ÕCANCERRESEARCH 27, 1306-1311,July 1967]
Influence of Pineal
in the Hamster1
TAPOSH
Gland on the Growth
and Spread of Melanoma
K. DAS GUPTA AND JOSE TERZ
Andre and Bella Meyer Laboratory of the Division of Experimental Surgery and Physiology of the Sloan-Kettering Institute for Cancer
Research, New York, New York, and the Department of Anatomy, Guy's Hospital Medical School, London S.E.Õ, England
SUMMARY
Pigmented malignant melanoma (M Mel No. 1) was trans
planted into the subcutaneous tissue of the dorsum of control,
sham-operated, and pinealectomized Syrian hamsters of both
sexes and weighing between 80 and 110 gm. Animals from each
group were sacrificed at regular intervals and autopsied. The
pinealectomized group of hamsters had significantly larger pri
mary tumors and more extensive métastasesthan the control or
sham-operated animals at every phase of the study.
It is felt that these findings are possible indications of a rela
tionship between the pineal gland and growth and spread of pigmented melanomata in hamsters.
INTRODUCTION
Biologists for a long time considered the pineal as a rudimen
tary organ. Marburg (11), at the beginning of this century, was
the first to postulate that the mammalian pineal could have an
endocrine function. The first comprehensive account on the
comparative anatomy along with some clinical considerations
was published by Gladstone and Wakeley in 1940 (9). Today it is
agreed that pineal»in all animals are derivatives of the dorsal
wall of the diencephalon and their microscopic anatomy varies
among adult forms of various vertebrate groups. For example, in
fish (18), amphibians (12), and reptiles (5, 6), the ultrastructure
of the principle cell is similar to photoreceptive cells of the verte
brate retina (4) and invertebrate light-sensitive organs (2). The
main cell types of the mammalian pineal, however, show no such
cytologie specializations (1).
The experimental work on the pineal remained inadequate un
til the 1950's. In 1954 Kitay and Altschule (13) reviewed the
literature and concluded that little was known about the function
of the mammalian pineal. Lerner et al. (14) generated the real
interest in the study of pineal in 1959 by successfully isolating
from cattle pineals a hormone, "Melatonin," which had a blanch
ing effect on amphibian skin (23). He and his associates gave a
further impetus by describing the structure of Melatonin later
(15). There is no question at present that mammalian pineal is a
functioning organ; the only problem is to define the function or
functions.
Histochemical studies on the pineal organ have shown that
1This work was partially supported by USPHS Grant
5110.
Received December 12, 1966; accepted March 28, 1967.
1306
CRT
pinealocytes have an abundance of dihydroxyphenylalaninepositive material, succinic dehydrogenase, and acid and alkaline
phosphatases, as well as nonspecific esterase and monoamine
oxidases. Axelrod et al. (3) have isolated hydroxyindole-0-methyl
transferase from rat pinealocytes. Quay (20) has demonstrated
that the 3 most abundant 5-hydroxy and 5-methoxy Ã-ndolesof the
rat pineal (serotonin, melatonin, and 5-hydroxyindole-3-acetic
acid) have different patterns of distribution according to the time
of day and pharmacologie treatment. It has also been shown that
highest concentration of serotonin, an amine with structure simi
lar to melatonin, is found in the rat pineal (23). This information
suggests a considerable metabolic rate and biochemical specificity
of adult pinealocytes in mammals, particularly in terms of
probable synthesis of secretory material.
The Syrian hamster is not only a pigmented animal but also
contains pigment granules in its pinealocytes. On the basis of
studies made by Santamarina and Meyer Arndt (22), these can
be assumed to be melanin pigment granules. The structure of the
pinealocytes in hamsters, because of the presence of smooth mem
brane system, is compatible with pigment production (19). Con
sequently, it is conceivable that the hamster pineal not only has a
structure suitable for pigment production but is possibly capable
of inducing some form of change in the pigmentation. No data is
available at present about the effects of pineal ablation on pig
mentation of hamsters. However, Syrian hamsters spontaneously
develop a highly pigmented malignant melanoma (7). The
hamster with this type of tumor was considered an ideal model in
the study of one of the functions of the pineal gland. Thus a study
was undertaken to observe the effects of pineal ablation on
growth and spread of transplantable pigmented melanotic mela
noma in Syrian hamsters.
