[CANCER RESEARCH 41, 1545-1548,
0008-5472/81
/0041-OOOOS02.00
April 1981]
Inappropriate Secretion of Antidiuretic Hormone in Nude Mice Bearing
a Human Bronchogenic Oat Cell Carcinoma1
Yukio Kondo,2 Yoshie Mizumoto, Shigehiro Katayama, Toshio Murase, Tohru Yamaji, Nakaaki Ohsawa, and
Kinori Kosaka
Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku,
ABSTRACT
A 58-year-old man with bronchogenic oat cell carcinoma
developed a typical syndrome of inappropriate secretion of
antidiuretic hormone. The tumor tissue obtained at autopsy
had been serially transplanted in nude mice for more than four
years with 20 passages. The levels of vasopressin were re
markably increased in the plasma of nude mice bearing this
tumor [24.4 ±18.3 (S.D.) pg/ml, n = 3] as well as in the tumor
tissues [134.3 ± 72.2 ng/g, n = 3]. Furthermore, human
nicotine-stimulated neurophysin was detected in both plasma
and tumor tissues (7.4 ±3.7 ng/ml, n = 3, and 2.28 ±0.90
jug/g, n = 3, respectively). On ad libitum intake of water, nude
mice bearing this tumor excreted significantly less urine with
higher sodium concentration than did controls, but serum so
dium concentrations did not differ from those of controls. When
tumor-bearing mice were hydrated with 2 ml of water twice a
day i.p., their diuretic response was found to be suppressed in
parallel with the tumor size. However, these mice did not
become hyponatremic because they drank less water. When a
larger amount of water was loaded which could not be com
pensated by restriction of water drinking, serum sodium con
centrations were markedly decreased. On the basis of these
results, the lung cancer, when transplanted into nude mice,
produced and secreted its own antidiuretic hormone, which
induced inappropriate secretion of antidiuretic hormone in the
mice. These mice may provide a useful experimental model for
the study of excessive secretion of antidiuretic hormone and
associated pathophysiological disorders.
INTRODUCTION
Since the first description
of SIADH3 in association
with
malignant neoplasms (16), an increasing amount of circum
stantial evidence that ADH is produced by neoplasms has been
accumulated. ADH as well as neurophysins, specific proteins
associated with posterior pituitary hormones, were demon
strated in the tumor tissues (1, 4, 7-9). However, it remains to
be established if these malignant tumors produce and secrete
ADH in vivo to induce SIADH.
Nude mice bearing transplants of malignant tumors have
been well recognized as a useful system for the study of
functioning tumors because functional activities of tumors are
well preserved in animals. Moreover, the high concentration of
' Supported
in part by the grant of Japan Ministries
of Education
and of
Welfare and Health.
2 To whom requests for reprints should be addressed.
3 The abbreviations used are: SIADH, syndrome of inappropriate secretion of
antidiuretic hormone; ADH, antidiuretic hormone; NSN. nicotine-stimulated neu
rophysin; ESN, estrogen-stimulated neurophysin.
Received July 31, 1980; accepted December 29, 1980.
Tokyo 113, Japan
released hormones in the bloodstream remarkably amplifies
the host responses. Occasionally, in mice even unexpected
humoral factors produced by tumors can be detected. Utilizing
this technique, we have identified lung cancer producing granulopoietic factor (2) and malignant melanoma excreting cachexia-producing principles (11).
The present paper gives an account of production and se
cretion of ADH by a lung cancer transplanted into nude mice
and also of SIADH induced in the mice.
MATERIALS
AND METHODS
Case Report. A 58-year-old man was admitted to Tokyo
University Hospital for the examination of cough and fever.
Laboratory data revealed severe hyponatremia; serum sodium
was 121 mEq/liter and chloride was 87 mEq/liter. Plasma
osmolality was consistently low (236 to 250 mOsmol/kg), while
plasma ADH levels were elevated (19.2 to 69.8 pg/ml). A water
load (20 ml/kg) resulted in a marked antidiuresis. Bronchoscopic examination showed a tumor mass on the fourth to the
tenth segmental bronchi of the right lung, and the pathological
diagnosis of the biopsied specimen was anaplastic small-cell
carcinoma. The biopsied tumor tissue contained 88 milliunits
of ADH per g of acetone, dry powder, by bioassay (19). He
died of massive hemoptysis. The autopsied tumor tissue con
tained 14.4 ng of arginine-vasopressin
per g wet tissue and
showed positive immunofluorescence with an antivasopressin
serum.
