Journal of Pediatric Surgery VOL 27, NO 8
AUGUST 1992
Autoregulation
of Neuroblastoma Growth by Vasoactive Intestinal Peptide By Jeffrey C. Pence and Nicholas A. Shorter Durham, North Carolina
l Elevated serum levels of vasoactive intestinal peptide (VIP) are associated with some cases of neuroblastoma and correlate with a favorable prognosis. VIP has previously been shown in our laboratory to cause the in vitro growth inhibition and morphological differentiation of the human neuroblastoma cell line, LA-N-5. It is now shown that LA-N-5 cells express immunoreactive VIP and bear specific VIP receptors. Antagonism of endogenous VIP, either by competitive inhibition or receptor blockade, increased cell proliferation, suggesting that VIP is operative in normal growth regulation. Intracellular and extracellular levels of VIP were also shown to increase significantly during the retinoic acid-induced differentiation of these cells. Furthermore, a concomitant marked increase in VIP receptor expression was demonstrated with cellular differentiation. These receptors remain functional as evidenced by a matching increase in the level of detectable CAMP generated in response to exogenous VIP. It is concluded that VIP is a normal autoregulator of neuroblastoma cell growth and differentiation, and that retinoic acidmediated differentiation may be, in part, due to endogenous VIP. Copyright o 7992 by W. 6. Saunders Company INDEX WORDS: Neuroblastoma; tide; retinoic acid.
vasoactive
intestinal
pep-
T
0 DATE, conventional forms of therapy have been relatively ineffective in improving the longterm survival rates for children with neurob1astoma.l This lack of success has prompted interest in other possible approaches to treatment, the most appealing of which is the use of differentiating agents to produce tumor maturation and/or regression. The basis of this approach is the well-known potential of neuroblastoma to show maturation over time in vivo, on occasion to a completely benign form but more commonly in focal areas of the tumor mass, and the rare cases of spontaneous complete tumor regression. Both phenomena suggest that pathogenesis of these tumors involves disruption of the normal embryologic process that results in the transformation of neuroblasts into the mature cells of the adrenal medulla or sympathetic nervous system. The behavior of early stage tumors and stage IVS tumors, and the identificaJournal of Pediatric Surgery, Vol 27, No 8 (August), 1992: pp 935-944
tion of in situ neuroblastoma,* indicates, in fact, that these cases may represent merely a delay in normal maturation rather than a true malignant process. Initial experimental support for this therapeutic approach came from the observation that many neuroblastoma cell lines will differentiate in vitro in response to certain substances. The most commonly used agent has been retinoic acid3a4(RA), but similar effects have been documented with other agents such as forskolin,5 phorbol esters,6,7 dibutyryl cAMP,~ and nerve growth factor.9 Although the original reports described changes in cellular proliferation and morphology alone, these effects have now been correlated with more specific indicators of cellular differentiation such as neurotransmitter synthesis,4,7 increased acetylcholinesterase activity,1° changes in oncogene expression, especially N-myc,” a decrease in levels of the P53 expression, l2 the presence of neurosecretory granules,7 and decreased tumorigenicity in nude mice.4 We have recently demonstrated that vasoactive intestinal peptide (VIP), a 28-amino acid compound originally identified in intestine, but predominantly found in neurons, can produce morphological differentiation of neuroblastoma cells in vitro to a degree comparable to that seen with RA.13 This finding is intriguing because VIP is the first substance shown to cause differentiation that also has a known clinical
From the Section of Pediatric Surgery, Department of Surgery, Duke UniversityMedical Center, Durham, NC. Supported in part by a grant from the Duke Children’s Miracle Network Telethon. First Place Winner in the Jens G. Rose&am Resident Competition. Presented at the 43rd Annual Meeting of the Surgical Section of the American Academy of Pediatrics, New Orleans, Louisiana, October 26-27, 1991. Address reprint requests to Nicholas A. Shorter, MD, Department of Surgery, Dartmouth-Hitchcock Medical Center, I Medical Center Dr, Lebanon, NH 03756. Copyright o 1992 by W B. Saunders Company 0022-3468192/2708-0001$03.OOlO 935
PENCE AND SHORTER
936
association with this tumor. It has been associated histologically with tumor cell differentiation,14 and there is a well-known small subset of neuroblastoma patients who present with high serum levels of this neuropeptide and secondary diarrhea and who have been found to have a favorable prognosis.15 These clinical observations prompted our initial work. Subsequently, we have shown that the differentiating activity requires the intact VIP molecule, is not shared by other members of the secretin-glucagon family of peptides, and is mediated through a specific receptor.16 On this basis, we have postulated that VIP may be responsible for the in vivo maturation that is seen in tumors and may also play an important role in the normal embryologic development of the adrenal medulla and sympathetic nervous system. VIP is produced by some neuroblastoma cells and the SH-SYSY line has previously been shown to bear specific VIP receptors,” suggesting an autocrine mechanism for the function of this neuropeptide. We now present additional evidence in support of an autoregulatory role for VIP. These data also indicate that at least part of the effect of RA may be mediated through endogenous VIP.
using bovine serum albumin as the standard (Bio-Rad, Richmond, CA).
VIP Zmmunofluorescence Exponentially growing cells (104) were plated in 9 cm2 SlideFlasks (Nunc, Kamstrup, Denmark). Growth medium containing 10 umol/L of RA was exchanged at 4 days. Cells were propagated for 14 days in parallel to untreated cultures. Growth medium was then removed, flasks discarded, and monolayers carefully washed with 0.1 mol/L of PBS. Rabbit polyclonal anti-VIP (1:200 in PBS with 0.3% Triton X-100; Peninsula) was applied to RA-treated and control slides and incubated at 4°C for 24 hours. Coincubation of primary antiserum with 10 umol/L of VIP (Peninsula) served as the negative control. Slides were subsequently washed and fluorescein-conjugated goat anti-rabbit IgG (1:lOO in PBS with 0.3% Triton X-100; Peninsula) applied for 45 minutes at 25°C. Slides were washed and coverslipped. Slides were examined and photographed under epifluorescence at 470 to 490 nm using a Nikon Diaphot phase contrast microscope (Nikon Inc, Garden City, NY).
VIP Receptor Expression
The human neuroblastoma cell line LA-N-5 was originally obtained from Dr Robert Seeger, Children’s Hospital of Los Angeles, and maintained in RPM1 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 IU/mL penicillin, and 100 p_g/mL streptomycin (Gibco, Grand Island, NY) at 37°C in a humidified incubator of 5% CO2 in 95% air. Cells were serially monitored for mycoplasma and were found to be free of contamination.