MATERIALS AND METHODS
One hundred fourteen Syrian hamsters of both sexes weighing
between 80 and 110 gm were used. The animals were divided into
3 groups. Thirty-eight hamsters were pinealectomized. Thirtysix hamsters were subjected to a comparative trauma and bleed
ing but the pineal gland and its stalk was left undamaged and
intact (sham pinealectomy). The remaining 40 animals were used
as straight controls. All animals were fed standard laboratory
diet and water ad libitum. Five weeks following either surgical
procedure, hamsters were considered ready for tumor inoculation.
These experiments were carried out in 4 stages, each of which
contained an equal proportion of animals from the original 3
groups receiving tumor transplant simultaneously.
CANCER RESEARCH VOL. 27
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Pineal Gland and Hamster Melanoma
Jnd stalk
CHART 1. Technic of pinealectomy. In /, a longitudinal incision about 2 cm long is made on the vertex and the incision deepened to
the periosteum. In 2 and 3, the periosteum is seen reflected laterally exposing the parietal and occipital bones. At this stage the straight
sinus and the lateral sinuses become visible underneath the bones. In 4, an electric saw helps raise the bone flap. 5, shows the actual
method of raising the flap —this is the most delicate step of the procedure. 6, the operative field after the bone flap has been raised —
at this stage the straight sinus and one of the lateral sinuses are doubly ligated and resected. In 7, the pineal gland and its stalk is
brought into view and can be easily removed by using a fine (watch maker's) forceps.
Melanotic Melanoma No. 1 (M Mel No. 1), kindly supplied
by J. G. Fortner, was chopped into very small pieces and injected
through a large-bore needle in the subcutaneous tissue of the
dorsum of the hamsters. In each transplant animal, 0.4 ml by
volume of tumor was used. After tumor inoculation, measure
ment of hamster weight, mean tumor volume, and time of ap
pearance of skin ulcérationswere recorded every 2 days, until the
animals were sacrificed. Calculation of tumor volume was based
on the method described by Mori et al. (16). All animals were
autopsied, and a detailed examination of the primary tumors as
well as of the metastatic deposits was made. In pinealectomized
hamsters the pineal area was looked into to assure completeness
of pinealectomy.
Technic
of Pinealectomy
(Chart 1)
The hamsters were anesthetized by intraperitoneal injection
of veterinary Nembutal. The dose used was 4 mg/kg body weight.
The head was shaved and the animals were held in position by
using the index and thumb of the left hand over the jaws, care
being taken to allow the animals to breathe freely. A longitudinal
incision was made on the vertex about 2 cm long. The skin inci-
JULY 1967
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1307
Taposh K. Das Gupta and Jose Terz
sion was deepened to the periosteum and the periosteum reflected
laterally—exposing a square area of the skull. The muscle attach
ments to the parietal bones were occasionally reflected to obtain
better exposure. A square-shaped bone flap encompassing parts
of both parietal and occipital bone was designed, and the bone
was actually cut on 3 sides using an electric saw. The posterior
boundary of the square was left intact; thus by applying mild
pressure the bone flap could be raised in front and held posteriorly
On the anterior boundary of the square, the saw usually trau
matized the straight sinus with some bleeding. This could easily
be controlled by gentle pressure. The flap was raised very care
fully without injuring the dura, and the confluence of sinuses was
•exposed
over the triangular space at the junction of the parietal
and occipital lobes. The lateral sinus of one side and the straight
sinus were ligated with seven zero silk. The dura was then cut and
reflected laterally. The pineal organ with its stalk was exposed
lying beneath the confluence of sinuses. The pineal was removed
without any difficulty. A few minutes of gentle pressure controlled
¡illbleeding. The bone flap was replaced and the wound closed
using four zero chromic atraumatic catgut. The use of electrocautery to control bleeding did not seem justifiable as it could
produce some damage to the surrounding area of the brain with
out the operator's being aware of it.
