[CANCER RESEARCH 47, 5620-5625, November 1, 1987]
Effects of Chemical Sympathectomy in Neonatal and Adult Mice on C-1300
Neuroblastoma Tumor Growth and Catecholamine Content1
Donald W. Fink, Jr.,2 and Bernard L. Mirkin3
Division of Clinical Pharmacology, Departments of Pediatrics and Pharmacology, University of Minnesota Health Sciences Center, Minneapolis, Minnesota 55455
ABSTRACT
The in situ C-1300 murine neuroblastoma (MM!) tumor model was
used to investigate the influence of 6-hydroxydopamine (6HD)-induced
sympathectomy on tumor growth and catecholamine concentration. One
week (adult) and 3 weeks (neonatal) after sympathectomy, mice were
implanted with 10' disaggregated MNB cells. The time interval between
implantation of MNB cells and detection of palpable tumor (tumor onset
time), transverse tumor diameter, tumor weight, tumor weight to body
weight ratio, and tumor catecholamine concentration were determined.
Sympathectomy following 6HD administration was confirmed by anal
ysis of catecholamine concentrations in the heart and spleen by highpressure liquid chromatography. Treatment of adult animals with 6HD
reduced the mean heart and spleen norepinephrine (NE) concentrations
to less than 20% of controls (vehicle treated). Neonatal sympathectomy
decreased the average heart and spleen NE concentrations to less than
10% of comparable control mice. Whole brain NE and dopamine concen
trations were not altered by treatment with 6HD in either age group.
Tumor onset time following implantation of MNB cells was signifi
cantly increased in animals sympathectomized as either neonates or as
adults. In contrast, MNB tumor growth rate following tumor onset was
significantly inhibited in animals sympathectomized as neonates but not
as adults.
The catecholamine concentrations of tumors removed from control and
sympathectomized mice 8 days after tumor onset were determined. Tumor
NE and dopamine concentrations were increased 9.09 ±2.8- (SE) and
7.03 ±1.8-fold, respectively, in mice sympathectomized as neonates.
There were no significant differences in the NE and dopamine concentra
tions of tumors obtained from sympathectomized and control adult mice.
Pretreatment with desmethylimipramine prior to 6HD administration
prevented destruction of sympathetic neurons, inhibition of tumor growth
rate, and the increase in tumor catecholamine concentration observed in
neonatally sympathectomized mice.
These data suggest that the influence of chemical sympathectomy on
MNB tumor growth and biochemical differentiation, as defined by cate
cholamine content, are age dependent.
INTRODUCTION
Neuroblastoma
glionic blocking agent, chlorisondamine, to neonatal mice ar
rests maturation of the SNS, resulting in an attenuation of C1300 MNB tumor growth in situ ( I ). Conversely, hypertrophy
of the SNS induced by treatment of neonatal mice with nerve
growth factor (NGF) augments C-1300 MNB growth in situ
(2). It has been reported that MNB tumor growth is markedly
suppressed in neonatal and adult mice that have been chemically
sympathectomized by administration of 6-hydroxydopamine
(6HD) (2, 3). In addition, coculturing MNB cell lines with
sympathetic ganglia or sympathetic ganglia-conditioned me
dium augments MNB cell replication (4-6).
The present investigation has demonstrated that 6HD-induced chemical sympathectomy of neonatal mice (4-10 days
old) inhibited MNB growth both prior to and following the
initial detection of palpable tumor. Chemical sympathectomy
of adult mice (58-66 days old) also caused an increase in tumor
onset time, however, MNB growth after tumor palpation was
not affected. The suppression of MNB growth in neonatally
sympathectomized mice was associated with an increase in
tumor catecholamine content, whereas sympathectomizing
adult mice did not alter tumor catecholamine content.
MATERIALS
AND METHODS
Animals
Litters of A/J mice were reared from a breeding colony maintained
in animal facilities assigned to the Department of Pharmacology at the
University of Minnesota. Litters were housed in individual cages and
pups remained with lactating mothers until weaned at approximately 4
weeks of age. Adult A/J mice, 56 days old, were obtained from The
Jackson Laboratory (Bar Harbor, ME). Mice were fed standard mouse
laboratory chow with water provided ad libitum. All animals were
maintained in a neutral thermal environment with carefully controlled
diurnal lighting cycles (12-h light, 12-h dark).
In Situ MNB Tumor Model
and precursor cells of the SNS4 derive from
a common embryological origin, the neural crest. This property
of shared ontogeny has led to speculation that the SNS exerts
a modulating influence on other neuroectodermal derivatives
such as neuroblastoma.