Cells were plated in 150 x 25 mm culture dishes at a density of 10’ cells/dish. Following an initial 4 days of culture, growth medium was exchanged twice weekly with medium containing 10 PmoliL of RA. Cells were mechanically harvested and pellets obtained as above following 0,8, and 16 days of RA exposure. Cells were washed in ice-cold PBS and resuspended in a binding medium of 10 mmol/L Hepes-buffered Hanks balanced salt solution containing 1% BSA, pH 7.4 (HHBSS; Sigma). Aliquots of approximately lo6 cells were incubated in suspension under gentle rotation for 2 hours at 4°C in 0.2 mL of binding medium containing 500 kallikrein inhibitor units/ml aprotinin, 50 pmol/L of [1251]-VIP (2,200 Ciimmol; New England Nuclear, Wilmington, DE), and increasing concentrations of unlabeled VIP. Cells were washed three times with HHBSS, and bound radioactivity within the pellets determined by gamma spectrometry. Specific binding was calculated as the difference between radioligand bound in the absence and presence of 1.0 pmol/L unlabeled VIP. Scatchard transformation’* was performed using the LIGAND software with a one-site model.19 Cell counts and protein determinations were performed as outlined above.
VIP Expression and Secretion
Covalent Cross-linking of [12sI]-VlP to Receptors
The measurement of intracellular VIP expression and its secretion into the growth medium was performed by radioimmunoassay (Peninsula Laboratories, Belmont, CA). Cells were plated onto 150 x 25 mm culture dishes (Becton Dickinson, Cockeysville, MD) in parallel at densities of lO’cells/dish. Upon reaching exponential growth on day 4 of culture, growth medium containing 10 umol/L all-truns RA (Sigma, St Louis, MO) was exchanged and maintained continuously for 8 days. Cells were harvested mechanically at the specified times in ice-cold phosphate-buffered saline (PBS), pH 7.4 containing 500 kallikrein inhibitor units/ml aprotinin (Sigma) and suspensions centrifuged at 2,000 rpm for 5 minutes. Cell pellets were resuspended in 1.0 mL of PBS at 4°C and dounce homogenized. This suspension was clarified by centrifugation as above, and O.l-mL aliquots sampled for VIP immunoreactivity. Concomitantly, aliquots (0.1 mL) of culture medium from the specified times were assayed directly for extracellular VIP. Following 8 continuous days of culture, RA-supplemented growth medium was then exchanged every 4 days. Cells and medium were assayed as described above at the designated times. Cell counts were determined by hemocytometer using erythrosin-b (Sigma) exclusion for viability determinations. Protein measurements were estimated
Cells (10’) were plated and exposed to RA as described above for the specified times. Radioligand binding was performed using 450 pmol/L [iasI]-VIP in 0.2 mL of binding medium in the absence and presence of 10 pmol/L unlabeled VIP. Covalent cross-linking was then performed using 1 mmol/Lof 3,3’-dithio-bis(succinimidy1 propioniate) (DTSP. in DMSO; Sigma) for 15 minutes at 4”C.*O The reaction was quenched by the addition of 60 mmol/L of ammonium acetate, pH 7.5. Cell suspensions were pelleted at 12,000 rpm for 15 minutes washed, and the subsequent pellets resuspended by 5 successive passages through a 27-gauge needle in a sample buffer of 60 mmol/L of Tris-HCl, pH 6.8, containing 10% (v/v) glycerol, 0.001% (w/v) bromophenol blue, and 3% (w/v) sodium dodecyl sulfate (SDS). Suspensions were heated at 60°C for 30 minutes, centrifuged at 12,000 rpm for 15 minutes, and aliquots of 0.05 mL were applied to a 10% SDS-polyacrylamide slab gel under nonreducing conditions. *t Gels were fixed, dried, and exposed for 3 days at -80°C between intensifying screens.
MATERIALS AND METHODS Cell Culture
VIP Receptor Function The assessment of intracellular CAMP accumulation was performed as previously described.” Cells (10’) were initially propa-
VIP AND NEUROBLASTOMA
gated 4 days followed by continuous exposure to 10 umol/L of RA for the indicated times as described above. Following harvest, cells were washed and resuspended in binding medium. Aliquots of 0.1 mL were incubated for 10 minutes in 0.2 mL of binding medium containing 0.5 mmol/L of 3-isobutyl-1-methylxanthine (IBMX; Sigma). Increasing concentrations of VIP were then added to the reaction mixtures and gently rotated for 20 minutes. Cells were then pelleted and washed as described. CAMP was extracted using 0.1 mol/L HCI for 15 minutes at 4°C with vortexing. The supernatants were clarified by centrifugation and assayed for CAMP by radioimmunoassay (Amersham, Arlington Heights, IL).
El m/e
RA+
O/O
RA-
VZPAntagonist Growth Studies Cells ( 104) were seeded in 24-well plates (Becton-Dickinson) in 1.0 mL of growth medium. At 48 hours of culture, fresh growth medium containing either the VIP competitive antagonist [N-AcTyr’, D-Phe’]-GRF-( 1-29)amide (N-GRF; Peninsula),22 or the VIP receptor binding inhibitor L-8-K (Peninsula),23 was exchanged in the absence or presence of 10 umol/L of RA. Growth modulation was assessed by [“HI-thymidine (86.4 Ciimmol; New England Nuclear) incorporation following a 24-hour pulse of 1.0 &i/well at day 7 of culture, as described previously.i3
VIPAntagonist Inhibition of Morphological Di@ererztiation Cells (105) were plated on to 60-mm culture dishes (BectonDickinson) for 48 hours in 5.0 mL of growth medium. Fresh medium was then exchanged containing 10 FmoliL of RA with increasing concentrations of N-GRF or L-8-K. Untreated cultures or cultures with RA alone were performed in parallel as controls. Morphological differentiation was documented by neurite extension following 8 days of culture as previously describedi and recorded using phase contrast photomicroscopy. RESULTS
The ability of LA-N-5 neuroblastoma cells to both synthesize and secrete immunoreactive VIP in response to RA-induced differentiation was determined by radioimmunoassay. Concomitant intracellular and extracellular VIP concentrations of approximately 430 pg of VIP per milligram of protein (22 pg VIP/million cells) and 10.4 pmol/L of VIP per milligram of protein (0.5 pmol/L VIP/million cells) were initially measured for exponentially growing, nontreated LA-N-5 cells (Figs 1 and 2). No immunoreactive VIP was detected in the growth medium prior to cell conditioning. RA treatment markedly increased both the intracellular and extracellular levels of VIP. Intracellular concentrations increased progressively throughout RA-mediated differentiation, achieving a 70-fold increase from nontreated cellular levels to approximately 30,000 pg of VIP per milligram of protein (1,143 pg VIP/million cells). Secreted VIP concentrations reached a maximum of 102 pmol/L of VIP per milligram of protein (3.6 pmol/L VIP/million cells throughout RA exposure, achieving a lo-fold increase above nontreated control levels over the same incubation period. These data clearly illustrate a time-dependent direct correlation
I
I
!