Transplantation of the Tumor. Female BbLB/c-nu/nu mice
weighing about 20 g were distributed at 4 weeks of age by
Central Institute for Experimental Animals, Kawasaki, Japan,
and maintained in specific-pathogen-free
conditions. Each
block of 5x5x5 cu mm of the autopsied tumor tissue was
transplanted s.c. to bilateral flanks of 3 nude mice with a trocar.
When the transplanted tumors grew large enough, mice were
sacrificed by decapitation, and the tumors were aseptically
removed for serial transplantation. The blood issuing from the
vessels of the trunk was collected in chilled heparinized tubes
for the subsequent determination of vasopressin and neuro
physins.
Analysis of Water Balance in Nude Mice. Experiments were
conducted in nude mice on ad libitum intake of food and water
at constant temperature and humidity. Each nude mouse was
transferred to a metabolic cage at 4 p.m. and maintained there
for the succeeding 24 hr unless otherwise specified. Volumes
of urine and drinking water were recorded. At the end of the
experiment, nude mice were sacrificed by decapitation, and
the trunk blood was collected for the determination of serum
sodium concentrations. Hydration was effected by i.p. injection
of 2 different doses of distilled water. In the first experiment, 2
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1545
Y. Kondo et al.
ml of water were injected into control and tumor-bearing mice
twice a day (4 p.m. and 10 a.m.). In the second experiment, 2
ml of water were administered 3 times (10 a.m., 1 p.m., and 4
p.m.), and the animals were sacrificed at 6 p.m.
Light and Electron Microscopy. The tissue specimens were
processed for the routine histopathology, and sections were
stained with hematoxylin and eosin. For the electron micro
scopic study, the tumor tissues were cut into small pieces,
fixed with 2% glutaraldehyde and then 1% osmium tetroxide,
dehydrated in graded ethanol, and embedded in Epon. Ultrathin
sections were doubly stained with uranyl acetate followed by
lead citrate.
Tumor Tissue Extraction. Tumors extirpated from the nude
mice were homogenized in 2 volumes of 0.1 N HCI with a
Potter-Elvehjem glass homogenizer. The homogenate was ad
justed to pH 1.5 with 1 N HCI followed by centrifugaron at
10,000 x g for 15 min. The supernatant was collected and
titrated to pH 7 at 4°. White precipitate was removed by
centrifugation, and the superantant was used for the assay of
vasopressin, oxytocin, and neurophysins.
Assay of Vasopressin, Oxytocin, and Neurophysins.
Plasma concentrations as well as tissue contents of vasopres
sin were estimated by a radioimmunoassay
previously de
scribed in detail (17). Oxytocin was measured by radioimmu
noassay utilizing 125l-labeled synthetic oxytocin (Sandoz,
Basel, Switzerland) and an antiserum raised against oxytocin
covalently linked to bovine serum albumin. NSN and ESN were
radioimmunoassayed according to the general method of Ro
binson (14) using the ¡mmunological materials kindly donated
by the National Institute of Arthritis, Metabolism, and Digestive
Diseases and the National Pituitary Agency, USPHS. Details of
the assay procedure were described elsewhere (18).
Statistical Analysis. Comparison of 2 samples were made
by an unpaired i test analysis. Linear regression analysis was
performed using the least-squares method.
RESULTS
In all nude mice bilaterally transplanted with the autopsied
tumor tissue, tumor growth was evident on at least one flank
within 5 weeks. Serial transplantation was successful in more
than 90% of the animals. Nude mice bearing this tumor seemed
to be quite healthy, and their body weight gradually increased
as tumors grew. Hematoxylin-eosin staining of the tumor tissue
transplanted into nude mice showed small round and oat cell
carcinoma with histology which was identical to the histology
of the tumor at autopsy. The electron microscopic findings in
the tumor tissue were similar to that described in previous
reports (7, 13). The major portion of the tumor tissue had
anaplastic features with few rough-surfaced endoplasmic re
tÃ-culaand no secretary granules. Only a small number of tumor
cells from the limited area possessed secretory granules with
developed rough-surfaced endoplasmic retÃ-cula.
As shown in Table 1, the mean plasma vasopressin concen
tration in nude mice bearing the tumor [24.4 ±18.3 (S.D.) pg/
ml] was significantly higher than that in control nude mice [4.6
± 3.1 pg/ml] (p < 0.05). Moreover, human NSN, which is
considered to be synthesized and released concomitantly with
vasopressin (5, 14, 15), was detected in the circulation of
tumor-bearing mice with the mean plasma concentration of 7.4
±3.7 ng/ml (range, 4.2 to 11.4 ng/ml), while it was undetectable in the plasma from all of the 4 control nude mice.