The effects of the SNS on neuroblastoma have been investi
gated in situ and in tissue culture. Administration of the ganReceived 2/13/87; revised 7/27/87; accepted 7/31/87.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1Supported by National Institute of Neurological and Communicative Disor
ders and Stroke Grant NS 17194, and by a grant from the Viking Children's
C-1300 MNB was originally provided by the E. G. and G. Mason
Research Institute, Worcester, MA. This tumor line has been main
tained by implantation into adult male A/J mice for 88 tumor genera
tions, each approximately 21 days long. Implantations were performed
by injection of disaggregated tumor cell suspensions by using a tech
nique previously described (7). Cell suspensions were prepared from
tumor specimens that had been dissected carefully and trimmed free of
necrotic tissue. The sample was minced in sterile 0.9% NaCl solution
under aseptic conditions, and 0.1 ml of the suspension containing 10"
viable MNB cells was inoculated s.c. into the abdominal area. Tumor
cell viability in suspension was determined by trypan blue dye exclusion.
Chemical Sympathectomy
Fund (Minneapolis, MN).
1 Recipient of a Predoctoral Fellowship from the Pharmaceutical Manufactur
ers Association.
3To whom requests for reprints should be addressed, at Division of Clinical
Pharmacology, Departments of Pediatrics and Pharmacology, University of Min
nesota Health Sciences Center, Mayo Box 87, Minneapolis, MN 55455.
4 The abbreviations used are: SNS, sympathetic nervous system; MNB, murine
neuroblastoma; 6HD, 6-hydroxydopamine; DMI, desmethylimipramine; NE,
norepinephrine; DA, dopamine; NGF, nerve growth factor, CI, confidence inter
val.
Neonates. Neonatal A/J mice were initially treated with 6HD (100
¿ig/gof body weight, s.c.) on day 4 postpartum. This dose was repeated
on days 6, 8, and 10 postpartum. Vehicle-treated animals received
injections of vehicle solution consisting of 0.9% NaCl solution and 1.0
mg/ml ascorbic acid (as an antioxidant). All solutions were prepared
prior to treatment. Destruction of adrenergic neurons was verified by
high-pressure liquid Chromatographie analysis of catecholamine con
centrations in sympathetic target organs. This regimen caused a marked
5620
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1987 American Association for Cancer Research.
MODULATION
OF NEUROBLASTOMA
depletion of norepinephrine in the heart and spleen that persisted for
at least 5 weeks after the final 6HD injection.
Adults. A/J mice, 56 days old, were administered 5 injections of
6HD ( 100 ng/g of body weight, i.p., every 48 h). Vehicle-treated animals
were administered 0.9% NaCl-ascorbic acid injections. Sympathectomy
was confirmed by analysis of tissue catecholamines.
Experimental Design
GROWTH
BY SNS
Statistics
Statistical evaluation of these data was performed using the unpaired
Student's t test for equal and unequal variances. Differences between
the experimental groups were considered to be significant at the P •_
0.05 level.
RESULTS
At 3 weeks (neonates) and 1 week (adult) following chemical sympathectomy, experimental animals were inoculated with IO6MNB cells.
Effect of Chemical Sympathectomy
Organ Weight
on Total Body and Specific
Animals in each experimental treatment group were examined daily for
The administration of 6HD (100 Mg/g) was used to sympathe appearance of palpable tumors. The transverse diameter of each
palpable mass was recorded for a period of 8 days after tumor onset (7,
thectomize adult and neonatal mice (see flow diagram, Fig. 1).
8). At the conclusion of the observation period animals were killed by The treatment schedules were designed to allow a period of 1
cervical dislocation. Immediately thereafter, brain, heart, spleen, and
tumor were excised, frozen on dry ice, weighed, and stored at —80"C week (adults) or 3 weeks (neonates) between the final 6HD
until assayed for catecholamines (see Fig. 1).
Analysis of Catecholamines
High-pressure liquid chromatography, coupled with electrochemical
detection, was used to determine NE, DA, and epinephrine tissue
concentrations. Frozen samples were thawed and homogenized in 0.1
M perchloric acid. A final homogenate volume of 1.0 ml was used to
perform the analysis. Catecholamines in the homogenate were adsorbed
onto alumina at alkaline pH and eluted with 0.1 M perchloric acid.
Methyldopamine was used as the internal standard. An aliquot of the
acid eluate was injected onto a cation exchange column (6 ft x 0.125
in., outside diameter) packed with Corasil C/X (Waters Associates, Inc.
Milioni. MA). The eluting buffer consisted of sodium acótaleII.<)
(11.5 g/liter). citric acid-H2O (13.6 g/liter), sodium hydroxide (4.8 g/
liter) and glacial acetic acid (2.1 ml/liter), pH 5.1, flowing at a rate of
1.0 ml Diiii under a pressure of 1000 psi.