I
0
2
4
6 culture
0
14
28
days
Fig 1. Intracellular concentrations of immunoreactive VIP during the growth of LA-N-5 cells in the presence or absence of 10 Pmol/L PA. Mean values are shown from two independent experiments standardized to picograms per milligram protein (solid lines) and picograms per million cells (dashed lines).
between RA-induced cellular differentiation and an increased cellular expression and secretion of immunoreactive VIP. Elevated concentrations of both intracellular and extracellular VIP noted with RA differentiation most likely reflect increased neuropeptide synthesis coupled with increased secretion; however, the possibility that these changes are due, in 120 -
O
loo-
20 -
o-
,
I
I
I
0
2
4
6
culture
a
14
28
days
Fig 2. Extracellular concentrations of immunoreactive VIP during the growth of LA-N-5 cells in the presence or absence of 10 Pmol/L RA. Mean values are shown from two independent experiments standardized to picomoles per milligram protein (solid lines) and picomoles per million cells (dashed lines).
PENCE AND SHORTER
Fig 3. Expression of VIP detected by immunofluorescence. (A) Phase contrast photomicrograph of untreated, exponentially-growing LA-N-5 cells. (B) Identical field as in (A) demonstrating VIP immunoreactivity. (C) Phase contrast photomicrograph of LA-N-5 cells exposed to 10 pmol/L of RA for 14 days demonstrating morphologicaldffferentiation to the neuronal phenotype. (D) Identical field as in (C) showing abundant expression of intracellular VIP extending from soma throughout interlacing neuritic extensions. (Original magnification x200.) Negative controls exhibited no detectable immunofluorescence.
part, to a prolonged half-life cannot be entirely excluded. Differential intracellular VIP expression with RA treatment was confirmed qualitatively by the immunofluorescent staining of both RA-treated and nontreated LA-N-S cells (Fig 3). Undifferentiated cells of the neuroblastic phenotype exhibited minimal, but detectable, immunofluorescence. In contrast, the development of the neuronal phenotype with RAmediated differentiation was associated with enhanced immunofluorescence. Significant immunoreactive VIP was readily observed within pseudoganglia and interconnecting neuritic extensions, indicative of intercellular communication and/or transport of this neuropeptide. VIP receptor expression and its modulation with RA-induced differentiation of LA-N-5 cells was investigated by the ability of both nontreated and RAtreated intact cells to bind monoiodinated VIP. The amount of radiolabeled VIP specifically bound at equilibrium increased 6.5-fold and 8.5-fold above nontreated cellular binding following 8 and 16 days of RA-induced differentiation, respectively (Fig 4). The competitive displacement curves for both nontreated and RA-treated cells exhibited displaceable binding of radioligand with increasing concentrations of unlabeled peptide. Scatchard transformation of these data showed a progressive lo-fold increase in VIP binding capacity by day 16 of RA-induced differentiation (Table 1). Binding kinetics demonstrated a minor decrease in receptor affinity from approximately 7 to 12 nmol/L with cellular differentiation.
These results demonstrate markedly enhanced VIP receptor expression of similarly retained affinity concomitant with RA-mediated differentiation of LAN-5 cells. Molecular characterization of the VIP binding site
5000
1000
0 0
11
10
9
8
7
6
-log IV/PI Fig 4. Competitive displacement of monoiodinated VIP bv increasing concentrations of unlabeled VIP in untreated LA-N-5 cells, and in cells exposed to 10 pmol/ L of RA for 6 and 16 days. Bound radioactivitieo are standardized per lo8 cells. Data are mean values f SEM for three independent experiments.
VIP AND NEUROBLASTOMA
939
Table 1. VIP Receptor Expression and Kinetics in Response to the WI-Induced Differentiation RA
Exposure Id) 0
of Human Neuroblastoma
Kd
hm.
(nmol/L)
7.3 c 0.2
(sites/cell)
18,557 * 413
LA-N-5 bn.x
(fmol/mg
protein)
173 + 4
6
6.4 2 2.2
91,625 2 38375
672 -t 365
16
11.6 k 4.8
202,941 2 72353
1,643 + 566
NOTE. Data given as mean ? SEM.
prior to and following RA-induced differentiation was performed by covalent cross-linking of radiolabeled VIP to its receptor (Fig 5). SDS-polyacrylamide gel electrophoresis and subsequent autoradiography demonstrated a single moiety of approximately 47 kd
1234
97 69
for both nontreated and RA-treated LA-N-5 cells. Specific binding was confirmed by displacement of radioligand binding by unlabeled VIP. Markedly enhanced expression of this component with differentiation is readily visualized. A corrected molecular size of approximately 43-44 kd was observed for this putative VIP receptor, which is similar in molecular size to the VIP binding site of neuroblastoma cell line, SH-EP(F), a subclone of SK-N-SH,24 but smaller than the 66-kd receptor identified on human colon adenocarcinoma cell membranes.*0 The difference in molecular size between these diverse cell types suggests the existence of tissue-specific populations of VIP receptors. The functional assessment of enhanced VIP receptor expression accompanying RA-induced differentiation was measured by intracellular CAMP accumulation in response to exogenous VIP stimulation (Fig 6). RA treatment was found to yield a 7-fold (9.8 pmol cAMP/mg protein) and 57-fold (79 pmol cAMP/mg protein) increase in unstimulated CAMP levels at 8 and 16 days of exposure, respectively, when compared with nontreated control cells (1.4 pmol cAMP/mg protein). Furthermore, increasing elevations in cellular CAMP accumulation were observed at increasing concentrations of exogenous VIP, yielding similar estimated half-maximal effective concentrations (EC& of 52, 86, and 220 nmol/L for non-
0 untreated
L-_--l A day n
6
day16
46 I
basal Fig 5. Molecular characterization of monoiodinated VIP covalently cross-linked to VIP receptors on LA-N-5 cells. (Lane 1) Untreated LA-N-5 cells colncubated in the presence of excess unlabeled VIP. (Lane 2) Untreated LA-N-5 cells incubated with radioligand alone. (Lane 3) LA-N-5 cells exposed to 16 days of 10 pmol/L of RA and coincubeted in the presence of excess unlabeled VIP. (Lane 4) Treated cells incubated with radioligand alone. Molecular weight markers are shown and expressed in kilodaltons.
11109
8 -log
[WI
7
6
5
forskolin
added
Fig 6. Generation of intracellular CAMP in response to exogenous VIP in untreated LA-N-5 cells or cells exposed to 10 pmol/L of RA for the specified times. Basal levels reflect intracellular cAMP in the absence of exogenous VIP stimulation. cAMP response was evaluated in response to 100 umol/L of forskolin in the absence of VIP and was found to increase proportionally to maximal VIP stimulation.