Tumor tissues extirpated from nude mice contained large
amount of vasopressin (134.3 ±72.2 ng/g wet tissue) as well
as NSN (2281 ± 898.2 ng/g wet tissue). The results are
consistent with the previous reports (7-9,12) and suggest that
vasopressin biosynthesis in the tumor is closely related to that
of NSN as demonstrated in the hypothalamus of the experi
mental animals (5, 6,15). Tumor contained also a small amount
of oxytocin (1.26 ±0.03 ng/g wet tissue) and ESN (12.35 ±
1.91 ng/g wet tissue). ESN thus determined may be overesti
mated in view of the cross-reaction of a large amount of NSN
in ESN radioimmunoassay (14). A control tumor tissue (a
human malignant melanoma) transplanted into a nude mouse
contained no detectable amount of vasopressin, NSN, and ESN
(Table 1). The amount of vasopressin in tumor varied consid
erably from tissue to tissue, but no diminution in vasopressin
activity was observed during serial transplantation of the tumor.
The results of the water balance study in nude mice are
shown in Table 2. When water was allowed ad libitum, no
significant difference was observed in the mean volumes of
drinking water for control and tumor-bearing mice. However,
tumor-bearing mice excreted significantly less urine with a
higher sodium concentration than did controls. When 2 ml of
water were administered i.p. twice a day, control nude mice
excreted significantly more urine (2.4 ± 0.4 ml/day) with a
Table 1
Hormones and their neurophysins in the plasma of nude mice and in tumor tissues
PlasmaMice
18.3*"4.6
±
cellcarcinoma
bearing oat
3.7<0.5OxytocinNDCNDESNNDND
±
3)Control (n =
mice (no tu
± 3.1NSNng/ml7.4
mor) (n = 4)Vasopressinpg/ml24.4
ng/g wet tissue
Tumor tissues
Oat cell carcinoma (n
-3)
Control tumor (n =
134.3 ±72.2
<1.7
2281.0
±898.2
<6.8
1.26 ±0.03
12.35 ±1.91
ND
<3.4
1)
8 p < 0.05 compared to control.
" Mean ±S.D.
c ND. not determined.
'' A human malignant melanoma transplanted into a nude mouse.
1546
CANCER
RESEARCH
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VOL. 41
SIADH in Nude Mice Bearing Human Lung Cancer
Table 2
Daily water balance in control and tumor-bearing nude mice with and without water load
of drink
ing water (ml/
day)6.3
Without water load
Control (n =- 10)
Tumor bearing (n = 10)
PWith
±1.1a
5.8 ±1.1
NS"6.1
volume
(ml/day)1.1
sodium con
sodium
centration (mEq/li
concentration
(mEq/liter)170.6
ter)147.0
±0.4
0.5 ±0.1
<0.052.4
±42.3
271.5 ±56.0
<0.0187.1
water load
±0.4tf
±16.8d
±0.4°
Control (n = 10)
1.0 ±0.5°
188.4 ±66.6e
3.6 ±0.4e
Tumor bearing (n —10)
<0.01Serum
<0.001Urine
<0.001Urine
PVolume
Mean ±S.D.
0 NS, not significant ( p £0.05).
c Not significant ( p ä0.05) compared to the same group without water load.
" p < 0.001 compared to the same group without water load.
8 p < 0.01 compared to the same group without water load.
lower sodium concentration (87.1 ±16.8 mEq/liter) than those
in the control study (1.1 ±0.4 ml/day, 170.6 ±42.3 mEq/
liter). In tumor-bearing mice, urine volume as well as urinary
sodium concentration was not changed significantly after the
water load. The volume of drinking water, on the other hand,
was significantly decreased after the water load in tumor-bear
ing mice but not in controls. In these experiments, no significant
difference was obtained in the mean serum sodium concentra
tions for control and tumor-bearing mice. A larger volume of
water was then loaded in the second experiment; 2 ml of water
were administered i.p. to both control and tumor-bearing mice
at 10 a.m., 1 p.m., and 4 p.m. Serum sodium concentrations at
6 p.m. in nude mice bearing the tumor (118.6 ±4.6 mEq/liter,
n = 3) were significantly lower than those in control nude mice
(146.6 ±3.1 mEq/liter, n = 3) (p< 0.001).