Estimation of Tumor Growth by the Method of Gompertz
MNB tumor growth kinetics was estimated by fitting transverse
tumor diameters to the Gompertzian function (9):
In D = ß
- fa'"
where D is the tumor diameter (cm), t is the time (days), and a, ß,
and
</>
are growth parameters estimated by utilizing a BASIC computor
program entitled GOMPERTZIAN run on a microcomputor.5 The
initial value of 0 is set as the asymptote of the tumor diameter. The
estimate is then refined by iteration with the use of Newton's method
until a convergence equivalent to 0.1% of ß
is achieved as follows:
0NEW= ßou—F/G
and
injection and implantation of MNB tumor cells. These intervals
were selected to ensure exclusion of a direct 6HD effect on the
tumor cells.
Treatment of adult mice with 6HD caused a transient reduc
tion in body weight. At the time of sacrifice, 8 days following
initial palpation of the tumor, total body and organ weights in
the vehicle and 6HD-treated groups did not differ significantly.
In contrast, neonatal mice receiving 6HD manifest a sustained
decrease in total body and organ weight. The total body and
tissue weights of neonatal mice treated with either vehicle
solution or 6HD were as follows (vehicle, 6HD): total body,
16.4 ±0.4 (SE) g, 11.6 ±0.4 g (P < 0.0005); brain, 339 ±4
mg, 297 ±4 mg (P < 0.0005); heart, 62 ±l mg, 47 ±l mg (P
< 0.005); and spieen, 81 ±4 mg, 48 ±4 mg (P < 0.005).
These effects on total body and organ weight were attributed
to the destruction of adrenergic neurons by 6HD. This hypoth
esis was tested in a group of five animals that were treated as
neonates with the adrenergic neuronal uptake blocking agent,
desmethylimipramine (DMI, 25 /ig/g) 45 min prior to 6HD.
The mice were sacrificed 35-40 days later and did not exhibit
any loss in total body or organ weight.
Catecholamine Concentrations of Tissues in Sympathectomized
Mice
Sympathectomy following treatment with 6HD was con
firmed by performing catecholamine assays on brains, hearts,
and spleens obtained from treated animals (Fig. 2). Adminis
tration of 6HD to neonatal or adult mice did not significantly
alter whole brain NE or DA concentrations. In contrast, the
heart and spleen exhibited a significant reduction in NE con
centration following treatment with 6HD in both age groups.
Tissue catecholamine concentrations were not depleted by 6HD
in animals pretreated with DMI as neonates (Fig. 2).
F = dS/dß
Influence of Chemical Sympathectomy on MNB Tumor Onset
Time and Growth Rate
G = dF/dß
S = I In D, - ß+ <t*-°"
Tumor Onset Time. The time to detection of palpable tumor
(tumor onset time) after inoculation with IO6 MNB cells was
i-1
The values of a and <t>were then estimated by linear regression of
In (ß
- In D) = In 0 - at
using the previously obtained estimate for ß.
Confidence intervals (95%)
of the estimated tumor diameters were calculated by applying a /
distribution to the residual values (i.e., the difference between the actual
and predicted diameters for each point). When the confidence intervals
for sets of predicted diameters did not overlap they were considered to
differ at the P < 0.05 level of significance.
' B. Bostrom, unpublished data.
determined in mice sympathectomized either as neonates or
adults (Fig. 3). Tumor onset time was significantly increased in
both experimental subgroups. The increase in tumor onset time
observed following implantation of MNB cells into sympathec
tomized mice suggested that cellular replication prior to devel
opment of palpable tumor had been inhibited. The tumor onset
times in vehicle-treated and sympathectomized adult mice were
5.7 ±0.3 days and 7.0 ±0.4 days (P < 0.005), respectively. In
mice sympathectomized as neonates, tumor onset time was 7.0
±0.3 days (N = 74) as compared to 5.8 ±0.2 days in control
mice (N = 82). Tumor onset time was increased by a factor of
5621
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1987 American Association for Cancer Research.
MODULATION
OF NEUROBLASTOMA
GROWTH BY SNS
NEONATAL
l
«HD TREATMENT
| [POST
TREATMENT
PERIODI
[106 MNB CELLS
MPLANTED]
TUMOR GROWTH OBSERVED I
I
30
TISSUES REMOVED
CATECHOLAMINES
ANALYZED BV MPIC
Fig. 1. Flow diagram summarizing the ex
perimental design and treatment schedule.