PENCE AND SHORTER
940
treated, 8-day, and 16-day RA-treated cells, respectively. CAMP accumulation at maximal VIP stimulation (10 umol/L) was measured at 86, 329, and 1,338 pmol cAMP/mg protein for nontreated, 8-day, and 16-day RA-treated cells, reflecting similar proportional increases in CAMP generation and VIP receptor expression with differentiation. A similar increase in receptor number and CAMP accumulation has been reported for the SH-SYSY neuroblastoma cell line.17 Finally, forskolin (100 lJ,mol/L) was used to achieve maximal CAMP accumulation for each differentiation state. Forskolin induction of near-equal levels of intracellular CAMP in comparison to maximal VIP stimulation suggests the functional coupling of differentially-expressed VIP receptors to the adenyl cyclase system. These data demonstrate retained functional coupling of enhanced VIP receptors to the stimulation of elevated intracellular CAMP levels. The role of the VIP system in normal undifferentiated LA-N-5 cell growth was examined by incubation of cells in the presence of either [N-AC-Tyrl, D-Phe*]growth hormone-releasing factor-(1-29)-amide, N-GRF, an inactive competitive antagonist of VIP, or L-8-K, a VIP receptor binding inhibitor. Cell proliferation increased to a maximum of 139% and 160% of nontreated control cultures at 1.0 umol/L and 10 pmol/L concentrations of N-GRF and L-8-K, respectively, as measured by [3H]-thymidine uptake (Fig 7). This supports the hypothesis that VIP normally functions as an autoregulator of neuroblastoma cell growth. Changes in VIP levels and VIP receptor expression seen during RA-induced differentiation suggest that a portion of the RA effect may, in actuality, be mediated through VIP. The elevation of CAMP levels seen with RA exposure in the absence of exogenous VIP supports this premise, as RA reportedly does not alter these levels directly. To test this hypothesis further, LA-N-5 cells were exposed to RA for 8 days in the presence of either L-8-K or N-GRF. Increasing concentrations of either antagonist were found to partially block the effects of RA on cellular proliferation (Fig 8). Cell growth achieved 64% and 57% of nontreated control cultures for cells exposed to 10 pmol/L of L-8-K and N-GRF, respectively. RA alone inhibited cell growth to 29% to 34% of nontreated values. Neurite outgrowth in response to RA was also partially blocked in the presence of either antagonist (Fig 9). Quantitative assessment of RA-induced differentiation, as measured by neurite outgrowth, demonstrated 66% to 68% phenotypically differentiated forms following 8 days of RA exposure (Fig 10). Coincubation with either L-8-K or N-GRF resulted in
1.7 1.6 1.5 .s 2 1.43 : % 1.30, 5 2 1.2z v) 1.1 -
0.9 -
9
6
7
6
5
4
-log ~antagonisrl Fig 7. Enhanced proliferation of LA-N-5 cells in response to VIP receptor antagonism as measured by [JH]-thymidine incorporation following 8 days of culture. Data are mean values f SEM of triplicate cultures. Stimulated/basal ratios are derived from the comparison of treated to untreated (control) cell growth. The concentration of antagonist at which statistically significant (P c .05, ANOVA) cell growth is reached above untreated control cultures is shown by asterisks (*, L-E-K; **, N-GRF).
35% and 45% differentiated forms, respectively, at maximal concentrations. Importantly, the antagonist N-GRF has been shown previously to be ineffective in altering intracellular CAMP levels in rat pancreatic cells and, therefore, could not directly account for the alterations in cellular proliferation or morphology by this mechanism. The antagonism of RA-induced growth inhibition and morphological differentiation of LA-N-5 cells by specific VIP antagonists as shown suggests an operative role for VIP in the regulation of neuroblastoma cell growth distinct and separable from those effects induced directly by RA. DISCUSSION
Despite its name, VIP has a very close association with neural tissue, functioning as a neurotransmitter in some neurons. Recent findings indicate that neurotransmitter molecules, in general, play an important role in neural development, and in the regulation of neuronal architecture.z,26 That VIP can induce the differentiation of neuroblastoma cells and has also been shown to have a similar effect on fetal neuroblastsz7 is consistent with this general principle.
941
VIP AND NEUAOBLASTOMA
80 -
z $
0 0
60
-
50
-
40
-
.:
30-
d 20
-
10
-
50-
s k
E 5
60 C .s
I
g
P
z 2 : Q
Q
403020 -
P
10 -
O-
O-
I,
RA
10
9
8 -log
7
6
5
[antagonist1
+lO,vM
/?A
Fig 8. Partial antagonism of RA-induced growth inhibition of LA-N-5 cells as measured by [3H]-thymidine incorporation following 8 days of treatment. Data are mean values f SEM of triplicate cultures. RA represents cells grown in the presence of 10 pmol/L of RA without antagonist. Percent control is derived from the comparison of treated to untreated (control) cell growth. The concentration of antagonist at which statistically significant (P c .05, ANOVA) cell growth is reached above RA-treated control cultures is shown by asterisks (*, L-8-K; **, N-GRF).
Fig 9. Partial antagonism of the F&induced morphological differentiation of LA-N-5 cells by VIP antagonists following 8 days of culture as demonstrated by phase contrast photomicrography. (A) Morphology of untreated control cultures. (B) Cells coincubated with 10 pmol/L of RA and 10 pmol/L of L-8-K. (C) Cells coincubated with IO pmol/L of RA and 10 &mot/L of N-GRF. (D) Morphology of 10 pmol/L FiAtreated cells. (Original magnification x200.)
I
RA
”
I
,
,
,
/
I
10
9
8
7
6
5
-log
iantagonis +70/M
.:
I
Control
tl
RA
Fig 10. Partial antagonism of the RA-induced morphological differentiation of LA-N-5 cells by VIP antagonists as assessed by neurite extension following 8 days of culture. F4Aand Control points indicate the percentage of cells exhibiting the differentiated neuronal phenotype in the presence or absence of 10 pmol/L of RA. Data are the means f SEM of triplicate experiments. The concentration of antagonist at which statistical signfficance (P < 95, ANOVA) is reached is shown by asterisks (*, L-8-K; **, N-GRF).
