glycol which, then, was diluted with water and neutralized.
The extracts thus obtained showed no poisonous action. The purer extract of
retine contained 0.01-0.05 mg dry weight per unit.
Discussion.-How far the active substances described are specific for the thymus
has not been established. Cancer-inhibiting substances have repeatedly been de-
scribed in various tissues. There are indications, suggesting that at least one of the
two observed activities has relation to the specific function of the gland. This is
suggested by the fact that growth is most active when the thymus is most developed
and that myasthenia is, in some way, connected with the thymus, since thymus ex-
tirpation often benefits patients. The mirror-image of myasthenia is myotonia.
We have found that retine, administered to myotonic goats, greatly aggravated
symptoms, while the opposite seemed to be true for promine.3 More detailed reports
on this line will be forthcoming later. Another observation, suggesting relations
to the physiological function of the gland, is that promine renders mice sterile.
The thymus is most active in sexually immature animals, unable to produce
progeny.
While promine may be a specific product of the gland retine seems to be a more
widely distributed tissue constituent. We have found similar activity in various
organs and tissues.
We believe that, at the moment, our preparations are still far from purity and
that the two substances have a high biological activity.
The experiments corroborate our earlier contention that the rate of growth is
dominated by the balance of the two substances described. The compensating
action of the two substances, their tendency to become adsorbed, and the lack
of a more acute pharmacological action may explain why they have hitherto been
overlooked.
Our observations give some substance to the hope that retine may be used in cancer
therapy, while promine may find various other medical applications. Calculated
from our observations on mice, the quantity needed to inhibit cancer in a full-
grown man seems unreasonably high. However, it should be mentioned that the
slower a cancer grows the easier it is inhibited and the tumor used by us is an ex-
tremely fast growing one.
Though promine promotes cancer growth, it seems to be unable to induce malig-
nancy.
*
Supported by Grant No. RG-9144(RI) of the National Institutes of Health and Grant No.
G-5836 of the National Science Founjdation. This work was initially supported by a grant from
the Commonwealth Fund and later by a grant from the Jane Coffin Childs Memorial Fund.
1 Szent-Gyorgyi, A., Introduction to a Submolecular Biology (New York: Academic Press, 1960),
p. 125.
2 The main impurity at this stage is choline and betaine, which can readily be crystallized and
were kindly identified for us by Merck and Company, Rahway, New Jersey.
3We have also had the impression that mice treated with retine showed an increased activity
and reflex irritability which supports the assumption that the substance decreases membrane
potentials, myotonia being probably due to a disturbed repolarization of the muscle membrane.
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