ADULT
i
6HD TREATMENT
I [POST
TREATMENT
PERIOD]
[IP6
MNB CELLS
|
IMPLANTED|
|TUMOR
GROWTH OBSERVED]
8 DAYS
n—i—i—rAGE (Days)
58
60
62
64
TISSUES REMOVED
CATECHOLAMINES
ANALYZED BY HPLC
Aqe at Tim«of
Sympathectomy:
A)
rh
ADULT
NEONATAL
6HD
B)
\:
I
0123456789
I VEH
2 «
6HD
i
6HO-C DM I
6
0
0
10
9
r »
8
0)
7
6
1
u
5
4
I
Z
3
0
ADULT
10
DAYS AFTEB TUMOR IMPLANTATION
NEONATE
Fig. 2. Norepinephrine concentration of heart, spleen, and brain after treat
ment of neonatal and adult mice with 6HD. Tissue concentrations of NE in ill
heart, (B) spleen, and i ( i brain were assayed in mice sympathectomized as
neonates or adults.
concentrations
determined
pressure mean;
liquid
chromatography
andNE
expressed
as ng/gwere
tissue
weight x byinhigh
•'.
Columns,
bars, SE.
21% in the neonatally sympathectomized animals and this
increment was highly significant (P < 0.001). The increase in
tumor onset time observed in sympathectomized neonates was
Fig. 3. Effect of chemical sympathectomy on MNB tumor onset time. Tumor
onset time was defined as the interval (days) between MNB cell implantation and
detection of palpable tumor. MNB tumor cells (10*) were implanted 1 week
(adult) and 3 weeks (neonatal) after chemical sympathectomy. Mice were treated
as adults or neonates with 6HD, vehicle solution ( VEH), or DMI. *, P £0.005,
adult-6HD compared to adult-Veh; **, /' - 0.02S, neonatal-6HD compared to
neonatal-Veh. Columns, mean; bars, SE.
prevented by pretreatment of neonatal animals with DMI prior
to the administration of 6HD. The tumor onset time of 5.0 ±
0.0 days noted in the DMI-treated group was similar to that
observed in both adult and neonatal vehicle-treated groups.
Sympathectomy did not alter MNB tumorigenicity or malig
nant expression. The incidence of tumor formation following
implantation of MNB cells was 100% and the survival time of
tumor-bearing mice was similar for vehicle-treated and sympathectomized animals (data not shown).
Tumor Growth. A profile of tumor growth in situ was obtained
by measurement of three major parameters, the transverse
tumor diameter determined over an 8-day period (Fig. 4), tumor
weight at the completion of the 8-day period (Fig. 5), and ratio
of tumor to body weight (Fig. 5). These data are presented
below.
Transverse tumor diameters were mathematically trans
formed and Gompertzian growth curves were generated. The
Gompertzian growth curves derived from measurements of
transverse tumor diameters over a fixed time period (8 days)
after tumor onset demonstrated that neonatal sympathectomy
5622
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1987 American Association for Cancer Research.
MODULATION
NEONATAL
ADULT
DAYS
OF NEUROBLASTOMA
SYMPATHECTOMY
SYMPATHECTOVY
AFTER
TUMOR
ONSET
Fig. 4. Effect of sympathectomy on MNB tumor growth. The transverse
diameter of each tumor was measured for 8 days following tumor onset. Trans
verse tumor diameters are presented in A (neonatal sympathectomy) and C (adult
sympathectomy). Points, mean; ears, SE. Symbols: vehicle treated (O), 6HD
treated (•),and 6HD plus DMI treated (»).Gompertzian transformation (See
"Materials and Methods") of transverse tumor diameters are presented in B and
D. Shaded areas represent 95% confidence intervals. Vehicle treated (•),6HD
treated (D), and 6HD plus DMI treated (D). *, P s 0.05 as determined by the
95% confidence intervals.
I
30
V
T
ADULT
GROWTH BY SNS
tumor growth was not inhibited in animals sympathectomized
during adult life (Fig. 4, C and D).
A corollary experiment designed to test the hypothesis that
inhibition of MNB tumor growth in neonatally sympathecto
mized mice was due principally to destruction of the sympa
thetic nervous system was performed. Treatment of neonatal
mice with DMI prior to administration of 6HD prevented the
destruction of their SNS. These animals did not exhibit any
inhibition of tumor growth (Fig. 4, A and B).
The mean tumor weight in each experimental subgroup 8
days after tumor onset was as follows (vehicle, 6HD): adult,
2.05 ±0.19 g, 2.64 ±0.35 g; neonatal, 1.87 ±0.07 g, 0.89 ±
0.08 g; and 6HD plus DMI, 1.69 ±0.14 g (Fig. 5). The mean
weight of tumors excised from adult animals treated with 6HD
was greater than the average weight of tumors removed from
vehicle-treated controls, but this difference was not significant
(P > 0.05). Tumors excised from animals sympathectomized
as neonates were significantly smaller than those of vehicletreated animals (P < 0.0005). The mean weights of tumors
obtained from vehicle- and 6HD plus DMI-treated animals
were not significantly different (P > 0.1).