942
VIP has all the necessary characteristics of an autocrine factor. It is produced by neuroblastoma cells, which themselves bear specific receptors for this neuropeptide. In this manner it appears to exert control over the rate of cell proliferation and the state of cellular differentiation. The onset of differentiation results in increased levels of VIP and increased expression of its receptor, which serve to amplify the initial effect. One criticism that can be made of this last point is that RA-induced differentiation and VIP-induced differentiation may not be identical processes, and therefore, findings from one may not be applicable to the other. This is certainly a valid concern, and we have not yet shown that endogenous VIP levels and VIP receptor expression increase during VIP-induced differentiation in the absence of RA. Evidence indicates that all differentiating agents must act through a final common pathway, but certainly some cellular effects have been observed which are not universal.5 ObviousIy further work wiIl be necessary before this question can be fully answered. However, given the synergy that has previously been demonstrated between these two agents,16 it seems likely that the majority of the effects of the two are, indeed, identical. All the evidence points to a fundamental role for VIP in the in vivo maturation of neuroblastoma cells. In support of this is the finding that local tumor concentrations of VIP can reach the levels needed to induce differentiation,28 even though these are much higher than normal serum levels (
AUGUST 1992
Autoregulation
of Neuroblastoma Growth by Vasoactive Intestinal Peptide By Jeffrey C. Pence and Nicholas A. Shorter Durham, North Carolina
l Elevated serum levels of vasoactive intestinal peptide (VIP) are associated with some cases of neuroblastoma and correlate with a favorable prognosis. VIP has previously been shown in our laboratory to cause the in vitro growth inhibition and morphological differentiation of the human neuroblastoma cell line, LA-N-5. It is now shown that LA-N-5 cells express immunoreactive VIP and bear specific VIP receptors. Antagonism of endogenous VIP, either by competitive inhibition or receptor blockade, increased cell proliferation, suggesting that VIP is operative in normal growth regulation. Intracellular and extracellular levels of VIP were also shown to increase significantly during the retinoic acid-induced differentiation of these cells. Furthermore, a concomitant marked increase in VIP receptor expression was demonstrated with cellular differentiation. These receptors remain functional as evidenced by a matching increase in the level of detectable CAMP generated in response to exogenous VIP. It is concluded that VIP is a normal autoregulator of neuroblastoma cell growth and differentiation, and that retinoic acidmediated differentiation may be, in part, due to endogenous VIP. Copyright o 7992 by W. 6. Saunders Company INDEX WORDS: Neuroblastoma; tide; retinoic acid.
vasoactive
intestinal
pep-
T
0 DATE, conventional forms of therapy have been relatively ineffective in improving the longterm survival rates for children with neurob1astoma.l This lack of success has prompted interest in other possible approaches to treatment, the most appealing of which is the use of differentiating agents to produce tumor maturation and/or regression. The basis of this approach is the well-known potential of neuroblastoma to show maturation over time in vivo, on occasion to a completely benign form but more commonly in focal areas of the tumor mass, and the rare cases of spontaneous complete tumor regression. Both phenomena suggest that pathogenesis of these tumors involves disruption of the normal embryologic process that results in the transformation of neuroblasts into the mature cells of the adrenal medulla or sympathetic nervous system. The behavior of early stage tumors and stage IVS tumors, and the identificaJournal of Pediatric Surgery, Vol 27, No 8 (August), 1992: pp 935-944
tion of in situ neuroblastoma,* indicates, in fact, that these cases may represent merely a delay in normal maturation rather than a true malignant process. Initial experimental support for this therapeutic approach came from the observation that many neuroblastoma cell lines will differentiate in vitro in response to certain substances. The most commonly used agent has been retinoic acid3a4(RA), but similar effects have been documented with other agents such as forskolin,5 phorbol esters,6,7 dibutyryl cAMP,~ and nerve growth factor.9 Although the original reports described changes in cellular proliferation and morphology alone, these effects have now been correlated with more specific indicators of cellular differentiation such as neurotransmitter synthesis,4,7 increased acetylcholinesterase activity,1° changes in oncogene expression, especially N-myc,” a decrease in levels of the P53 expression, l2 the presence of neurosecretory granules,7 and decreased tumorigenicity in nude mice.4 We have recently demonstrated that vasoactive intestinal peptide (VIP), a 28-amino acid compound originally identified in intestine, but predominantly found in neurons, can produce morphological differentiation of neuroblastoma cells in vitro to a degree comparable to that seen with RA.13 This finding is intriguing because VIP is the first substance shown to cause differentiation that also has a known clinical
From the Section of Pediatric Surgery, Department of Surgery, Duke UniversityMedical Center, Durham, NC. Supported in part by a grant from the Duke Children’s Miracle Network Telethon. First Place Winner in the Jens G. Rose&am Resident Competition. Presented at the 43rd Annual Meeting of the Surgical Section of the American Academy of Pediatrics, New Orleans, Louisiana, October 26-27, 1991. Address reprint requests to Nicholas A. Shorter, MD, Department of Surgery, Dartmouth-Hitchcock Medical Center, I Medical Center Dr, Lebanon, NH 03756. Copyright o 1992 by W B. Saunders Company 0022-3468192/2708-0001$03.OOlO 935
PENCE AND SHORTER
936
association with this tumor. It has been associated histologically with tumor cell differentiation,14 and there is a well-known small subset of neuroblastoma patients who present with high serum levels of this neuropeptide and secondary diarrhea and who have been found to have a favorable prognosis.15 These clinical observations prompted our initial work. Subsequently, we have shown that the differentiating activity requires the intact VIP molecule, is not shared by other members of the secretin-glucagon family of peptides, and is mediated through a specific receptor.16 On this basis, we have postulated that VIP may be responsible for the in vivo maturation that is seen in tumors and may also play an important role in the normal embryologic development of the adrenal medulla and sympathetic nervous system. VIP is produced by some neuroblastoma cells and the SH-SYSY line has previously been shown to bear specific VIP receptors,” suggesting an autocrine mechanism for the function of this neuropeptide. We now present additional evidence in support of an autoregulatory role for VIP. These data also indicate that at least part of the effect of RA may be mediated through endogenous VIP.
using bovine serum albumin as the standard (Bio-Rad, Richmond, CA).
VIP Zmmunofluorescence Exponentially growing cells (104) were plated in 9 cm2 SlideFlasks (Nunc, Kamstrup, Denmark). Growth medium containing 10 umol/L of RA was exchanged at 4 days. Cells were propagated for 14 days in parallel to untreated cultures. Growth medium was then removed, flasks discarded, and monolayers carefully washed with 0.1 mol/L of PBS. Rabbit polyclonal anti-VIP (1:200 in PBS with 0.3% Triton X-100; Peninsula) was applied to RA-treated and control slides and incubated at 4°C for 24 hours. Coincubation of primary antiserum with 10 umol/L of VIP (Peninsula) served as the negative control. Slides were subsequently washed and fluorescein-conjugated goat anti-rabbit IgG (1:lOO in PBS with 0.3% Triton X-100; Peninsula) applied for 45 minutes at 25°C. Slides were washed and coverslipped. Slides were examined and photographed under epifluorescence at 470 to 490 nm using a Nikon Diaphot phase contrast microscope (Nikon Inc, Garden City, NY).
VIP Receptor Expression
The human neuroblastoma cell line LA-N-5 was originally obtained from Dr Robert Seeger, Children’s Hospital of Los Angeles, and maintained in RPM1 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 IU/mL penicillin, and 100 p_g/mL streptomycin (Gibco, Grand Island, NY) at 37°C in a humidified incubator of 5% CO2 in 95% air. Cells were serially monitored for mycoplasma and were found to be free of contamination.