It is felt that atraumatic raising of the bone flap held the key to
a successful operation. Rough handling at this stage led to a tear
in the dura with injury to the confluence of sinuses. The bleeding
became brisk and often fatal. Moreover, the hamster pineals are
intimately adherent to the undersurface of the confluence and
the pineals were occasionally lost while attempts to control the
bleeding were made.
The postoperative period was usually quite smooth and the
animals started to eat and drink 8 hours after the operation. The
majority of poetoperative deaths occurred within 48 hours and
have been invariably due to continued bleeding. A small number
died during the later part due to infection of the operative site.
No antibiotics have been used postoperatively. The use of gelfoam to stop bleeding was found unsatisfactory because it led to
the development of brain abscesses.
The technic described here is easy and can be performed in any
laboratory. The initial mortality was quite high but as experience
grew the operative mortality has come down to less than 5%.
The operation takes about 10-15 minutes to perform and can be
used for all rodents. In cases where a sham operation was desired,
the pineal body with the stalk was exposed as described above,
but left intact. The bone flap was replaced and the wound closed.
RESULTS
The growth of tumors in pinealectomized hamsters did not
differ from that in the control animals during the 1st week of
tumor inoculation. However, the progressive increase in the size of
the primary tumors and visceral métastasesin pinealectomized
hamsters after the 1st week of tumor transplantation compared
to the sham-operated and control animals were the striking fea
tures in this study.
Table 1 describes the observations at autopsy on the 14th day
of tumor inoculation. The increase in volume of the tumors in
pinealectomized hamsters as opposed to the other 2 groups is
highly significant (P «0.001). Metastatic deposits in the lung
were found in 7 of 10 pinealectomized hamsters whereas only in
2 of 10 in sham-operated and 1 of 10 in control animals. This in
crease in pinealectomized animals compared to the control and
sham-operated groups was significant (P < 0.001 and < 0.02,
respectively). None of the other viscera was involved in either the
control or sham-operated group. On the other hand, in the pin
ealectomized group metastatic melanoma was found in the liver,
kidney, and spleen. The axillary lymph nodes were the site of
metastatic melanoma on 8 occasions in the pinealectomized group
and were involved only 3 times in both the control or shamoperated group (P < 0.02).
A similar trend for rapid progression of the primary tumor and
métastases,as in the first 14-day period, was noted in the 2nd
group (Table 2). The progressive increase in the tumor volume in
the pinealectomized group was consistently maintained (Chart 2).
With increasing tumor volume, the incidence of skin ulcération
increased and 11 of 12 pinealectomized hamsters showed some
form of skin ulcérationover the tumors. The lung was again the
principal site of increased visceral métastases.Nine of 12 in the
pinealectomized group had lung métastases.All the remaining
major viscera in the pinealectomized group had metastatic de
posits compared to the other 2 groups who had none. The regional
lymphatic métastasesincreased in all groups with passage of time.
TABLE 1
Observations at Autopsy Fourteen Days following Tumor Inoculation
The difference in tumor volume in pinealectomized compared to sham-operated and control hamsters
was significant (P < 0.001), whereas the difference between control and sham-operated
animals was
insignificant.
pattern"Lung721Liver200Kidney600Spleen200Axillary
No.
groupPinealectomized
of hamsters in each
of
tumor volume ulcérationsof
S.E.1.736
(ml) ±
primarytumor831Metastatic
nodes833
[10]Sham
[10]Control
-operated
[10]Mean
0.1270.172
±
0.0260.110
±
± 0.019No.