The ratio of tumor to body weight was also used as an
expression of tumor growth (Fig. 5). This parameter was in
agreement with results obtained by using tumor diameter and
tumor weight to elucidate any age-dependent effects of sympa
thectomy on tumor growth. The ratios of tumor to body weight
(expressed as percentage) in vehicle-treated and sympathecto
mized adult mice were 10.0± 1.0 and 13.7 ±2.1%, respectively,
and did not differ significantly (P > 0.05); whereas ratios
obtained in neonatally treated animals differed significantly (/'
< 0.0005) and were 13.2 ±0.5% (vehicle) and 8.2 ±0.7%
(6HD). The reduction in ratio of tumor to body weight of
neonatally sympathectomized
mice was prevented by the
administration of DMI prior to 6HD. The ratio in 6HD plus
DMI-treated neonates was 12.7 ±1.0% and did not differ
significantly from vehicle-treated mice (P > 0.375).
Another index of tumor growth generated from these data
was the tumor growth rate constant (transverse tumor diameter
at 8 days minus initial diameter, divided by days of observation).
It provided an estimate of the average daily increment in tumor
diameter during the 8 days following tumor onset. Values for
tumor growth rate constants, expressed as mm/day, were as
follows (vehicle, 6HD): adult, 1.9 ±0.07, 1.9 ±0.08 (P> 0.1);
neonatal, 2.1 ±0.05, 1.6 ±0.08 (P < 0.0005). The decrease in
tumor growth rate constant observed in neonatally sympathec
tomized mice did not occur in DMI-treated animals.
Effect of Nerve Growth Factor on MNB Tumor Growth in
Nonsympathectomized
and Neonatally Sympathectomized Ani
mals
NEONATAL
Fig. 5. Effect of chemical sympathectomy on tumor weight and tumor weight
to body weight ratio. Tumor and body weights were determined 8 days after
tumor onset in animals implanted with to' MNB cells. Net body weight was
determined by subtracting the weight of dissected tumors from total body weight
measured prior to tumor excision. Mice were treated as adults or neonates with
6HD, vehicle solution (Veh), or DMI. *, P < 0.0005 for both tumor weight and
ratio of tumor weight to body weight when 6HD- and vehicle-treated groups were
compared. Columns, mean; bars, SE.
inhibited MNB tumor growth (Fig. 44). Inspection of the 95%
CIs for each point on the Gompertzian growth curve indicated
that suppression of tumor growth in neonatally sympathectomized mice occurred as early as the third day following tumor
onset (Fig. 4Ä); mean transverse tumor diameters were 5.59
mm (CI, 5.56-6.19) and 6.79 mm (CI, 6.58-7.18) in sympathectomized and vehicle-treated mice, respectively. In contrast,
The administration of murine NGF to mice at the time of
MNB tumor implantation effectively antagonized the suppressive effect of neonatal sympathectomy on tumor growth. In this
study, animals were administered NGF (15 Mg/day) for a period
of 8 days, following which tumors were excised and weighed.
The mean tumor weights in each experimental group were as
follows: nonsympathectomized mice (TV= 83), 1.99 ±0.1 g;
nonsympathectomized mice administered NGF (N = 24), 2.02
±0.1 g; sympathectomized mice (N = 81), 0.95 ±0.1 g; and
sympathectomized mice administered NGF (N = 39), 1.52 ±
0.1 g. The administration of NGF to control (nonsympathec
tomized) mice did not produce any effect, whereas tumor weight
was restored to 75% of nonsympathectomized
controls by
administration of NGF to sympathectomized animals.
5623
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1987 American Association for Cancer Research.
MODULATION
OF NEUROBLASTOMA
GROWTH BY SNS
The ratio of tumor to body weight provided a more precise
index by which to assess the effects of NGF upon tumor growth.
In nonsympathectomized mice this ratio was 13.6 ±0.4%, in
nonsympathectomized mice administered NGF it was 14.6 ±
0.9%, in sympathectomized mice it was 8.9 ±0.5%, and in
sympathectomized mice administered NGF it was 14.2 ±0.8%.
Thus, the administration of nerve growth factor to neonatally
sympathectomized mice restored the ratio of tumor to body
weight to that of control (nonsympathectomized) animals, sug
gesting that NGF enhanced tumor growth rate only after sympathectomy.
physiological signals generated during development may regu
late the expression of tumorigenicity. It has been proposed that
the interaction between neuroblastoma cells and the sympa
thetic nervous system plays a pivotal role in modulating the
growth and differentiation of this tumor. The present study has
utilized the C-1300 MNB tumor model to test the hypothesis
that the effects of chemical sympathectomy on MNB tumor
growth and biochemical differentiation are host-age dependent.