Cells were plated in 150 x 25 mm culture dishes at a density of 10’ cells/dish. Following an initial 4 days of culture, growth medium was exchanged twice weekly with medium containing 10 PmoliL of RA. Cells were mechanically harvested and pellets obtained as above following 0,8, and 16 days of RA exposure. Cells were washed in ice-cold PBS and resuspended in a binding medium of 10 mmol/L Hepes-buffered Hanks balanced salt solution containing 1% BSA, pH 7.4 (HHBSS; Sigma). Aliquots of approximately lo6 cells were incubated in suspension under gentle rotation for 2 hours at 4°C in 0.2 mL of binding medium containing 500 kallikrein inhibitor units/ml aprotinin, 50 pmol/L of [1251]-VIP (2,200 Ciimmol; New England Nuclear, Wilmington, DE), and increasing concentrations of unlabeled VIP. Cells were washed three times with HHBSS, and bound radioactivity within the pellets determined by gamma spectrometry. Specific binding was calculated as the difference between radioligand bound in the absence and presence of 1.0 pmol/L unlabeled VIP. Scatchard transformation’* was performed using the LIGAND software with a one-site model.19 Cell counts and protein determinations were performed as outlined above.
VIP Expression and Secretion
Covalent Cross-linking of [12sI]-VlP to Receptors
The measurement of intracellular VIP expression and its secretion into the growth medium was performed by radioimmunoassay (Peninsula Laboratories, Belmont, CA). Cells were plated onto 150 x 25 mm culture dishes (Becton Dickinson, Cockeysville, MD) in parallel at densities of lO’cells/dish. Upon reaching exponential growth on day 4 of culture, growth medium containing 10 umol/L all-truns RA (Sigma, St Louis, MO) was exchanged and maintained continuously for 8 days. Cells were harvested mechanically at the specified times in ice-cold phosphate-buffered saline (PBS), pH 7.4 containing 500 kallikrein inhibitor units/ml aprotinin (Sigma) and suspensions centrifuged at 2,000 rpm for 5 minutes. Cell pellets were resuspended in 1.0 mL of PBS at 4°C and dounce homogenized. This suspension was clarified by centrifugation as above, and O.l-mL aliquots sampled for VIP immunoreactivity. Concomitantly, aliquots (0.1 mL) of culture medium from the specified times were assayed directly for extracellular VIP. Following 8 continuous days of culture, RA-supplemented growth medium was then exchanged every 4 days. Cells and medium were assayed as described above at the designated times. Cell counts were determined by hemocytometer using erythrosin-b (Sigma) exclusion for viability determinations. Protein measurements were estimated
Cells (10’) were plated and exposed to RA as described above for the specified times. Radioligand binding was performed using 450 pmol/L [iasI]-VIP in 0.2 mL of binding medium in the absence and presence of 10 pmol/L unlabeled VIP. Covalent cross-linking was then performed using 1 mmol/Lof 3,3’-dithio-bis(succinimidy1 propioniate) (DTSP. in DMSO; Sigma) for 15 minutes at 4”C.*O The reaction was quenched by the addition of 60 mmol/L of ammonium acetate, pH 7.5. Cell suspensions were pelleted at 12,000 rpm for 15 minutes washed, and the subsequent pellets resuspended by 5 successive passages through a 27-gauge needle in a sample buffer of 60 mmol/L of Tris-HCl, pH 6.8, containing 10% (v/v) glycerol, 0.001% (w/v) bromophenol blue, and 3% (w/v) sodium dodecyl sulfate (SDS). Suspensions were heated at 60°C for 30 minutes, centrifuged at 12,000 rpm for 15 minutes, and aliquots of 0.05 mL were applied to a 10% SDS-polyacrylamide slab gel under nonreducing conditions. *t Gels were fixed, dried, and exposed for 3 days at -80°C between intensifying screens.
MATERIALS AND METHODS Cell Culture
VIP Receptor Function The assessment of intracellular CAMP accumulation was performed as previously described.” Cells (10’) were initially propa-
VIP AND NEUROBLASTOMA
gated 4 days followed by continuous exposure to 10 umol/L of RA for the indicated times as described above. Following harvest, cells were washed and resuspended in binding medium. Aliquots of 0.1 mL were incubated for 10 minutes in 0.2 mL of binding medium containing 0.5 mmol/L of 3-isobutyl-1-methylxanthine (IBMX; Sigma). Increasing concentrations of VIP were then added to the reaction mixtures and gently rotated for 20 minutes. Cells were then pelleted and washed as described. CAMP was extracted using 0.1 mol/L HCI for 15 minutes at 4°C with vortexing. The supernatants were clarified by centrifugation and assayed for CAMP by radioimmunoassay (Amersham, Arlington Heights, IL).
El m/e
RA+
O/O
RA-
VZPAntagonist Growth Studies Cells ( 104) were seeded in 24-well plates (Becton-Dickinson) in 1.0 mL of growth medium. At 48 hours of culture, fresh growth medium containing either the VIP competitive antagonist [N-AcTyr’, D-Phe’]-GRF-( 1-29)amide (N-GRF; Peninsula),22 or the VIP receptor binding inhibitor L-8-K (Peninsula),23 was exchanged in the absence or presence of 10 umol/L of RA. Growth modulation was assessed by [“HI-thymidine (86.4 Ciimmol; New England Nuclear) incorporation following a 24-hour pulse of 1.0 &i/well at day 7 of culture, as described previously.i3
VIPAntagonist Inhibition of Morphological Di@ererztiation Cells (105) were plated on to 60-mm culture dishes (BectonDickinson) for 48 hours in 5.0 mL of growth medium. Fresh medium was then exchanged containing 10 FmoliL of RA with increasing concentrations of N-GRF or L-8-K. Untreated cultures or cultures with RA alone were performed in parallel as controls. Morphological differentiation was documented by neurite extension following 8 days of culture as previously describedi and recorded using phase contrast photomicroscopy. RESULTS
The ability of LA-N-5 neuroblastoma cells to both synthesize and secrete immunoreactive VIP in response to RA-induced differentiation was determined by radioimmunoassay. Concomitant intracellular and extracellular VIP concentrations of approximately 430 pg of VIP per milligram of protein (22 pg VIP/million cells) and 10.4 pmol/L of VIP per milligram of protein (0.5 pmol/L VIP/million cells) were initially measured for exponentially growing, nontreated LA-N-5 cells (Figs 1 and 2). No immunoreactive VIP was detected in the growth medium prior to cell conditioning. RA treatment markedly increased both the intracellular and extracellular levels of VIP. Intracellular concentrations increased progressively throughout RA-mediated differentiation, achieving a 70-fold increase from nontreated cellular levels to approximately 30,000 pg of VIP per milligram of protein (1,143 pg VIP/million cells). Secreted VIP concentrations reached a maximum of 102 pmol/L of VIP per milligram of protein (3.6 pmol/L VIP/million cells throughout RA exposure, achieving a lo-fold increase above nontreated control levels over the same incubation period. These data clearly illustrate a time-dependent direct correlation
I
I
!