" The figures shown represent the number of times each viscus has been involved. Comparison of
percentages of metastatic involvement of any site in pinealectomized hamsters with the other groups
were significant (e.g. in lung, P < 0.001).
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Pineal Gland and Hamster Melanoma
TABLE 2
Observations al Autopsy Twenty-one Days following Tumor Inoculation
The difference in tumor volume in pinealectomized compared to sham-operated and control hamsters
was significant (P < 0.001). Not so in the other 2 groups.
No.
groupPinealectomized
of hamsters in each
tumor volume
S.E.20.3
(ml) ±
pattern"Lung911Liver400Kidney700Spleen200Axillary
of
ulcérationsof
primary
tumor1183Metastatic
nodes843
[12]Sham-operated
[10]Control
[10]Mean
0.474.50
±
0.552.390
±
db 0.085No.
" The figures shown represent the number of times each viscus has been involved. Comparison of
percentages of metastatic involvement of any site in pinealectomized hamsters with the other groups
were significant (e.g. in lung, P < 0.001).
The 3rd group of hamsters were sacrificed on the 28th day of
tumor inoculation and Table 3 summarizes the results at autopsy.
The marked difference in tumor volume between pinealectomized
hamsters and the sham-operated and control groups is obvious
(Chart 2). Incidence of visceral métastaseswas high in all the 3
groups of hamsters. All animals had lung métastasesin the pinealablated group, whereas in sham-operated and controls the in
cidence of pulmonary métastaseswas 40% and 30%, respectively.
Pinealectomized hamsters were overwhelmed with multivisceral
métastases,whereas the 2 control groups had negligible incidence
of metastatic deposits in other viscera.
In the terminal stage of the experiment (Table 4), the rapid
progression of melanoma was maintained in the pinealectomized
animals. However, it was also evident that in the other 2 groups
the melanoma was progressing at a rapid pace and the incidence
of lung métastaseswas high in these 2 groups, 6 of 10 in control
animals and 4 of 6 in sham-operated animals. Kidney métastases
were also recorded in these 2 control groups.
Histologie study of the primary tumors and the metastatic
deposits did not show any significant differences in any of the 3
groups studied. Detailed examination of the pineal area in the
pinealectomized hamsters demonstrated that removal of the
gland was complete in each case.
DISCUSSION
The results obtained in this study suggest that pinealectomy
alters the course of pigmented melanoma in Syrian hamsters.
The validity of these experimental results depend on the accuracy
of the measurement of different parameters. All the animals in
each group were transplanted and sacrificed at the same time so
the question of time interval and appearance of métastasesor the
size of the tumors could be accurately checked. Inclusion of a
sham-operated group automatically allowed a further check on
the effects of pinealectomy in these experiments. The interval of
5 weeks between any surgical trauma and tumor inoculation
obviates the possible effects of trauma.
The pinealectomized animals weighed more than the other 2
groups of hamsters at the time of sacrifice. This could be either
due to pinealectomy alone or the increased tumor volume, or
both. From these experiments no statement can be made.
Fortner et al. (8) have reported a current host survival time
JULY 1967
14
21
28
35
Days after transplantation
CHART2. Graph showing the progressive increase of tumor
volume in the pinealectomized, sham-operated, and control
animals at 14-, 21-, 28-, and 35-day intervals.
varying from 34 to 82 days after inoculation of these tumors.
They (8) further reported that there was always a high incidence
of lung and other visceral métastases,from these tumors at the
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Taposh K. Dos Gupta and Jose Terz
TABLE 3
Observations at Autopsy Twenty-eight Days following Tumor Inoculation
The difference in tumor volume in pinealectomized compared to sham-operated and control hamsters
was significant (P < 0.001), whereas the difference between control and sham-operated
animals was
insignificant.