Data obtained in this study have demonstrated a relationship
between animal age at the time of sympathetic nervous system
ablation and modulation of MNB tumor growth and biochem
ical differentiation. An increase in tumor onset time was ob
Effect of Chemical Sympathectomy on the Catecholamine Con
served after implanting MNB cells into mice that had been
tent of MNB Tumors
sympathectomized as adults. However, growth rate following
tumor onset was similar to that of vehicle-treated animals. The
Catecholamine content was determined in tumors excised on
state of biochemical differentiation, as characterized by tumor
day 8 following tumor onset from mice that had been sympa
thectomized either as neonates or adults. This parameter was Catecholamine content, was not altered by the treatment of
used to assess Catecholamine metabolism in MNB tumors in adult mice with 6HD. In contrast, sympathectomy of neonatal
mice caused an inhibition in MNB tumor growth rate that was
order to ascertain whether alteration in biochemical function
had occurred as a consequence of implanting MNB tumor cells manifest both as an increase in tumor onset time and an
in animals that lacked an intact SNS. NE and/or DA was inhibition in the rate of tumor enlargement following initial
present in all specimens (Fig. 6). Treatment of adult mice with detection. Furthermore, a significant increase in the Catechol
amine content of tumors obtained from neonatally sympathec
6HD did not alter tumor NE or DA concentrations, whereas
tomized mice suggested that biochemical differentiation of the
tumors obtained from mice receiving 6HD as neonates exhib
MNB tumors may have occurred. The apparent age-dependent
ited a marked increase in NE and DA concentrations. In these
differences
in the responses of these two experimental sub
animals, the elevation in NE and DA were 9.09 ±2.8-fold (P
groups may be attributed to the following: (a) 6HD administra
< 0.005) and 7.03 ±1.8-fold (P < 0.0005) above controls.
tion to neonatal animals not only ablates neurons of the sym
Pretreatment of neonatal mice with DMI prior to 6HD pre
pathetic nervous system, but causes depletion of an endogenous
vented the increment in Catecholamine concentrations.
factor(s) required to facilitate MNB tumor growth; and (b) the
inhibition of MNB tumor growth observed in mice sympathec
DISCUSSION
tomized as neonates may occur in association with biochemical
differentiation.
Neuroblastoma is a malignant tumor of neural crest origin
One potential candidate for an endogenous factor involved
that may undergo spontaneous regression (10, 11) and differ
in
the regulation of MNB tumor growth in neonatal and adult
entiation to a benign form in the human (10, 12-17). This
animals is the neurotrophic polypeptide, NGF. NGF exerts
phenomenon appears to be age dependent and suggests that
several critical influences on cells derived from the neural crest.
Developing sympathetic neurons require NGF for survival and
NEONATAL
SYMPATHECTOMY
ADULT SYMPATHECTOMY
differentiation (18, 19). It has beer observed that human neu
roblastoma cells also exhibit a marked dependency on NGF for
500
survival in primary cultures (20). The secretion of a substance
from cultured MNB cells that is immunochemically similar to
50
NGF and exhibits comparable biological activity has been re
ported (21). Furthermore, C-1300 MNB cells are able to spe
400
cifically bind NGF, indicating the presence of NGF membrane
receptors (22). Several cell lines of neuroectodermal origin,
namely, neuroblastoma, adrenal chromaffin, and rat pheochro300
mocytoma have been shown to respond mitogenically to NGF
(23-25). A 2- to 4-fold increase in the NGF levels of tissues
SO
that are target organs for sympathetic nerve innervation and a
corresponding
decrease in sympathetic ganglia have been re
200
ported following chemical sympathectomy of adult mice with
SO
6HD (26).
In view of the above findings we hypothesize that the main
100
tenance of MNB tumor growth observed in chenically sympa
thectomized adult mice may be due to an enhanced availability
50
of NGF. Conversely, the inhibition of tumor growth observed
following implantation of MNB cells into neonatally sympa
VEH
6HD
VEH
6HD
6HD + DMI
thectomized mice could reflect a decrease in NGF. Administra
Fig. 6. Effect of chemical sympathectotny on norepinephrine and dopamine
tion of syngeneic NGF to neonatally sympathetomized mice
concentration of MNB tumors. Animals sympathectomized either as neonates or
adults were inoculated with IO" MNB cells. Tumors were surgically dissected 8
concomitant with implantation of MNB cells and continuation
days following tumor onset. Catecholamine concentrations (ng/g tumor wt):
until termination of the experiment 8 days after tumor onset,
Norepinephrine (D) and dopamine (•).*, P s 0.005 (norepinephrine) and **, P
produced a 75% restoration of tumor growth rate based on
•
0.0005 (dopamine) when neonatal 6HD-treated group was compared to vehicletumor weight and a 100% restoration based on the ratio of
treated group.
5624
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1987 American Association for Cancer Research.
MODULATION
OF NEUROBLASTOMA
tumor to body weight. These results support the hypothesis
that neonatal sympathectomy causes a decrease in NGF and
suggest that circulating titers of NGF may be critical in deter
mining MNB tumor growth rate.