I
0
2
4
6 culture
0
14
28
days
Fig 1. Intracellular concentrations of immunoreactive VIP during the growth of LA-N-5 cells in the presence or absence of 10 Pmol/L PA. Mean values are shown from two independent experiments standardized to picograms per milligram protein (solid lines) and picograms per million cells (dashed lines).
between RA-induced cellular differentiation and an increased cellular expression and secretion of immunoreactive VIP. Elevated concentrations of both intracellular and extracellular VIP noted with RA differentiation most likely reflect increased neuropeptide synthesis coupled with increased secretion; however, the possibility that these changes are due, in 120 -
O
loo-
20 -
o-
,
I
I
I
0
2
4
6
culture
a
14
28
days
Fig 2. Extracellular concentrations of immunoreactive VIP during the growth of LA-N-5 cells in the presence or absence of 10 Pmol/L RA. Mean values are shown from two independent experiments standardized to picomoles per milligram protein (solid lines) and picomoles per million cells (dashed lines).
PENCE AND SHORTER
Fig 3. Expression of VIP detected by immunofluorescence. (A) Phase contrast photomicrograph of untreated, exponentially-growing LA-N-5 cells. (B) Identical field as in (A) demonstrating VIP immunoreactivity. (C) Phase contrast photomicrograph of LA-N-5 cells exposed to 10 pmol/L of RA for 14 days demonstrating morphologicaldffferentiation to the neuronal phenotype. (D) Identical field as in (C) showing abundant expression of intracellular VIP extending from soma throughout interlacing neuritic extensions. (Original magnification x200.) Negative controls exhibited no detectable immunofluorescence.
part, to a prolonged half-life cannot be entirely excluded. Differential intracellular VIP expression with RA treatment was confirmed qualitatively by the immunofluorescent staining of both RA-treated and nontreated LA-N-S cells (Fig 3). Undifferentiated cells of the neuroblastic phenotype exhibited minimal, but detectable, immunofluorescence. In contrast, the development of the neuronal phenotype with RAmediated differentiation was associated with enhanced immunofluorescence. Significant immunoreactive VIP was readily observed within pseudoganglia and interconnecting neuritic extensions, indicative of intercellular communication and/or transport of this neuropeptide. VIP receptor expression and its modulation with RA-induced differentiation of LA-N-5 cells was investigated by the ability of both nontreated and RAtreated intact cells to bind monoiodinated VIP. The amount of radiolabeled VIP specifically bound at equilibrium increased 6.5-fold and 8.5-fold above nontreated cellular binding following 8 and 16 days of RA-induced differentiation, respectively (Fig 4). The competitive displacement curves for both nontreated and RA-treated cells exhibited displaceable binding of radioligand with increasing concentrations of unlabeled peptide. Scatchard transformation of these data showed a progressive lo-fold increase in VIP binding capacity by day 16 of RA-induced differentiation (Table 1). Binding kinetics demonstrated a minor decrease in receptor affinity from approximately 7 to 12 nmol/L with cellular differentiation.
These results demonstrate markedly enhanced VIP receptor expression of similarly retained affinity concomitant with RA-mediated differentiation of LAN-5 cells. Molecular characterization of the VIP binding site
5000
1000
0 0
11
10
9
8
7
6
-log IV/PI Fig 4. Competitive displacement of monoiodinated VIP bv increasing concentrations of unlabeled VIP in untreated LA-N-5 cells, and in cells exposed to 10 pmol/ L of RA for 6 and 16 days. Bound radioactivitieo are standardized per lo8 cells. Data are mean values f SEM for three independent experiments.
VIP AND NEUROBLASTOMA
939
Table 1. VIP Receptor Expression and Kinetics in Response to the WI-Induced Differentiation RA
Exposure Id) 0
of Human Neuroblastoma
Kd
hm.
(nmol/L)
7.3 c 0.2
(sites/cell)
18,557 * 413
LA-N-5 bn.x
(fmol/mg
protein)
173 + 4
6
6.4 2 2.2
91,625 2 38375
672 -t 365
16
11.6 k 4.8
202,941 2 72353
1,643 + 566
NOTE. Data given as mean ? SEM.
prior to and following RA-induced differentiation was performed by covalent cross-linking of radiolabeled VIP to its receptor (Fig 5). SDS-polyacrylamide gel electrophoresis and subsequent autoradiography demonstrated a single moiety of approximately 47 kd
1234
97 69
for both nontreated and RA-treated LA-N-5 cells. Specific binding was confirmed by displacement of radioligand binding by unlabeled VIP. Markedly enhanced expression of this component with differentiation is readily visualized. A corrected molecular size of approximately 43-44 kd was observed for this putative VIP receptor, which is similar in molecular size to the VIP binding site of neuroblastoma cell line, SH-EP(F), a subclone of SK-N-SH,24 but smaller than the 66-kd receptor identified on human colon adenocarcinoma cell membranes.*0 The difference in molecular size between these diverse cell types suggests the existence of tissue-specific populations of VIP receptors. The functional assessment of enhanced VIP receptor expression accompanying RA-induced differentiation was measured by intracellular CAMP accumulation in response to exogenous VIP stimulation (Fig 6). RA treatment was found to yield a 7-fold (9.8 pmol cAMP/mg protein) and 57-fold (79 pmol cAMP/mg protein) increase in unstimulated CAMP levels at 8 and 16 days of exposure, respectively, when compared with nontreated control cells (1.4 pmol cAMP/mg protein). Furthermore, increasing elevations in cellular CAMP accumulation were observed at increasing concentrations of exogenous VIP, yielding similar estimated half-maximal effective concentrations (EC& of 52, 86, and 220 nmol/L for non-
0 untreated
L-_--l A day n
6
day16
46 I
basal Fig 5. Molecular characterization of monoiodinated VIP covalently cross-linked to VIP receptors on LA-N-5 cells. (Lane 1) Untreated LA-N-5 cells colncubated in the presence of excess unlabeled VIP. (Lane 2) Untreated LA-N-5 cells incubated with radioligand alone. (Lane 3) LA-N-5 cells exposed to 16 days of 10 pmol/L of RA and coincubeted in the presence of excess unlabeled VIP. (Lane 4) Treated cells incubated with radioligand alone. Molecular weight markers are shown and expressed in kilodaltons.
11109
8 -log
[WI
7
6
5
forskolin
added
Fig 6. Generation of intracellular CAMP in response to exogenous VIP in untreated LA-N-5 cells or cells exposed to 10 pmol/L of RA for the specified times. Basal levels reflect intracellular cAMP in the absence of exogenous VIP stimulation. cAMP response was evaluated in response to 100 umol/L of forskolin in the absence of VIP and was found to increase proportionally to maximal VIP stimulation.