Chart 1 illustrates the correlation between tumor sizes and
urine sodium concentrations when 2 ml of water were admin
istered i.p. to mice twice a day. The coefficient of correlation
was 0.74 (n = 15), which shows a significant positive correla
tion (p < 0.01) between these 2 variables.
DISCUSSION
SIADH is frequently encountered in patients with malignant
neoplasms and is clinically characterized by hyponatremia and
antidiuresis (3). It has not been shown, however, that a neo
plasm removed from a patient with SIADH was successfully
transplanted to experimental animals to develop SIADH. Kameya et al. (10) detected ADH ranging from 3.8 microunits to
5.8 milliunits per g in tumor tissues from 3 bronchogenic oat
cell carcinomas transplanted into nude mice. Whether ADH
secreted from the tumors circulated in nude mice was not
determined in their study. Furthermore, the host response to
the ectopically produced ADH was not established. In this
report, we described successful transplantation of a vasopressin-producing human bronchogenic oat cell carcinoma into
nude mice, which has been maintained more than 4 years with
20 passages. High levels of vasopressin and also human NSN
were detected in the plasma of tumor-bearing animals as well
as in tumor tissues removed from mice. Moreover, it was
demonstrated that the transplanted tumor could induce SIADH
in mice.
Although the foregoing results indicate that the tumor trans
planted into nude mice does produce and secrete ADH (Table
±7.4
147.6 ±23.0
NS144.7
±7.4C
144.2 ±3.0C
NS
300-
200-
100-
0
200 400 600 800 1000
TUMOR
SIZE (sq mm)
Chart 1. Relationship between tumor sizes and urinary sodium concentrations
in nude mice bearing the tumor when 2 ml of water were administered i.p. twice
a day. Solid lines connect the values obtained in the same nude mice at different
occasions. Stippled area, range of urine sodium concentrations of control nude
mice without tumors when the same amount of water was loaded, r = 0.74; p <
0.01.
1), these mice did not develop hyponatremia on ad libitum
water intake (Table 2). When 2 ml of water were loaded twice
a day, a remarkable diuresis was observed in control mice. In
tumor-bearing mice, on the other hand, the same maneuver
resulted in impaired diuresis. In addition, the magnitude of
suppressed diuresis was significantly correlated to the tumor
size (Chart 1). However, serum sodium concentrations deter
mined 6 hr after the second injection were not decreased. Of
importance in this regard is the fact that drinking water was
significantly less in tumor-bearing mice than in controls (Table
2). These results suggest that ADH produced by the tumor
suppressed diuretic response but the resulting positive water
balance was compensated by self-restriction of water drinking
and that ingested water was mainly lost through the skin and
the lung, which is not directly regulated by ADH. The thirst
center may be more sensitive in mice than in humans, which
results in self-restriction of water intake in response to a minute
decrease in plasma osmolality. Habitual water drinking, in fact,
is known to be an important trigger for the development of
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1547
Y. Koncto ef al.
hyponatremia in cancer patients.
If our assumption is correct, then a typical SIADH may
develop in these tumor-bearing mice by forced water intake of
more than 6 ml/day, because water excretion through the skin
and the lung may not be able to compensate such a large water
ingestion. In an attempt to test this possibility, we performed
the second experiment in which 2 ml of water were adminis
tered i.p. to control and tumor-bearing mice 3 times in 8 hr. As
expected, a remarkable decrease in serum sodium concentra
tion appeared only in tumor-bearing mice. It was concluded
from these results that inappropriate secretion of ADH alone is
not sufficient for the appearance of hyponatremia in nude mice
and that a large amount of forced water intake or disturbed
thirst center, in addition, may be required for the development
of hyponatremia. This lung cancer transplanted into nude mice
may provide a useful experimental tool for the investigation of
synthesis of ADH as well as a good experimental model for
SIADH.
ACKNOWLEDGMENTS
We are grateful to Drs. N. Aoki and N. Urano (Department of Pathology,
Faculty of Medicine, University of Tokyo) for their comments on pathology. We
are also indebted to Dr. A. Urabe (The Third Department of Internal Medicine,
Faculty of Medicine, University of Tokyo) for his encouragement throughout the
work.
5.
6.
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8.
9.
10.
11.
12.
13.
14.
15.
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CANCER
RESEARCH
VOL. 41
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Inappropriate Secretion of Antidiuretic Hormone in Nude Mice
Bearing a Human Bronchogenic Oat Cell Carcinoma
Yukio Kondo, Yoshie Mizumoto, Shigehiro Katayama, et al.
Cancer Res 1981;41:1545-1548.
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