It is possible that neurotrophic factors other than NGF are
involved in sustaining MNB growth in mice sympathectomized
as adults. Treatment of adult mice with 6HD results in a
reversible sympathectomy. Eventually, target organs deprived
of their adrenergic innervation following treatment with 6HD
will be reinnervated by the ingrowth of new sympathetic nerve
endings. Within the time period of the experiments reported
involving tumor growth in sympathectomized adult mice, par
tial recovery of sympathetic innervation in the heart and spleen
was noted (data not shown). Increases in the titers of circulating
trophic factors responsible for supporting the process of adre
nergic reinnervation may also affect the growth rate of im
planted MNB cells.
Another plausible explanation for the age-dependent differ
ences observed in tumor growth may be that MNB tumors
grown in neonatally sympathectomized mice undergo biochem
ical differentiation that is both associated with and responsible
for the reduction in tumor growth rate. The presence of NE
and DA in MNB tumors confirms the predominantly adrenergic
nature of this neoplasm, and is consistent with previous reports
demonstrating the synthesis and release of catecholamines from
MNB tumor (7) as well as the presence of catecholamine
synthesizing and degrading enzymes (27-30). Marked increases
in the activity of catecholamine synthesizing enzymes and an
inhibition of cellular replication have been associated with the
differentiation of cultured neuroblastoma cells (31). The turn
over rate of radiolabeled tyrosine following uptake by MNB
tumors was found to be significantly greater in tumors excised
from neonatally sympathectomized mice when compared to
vehicle-treated controls.6 These preliminary findings suggest
that an increase in the activity of catecholamine synthesizing
enzymes may underlie the elevation in MNB tumor catechol
amine concentrations observed after implantation of MNB cells
in mice sympathectomized as neonates. While the increment in
catecholamine concentration/alone is not sufficient evidence to
conclude that MNB cells have differentiated further, the coin
cidental suppression of tumor growth does support this hypoth
esis. Changes in cellular morphology and tumorigenic potential
must also be demonstrated. MNB tumors obtained from sym
pathectomized adult animals did not exhibit any change in
catecholamine concentrations, and, together with the lack of
effect of sympathectomy on tumor growth rate, suggests that
biochemical differentiation did not occur.
These data have confirmed previous reports demonstrating a
modulating effect of the sympathetic nervous system on MNB
tumor growth (1-6). In addition, we have elucidated a hereto
fore unreported host age-dependent relationship that deter
mines the effects of sympathectomy on MNB tumor growth
and catecholamine concentrations. These age-dependent phe
nomena can be reversed by exogenous NGF and may, therefore,
reflect perturbations in NGF synthesis and/or secretion result
ing from neonatal sympathectomy.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
REFERENCES
30.
1. Chelmicka-Schorr, E., and Arnason, B. G. W. Suppression of growth of
mouse neuroblastoma and AIO adenocarcinoma in newborn mice treated
31.
6 B. L. Mirkin and H. Wu, unpublished data.
GROWTH
BY SNS
with the ganglionic blocking agent chlorisondamine. Eur. J. Cancer, 75:533535, 1979.
Chelmicka-Schorr, E., and Arnason, B. G. W. Modulatory effect of the
sympathetic nervous system on neuroblastoma tumor growth. Cancer Res.,
38: 1374-1375, 1978.
Chelmicka-Schorr, E., and Arnason, B. G. W. Effect of 6-hydroxydopamine
on tumor growth. Cancer Res., 36: 2382-2384, 1976.
Chelmicka-Schorr, E., Yu, R., Sportiello, M., and Arnason, B. G. W. Sym
pathetic ganglia augment growth of neuroblastoma in vitro. Eur. J. Cancer,
27:957-964, 1985.
Chelmicka-Schorr, E., Jones, K., Checinski, M., Yu, R., and Arnason, B. G.
W. Influence of the sympathetic nervous on the growth of neuroblastoma in
vivo and in vitro. Cancer Res., 45:6213-6215, 1985.
Chelmicka-Schoor, E., Checinski, M., Jones, D., Yu, R., and Arnason, B. G.
W. Sympathetic nervous system trophism for neuroblastoma and its age
dependence in rats. Cancer Res., 46: 5504-5506, 1986.
Mirkin, B., Pons, G., and O'Dea, R. Biological characterization of the C1300 murine neuroblastoma: an in vivo neural crest model. Cancer Res., 42:
3719-3723, 1982.
Simpson-Herren, L., and Lloyd, H. Kinetic parameters and growth curves
for experimental tumor systems. Cancer Chemother. Rep., 54: 143-174,
1970.