Xo.
groupPinealectomized
of hamsters in each
tumor volume
S.E.33.61
(ml) ±
pattern"Lung843Liver300Kidney510Spleen100Axillary
of
ulcérationsof
primarytumor875Metastatic
nodes745
[8]Sham-operated
[10]Control
[10]Mean
2.76.887±
1.07.45±
0.891Xo. ±
0 The figures shown represent
the number of times each viscus has been involved.
TABLE 4
Observations at Autopsy Thirty-five Days following Tumor Inoculation
The difference in tumor volume in pinealectomized compared to sham-operated and control hamsters
was significant (P < 0.001). The difference between the other 2 groups was insignificant.
Xo.
groupPinealectomized
of hamsters in each
tumor volume
S.E.80.22
(ml) ±
pattern"Lung846Liver300Kidney743Spleen200Aiilliry
of
ulcérationsof
primarytumor857Metastatic
nodes867
[8]Sham-operated
[6]Control
[10]Mean
2.525.209±
2.222.74±
± 0.65Xo.
' The figures shown represent
the number of times each viscus has been involved.
time of death of these hamsters. But this remarkable rapidity of
increase in tumor volume as well as metastatic spread after 1
week of tumor inoculation as seen in this experiment following
pinealectomy was not observed on any occasion by Fortner
el al. (8). These results were further corroborated by using a
sham-operated group along with a standard control group in the
present study.
The data obtained here tends to support a concept that the
pineal body exerts some control on the growth and spread of pigmented neoplasms in hamsters. The changes noted in the pinealocytes in pineals of the animals dying of melanoma suggest a
possible increase in function of the gland as proposed by Rodin
(21). These experiments, however, do not explain whether the
control is mediated via a pineal hormone acting on melanophores
or whether ablation of the pineal depressed host resistance to
tumors and homografts in general. Rodin (21), reporting a small
series of pinealectomized albino rats, observed an increased trend
in the rate of growth and spread of Walker 256 carcinoma. This
trend observed by Rodin (21), though considerably less in magni
tude than that which has been demonstrated here, nevertheless
was statistically significant. Consequently, the question of
whether ablation of the pineal gland depresses host resistance to
tumors becomes an interesting problem and will need further
investigation. Similar reports of marked accentuation of growth
of sarcoma in pinealectomized animals have come from Japan
(10, 17) but these experiments have been performed without
adequate control and should be viewed with caution. On the
other hand, Kitay and Altschule (13) collected reports on the
effect of pineal extract administration on tumors and other
1310
diseases. These authors after a review of these papers concluded,
"Reports on the use of pineal extract in various diseases afford
no information on the physiologic role of the gland. This is owing
to incompleteness or poor quality of most of the reported clinical
studies." Thus it seems that the role of the pineal gland on growth
and spread of tumor is still open to several interpretations. How
ever, the fact that the hamster pinealocyte is capable of pigment
production, and ablation of the gland increases the growth and
spread of malignant melanoma in such a striking fashion tempts
one to postulate a probable cause and effect relationship.
To what extent these results obtained in hamsters under
laboratory conditions could be applied to human melanoma is
highly conjectural. However, one might feel encouraged to in
vestigate the status of the pineal gland in patients suffering from
malignant melanoma, especially those with diffuse metastatic
disease.
ACKNOWLEDGMENTS
The authors are grateful for the advice and constructive criti
cisms received from Dr. Walter Lawrence, Jr., of the SloanKettering Institute, New York, and Professor R. Warwick of the
Department of Anatomy, Guy's Hospital Medical School, London,
throughout this study.
The technical assistance of Mr. Serge Denecko of the SloanKettering Institute, New York, and Mr. Graham Stevens and
Miss Linda Rogers of the Department of Anatomy, Guy's Hospital
Medical School, London, are highly appreciated.
CANCER
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VOL. 27
Pineal Gland and Hamster Melanoma
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JULY 19f)7
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Influence of Pineal Gland on the Growth and Spread of
Melanoma in the Hamster
Taposh K. Das Gupta and Jose Terz
Cancer Res 1967;27:1306-1311.
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