PENCE AND SHORTER
940
treated, 8-day, and 16-day RA-treated cells, respectively. CAMP accumulation at maximal VIP stimulation (10 umol/L) was measured at 86, 329, and 1,338 pmol cAMP/mg protein for nontreated, 8-day, and 16-day RA-treated cells, reflecting similar proportional increases in CAMP generation and VIP receptor expression with differentiation. A similar increase in receptor number and CAMP accumulation has been reported for the SH-SYSY neuroblastoma cell line.17 Finally, forskolin (100 lJ,mol/L) was used to achieve maximal CAMP accumulation for each differentiation state. Forskolin induction of near-equal levels of intracellular CAMP in comparison to maximal VIP stimulation suggests the functional coupling of differentially-expressed VIP receptors to the adenyl cyclase system. These data demonstrate retained functional coupling of enhanced VIP receptors to the stimulation of elevated intracellular CAMP levels. The role of the VIP system in normal undifferentiated LA-N-5 cell growth was examined by incubation of cells in the presence of either [N-AC-Tyrl, D-Phe*]growth hormone-releasing factor-(1-29)-amide, N-GRF, an inactive competitive antagonist of VIP, or L-8-K, a VIP receptor binding inhibitor. Cell proliferation increased to a maximum of 139% and 160% of nontreated control cultures at 1.0 umol/L and 10 pmol/L concentrations of N-GRF and L-8-K, respectively, as measured by [3H]-thymidine uptake (Fig 7). This supports the hypothesis that VIP normally functions as an autoregulator of neuroblastoma cell growth. Changes in VIP levels and VIP receptor expression seen during RA-induced differentiation suggest that a portion of the RA effect may, in actuality, be mediated through VIP. The elevation of CAMP levels seen with RA exposure in the absence of exogenous VIP supports this premise, as RA reportedly does not alter these levels directly. To test this hypothesis further, LA-N-5 cells were exposed to RA for 8 days in the presence of either L-8-K or N-GRF. Increasing concentrations of either antagonist were found to partially block the effects of RA on cellular proliferation (Fig 8). Cell growth achieved 64% and 57% of nontreated control cultures for cells exposed to 10 pmol/L of L-8-K and N-GRF, respectively. RA alone inhibited cell growth to 29% to 34% of nontreated values. Neurite outgrowth in response to RA was also partially blocked in the presence of either antagonist (Fig 9). Quantitative assessment of RA-induced differentiation, as measured by neurite outgrowth, demonstrated 66% to 68% phenotypically differentiated forms following 8 days of RA exposure (Fig 10). Coincubation with either L-8-K or N-GRF resulted in
1.7 1.6 1.5 .s 2 1.43 : % 1.30, 5 2 1.2z v) 1.1 -
0.9 -
9
6
7
6
5
4
-log ~antagonisrl Fig 7. Enhanced proliferation of LA-N-5 cells in response to VIP receptor antagonism as measured by [JH]-thymidine incorporation following 8 days of culture. Data are mean values f SEM of triplicate cultures. Stimulated/basal ratios are derived from the comparison of treated to untreated (control) cell growth. The concentration of antagonist at which statistically significant (P c .05, ANOVA) cell growth is reached above untreated control cultures is shown by asterisks (*, L-E-K; **, N-GRF).
35% and 45% differentiated forms, respectively, at maximal concentrations. Importantly, the antagonist N-GRF has been shown previously to be ineffective in altering intracellular CAMP levels in rat pancreatic cells and, therefore, could not directly account for the alterations in cellular proliferation or morphology by this mechanism. The antagonism of RA-induced growth inhibition and morphological differentiation of LA-N-5 cells by specific VIP antagonists as shown suggests an operative role for VIP in the regulation of neuroblastoma cell growth distinct and separable from those effects induced directly by RA. DISCUSSION
Despite its name, VIP has a very close association with neural tissue, functioning as a neurotransmitter in some neurons. Recent findings indicate that neurotransmitter molecules, in general, play an important role in neural development, and in the regulation of neuronal architecture.z,26 That VIP can induce the differentiation of neuroblastoma cells and has also been shown to have a similar effect on fetal neuroblastsz7 is consistent with this general principle.
941
VIP AND NEUAOBLASTOMA
80 -
z $
0 0
60
-
50
-
40
-
.:
30-
d 20
-
10
-
50-
s k
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60 C .s
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g
P
z 2 : Q
Q
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P
10 -
O-
O-
I,
RA
10
9
8 -log
7
6
5
[antagonist1
+lO,vM
/?A
Fig 8. Partial antagonism of RA-induced growth inhibition of LA-N-5 cells as measured by [3H]-thymidine incorporation following 8 days of treatment. Data are mean values f SEM of triplicate cultures. RA represents cells grown in the presence of 10 pmol/L of RA without antagonist. Percent control is derived from the comparison of treated to untreated (control) cell growth. The concentration of antagonist at which statistically significant (P c .05, ANOVA) cell growth is reached above RA-treated control cultures is shown by asterisks (*, L-8-K; **, N-GRF).
Fig 9. Partial antagonism of the F&induced morphological differentiation of LA-N-5 cells by VIP antagonists following 8 days of culture as demonstrated by phase contrast photomicrography. (A) Morphology of untreated control cultures. (B) Cells coincubated with 10 pmol/L of RA and 10 pmol/L of L-8-K. (C) Cells coincubated with IO pmol/L of RA and 10 &mot/L of N-GRF. (D) Morphology of 10 pmol/L FiAtreated cells. (Original magnification x200.)
I
RA
”
I
,
,
,
/
I
10
9
8
7
6
5
-log
iantagonis +70/M
.:
I
Control
tl
RA
Fig 10. Partial antagonism of the RA-induced morphological differentiation of LA-N-5 cells by VIP antagonists as assessed by neurite extension following 8 days of culture. F4Aand Control points indicate the percentage of cells exhibiting the differentiated neuronal phenotype in the presence or absence of 10 pmol/L of RA. Data are the means f SEM of triplicate experiments. The concentration of antagonist at which statistical signfficance (P < 95, ANOVA) is reached is shown by asterisks (*, L-8-K; **, N-GRF).
942
VIP has all the necessary characteristics of an autocrine factor. It is produced by neuroblastoma cells, which themselves bear specific receptors for this neuropeptide. In this manner it appears to exert control over the rate of cell proliferation and the state of cellular differentiation. The onset of differentiation results in increased levels of VIP and increased expression of its receptor, which serve to amplify the initial effect. One criticism that can be made of this last point is that RA-induced differentiation and VIP-induced differentiation may not be identical processes, and therefore, findings from one may not be applicable to the other. This is certainly a valid concern, and we have not yet shown that endogenous VIP levels and VIP receptor expression increase during VIP-induced differentiation in the absence of RA. Evidence indicates that all differentiating agents must act through a final common pathway, but certainly some cellular effects have been observed which are not universal.5 ObviousIy further work wiIl be necessary before this question can be fully answered. However, given the synergy that has previously been demonstrated between these two agents,16 it seems likely that the majority of the effects of the two are, indeed, identical. All the evidence points to a fundamental role for VIP in the in vivo maturation of neuroblastoma cells. In support of this is the finding that local tumor concentrations of VIP can reach the levels needed to induce differentiation,28 even though these are much higher than normal serum levels (