McCreedie, J., Inch, W., Krouv, J., and Watson, T. The rate of tumor growth
in animals. Growth, 29: 331-347, 1965.
t iritti ».M., and Bolande, R. Familial neuroblastoma with regression and
maturation to ganglioneurofibroma. Pediatrics, 43: 377-382, 1969.
Everson, T. Spontaneous regression of cancer. Ann. NY Acad. Sci., 114:
721-735, 1964.
Cushing, H., and Wolbach, S. The transformation of a malignant paravertebral sympathicoblastoma into a benign ganglioneuroma. Am. J. Pathol., .v
203-206, 1927.
Aterman, K., and Schnellner, E. Maturation of neuroblastoma to gangli
oneuroma. Am. J. Dis. Child., ¡20:217-222, 1970.
Dyke, P., and Mulkey, D. Maturation of ganglioneuroblastoma to gangli
oneuroma. Cancer (Phila.), 20: 1343-1349, 1967.
Bolande, R. Benignity of neonatal tumors and concept of cancer repression
in early life. Am. J. Dis. Child., 122: 12-14, 1971.
Sitara, A., Santulli, T., Wigger, J., and Berndon, W. Complete maturation
of neuroblastoma with bone metastasis in documented stages. J. Pediatr.
Surg., 10: 533-536, 1975.
Goldman, R., Winterling, A., and Winterling, C. Maturation of tumors of
the sympathetic nervous system. Cancer (Phila.), /;.- 1510-1516, 1965.
Harper, G., and Thoenen, H. Target cells, biological effects, and mechanism
of action of nerve growth factor and its antibodies. Annu. Rev. Pharmacol.
Toxicol., 21: 205-229, 1981.
Yanker, B., and Shooter, E. The biology and mechanism of action of nerve
growth factor. Annu. Rev. Biochem., 51: 845-868, 1982.
Tischler, A., Slayton, V., Costopoulos, D., Leape, L., DeLellis, R., and
Wolfe, H. Nerve growth factor may function as a survival factor for human
neuroblastoma cells in culture. Cancer (Phila.), 54: 1344-1347, 1984.
Young, M., Murphy, R., Pantazis, N., and Arnason, B. G. W. Secretion of a
nerve growth factor by mouse neuroblastoma cells in culture. Proc. Nati.
Acad. Sci. USA, 72: 1895-1898, 1975.
Revoltella, R., Bertolini, L., Pediconi, M., and Vigneti, E. Specific binding
of nerve growth factor (NGF) by murine C-1300 neuroblastoma cells. J. Exp.
Med., 140:437-451, 1974.
Lillien, L., and Claude, P. Nerve growth factors is a mitogen for cultured
chromaffin cells. Nature (Lond.), 317:632-634, 1985.
Boonstra, J., Moolenaar, W., Harrison, P., Moed, P., Van DerSaag, P., and
DeLaat, S. Ionic responses and growth stimulation induced by nerve growth
factor and epidermal growth factor in rat pheochromocytoma (PC 12) cells.
J. Cell Biol., 97:92-98, 1983.
Revoltella, R., and Butler, R. Nerve growth factor may stimulate either
division or differentiation of cloned C1300 neuroblastoma cells in serumfree cultures. J. Cell. Physiol., ¡04:27-33, 1980.
Korsching, S., and Thoenen, H. Treatment with 6-hydroxydopamine and
colchicine decreases nerve growth factor levels in sympathetic ganglia and
increases them in the corresponding target tissues. J. Neurosci., 5: 10581061, 1985.
Amano. T., Richelson, E., and Nirenberg, M. Neurotransmitter synthesis by
neuroblastoma clones. Proc. Nati. Acad. Sci. USA, 69: 258-263, 1972.
Anagnoste, B., Freedman, L., Goldstein, M., Broome, J., and Fuxe, K.
Dopamine-/3-hydroxylase activity in mouse neuroblastoma tumors and in cell
cultures. Proc. Nati. Acad. Sci. USA, 69: 1883-1886, 1972.
Richelson, E. Regulation of tyrosine hydroxylase activity in mouse neuro
blastoma clone, NIE-115. J. Neurochem., 21: 1139-1145, 1973.
Skaper, S., Adelson, G., and Seegmiller, J. Metabolism of biogenic amines
in neuroblastoma and glioma cells in culture. J. Neurochem., 27:1065-1070,
1976.
Prasad, K. Differentiation of neuroblastoma cells in culture. Biol. Rev. Camb.
Philos. Soc., 5ft- 129-165, 1975.
5625
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1987 American Association for Cancer Research.
Effects of Chemical Sympathectomy in Neonatal and Adult Mice
on C-1300 Neuroblastoma Tumor Growth and Catecholamine
Content
Donald W. Fink, Jr. and Bernard L. Mirkin
Cancer Res 1987;47:5620-5625.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/47/21/5620
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1987 American Association for Cancer Research.