Chemotherapy-induced apoptosis in a transgenic model of neuroblastoma proceeds through p53 induction.
ABSTRACT Chemoresistance in neuroblastoma is a significant issue complicating treatment of this common pediatric solid tumor. MYCN-amplified neuroblastomas are infrequently mutated at p53 and are chemosensitive at diagnosis but acquire p53 mutations and chemoresistance with relapse. Paradoxically, Myc-driven transformation is thought to require apoptotic blockade. We used the TH-MYCN transgenic murine model to examine the role of p53-driven apoptosis on neuroblastoma tumorigenesis and the response to chemotherapy. Tumors formed with high penetrance and low latency in p53-haploinsufficient TH-MYCN mice. Cyclophosphamide (CPM) induced a complete remission in p53 wild type TH-MYCN tumors, mirroring the sensitivity of childhood neuroblastoma to this agent. Treated tumors showed a prominent proliferation block, induction of p53 protein, and massive apoptosis proceeding through induction of the Bcl-2 homology domain-3-only proteins PUMA and Bim, leading to the activation of Bax and cleavage of caspase-3 and -9. Apoptosis induced by CPM was reduced in p53-haploinsufficient tumors. Treatment of MYCN-expressing human neuroblastoma cell lines with CPM induced apoptosis that was suppressible by siRNA to p53. Taken together, the results indicate that the p53 pathway plays a significant role in opposing MYCN-driven oncogenesis in a mouse model of neuroblastoma and that basal inactivation of the pathway is achieved in progressing tumors. This, in part, explains the striking sensitivity of such tumors to chemotoxic agents that induce p53-dependent apoptosis and is consistent with clinical observations that therapy-associated mutations in p53 are a likely contributor to the biology of tumors at relapse and secondarily mediate resistance to therapy.
Article: Neuroblastoma and MYCN.[Show abstract] [Hide abstract]
ABSTRACT: Neuroblastoma, the most common extracranial solid tumor of childhood, is thought to originate from undifferentiated neural crest cells. Amplification of the MYC family member, MYCN, is found in ∼25% of cases and correlates with high-risk disease and poor prognosis. Currently, amplification of MYCN remains the best-characterized genetic marker of risk in neuroblastoma. This article reviews roles for MYCN in neuroblastoma and highlights recent identification of other driver mutations. Strategies to target MYCN at the level of protein stability and transcription are also reviewed.Cold Spring Harbor Perspectives in Medicine 10/2013; 3(10). · 7.56 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: PURPOSE: MYCN amplification and p53 inactivation are two typical characteristics of aggressive neuroblastomas and are strongly associated with cancer progression and treatment failure. In an effort to develop new therapeutic agents to treat the aggressive neuroblastomas, we constructed ZD55-shMYCN, an oncolytic adenovirus ZD55 carrying short hairpin RNA (shRNA) targeting MYCN gene, and investigated the effects on proliferation of the p53-null and MYCN-amplified neuroblastoma cell line LA1-55N in vitro and in vivo by ZD55-shMYCN. METHODS: In this study, we used ZD55-shMYCN to treat p53-null and MYCN-amplified neuroblastoma cells. To confirm the ability of selective replication of the ZD55-shMYCN, we examined the expression of E1A protein by western blotting. We used quantitative real-time PCR analysis and western blotting analysis to determine the inhibitory effect of ZD55-shMYCN on MYCN expression. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cell proliferation assay and xenograft mouse model were used to test the antigrowth efficacy of ZD55-shMYCN. RESULTS: The results showed that ZD55-shMYCN selectively replicated and significantly downregulated the MYCN expression in LA1-55N cells. ZD55-shMYCN effectively inhibited the proliferation in LA1-55N cells in vitro and significantly inhibited tumor growth in vivo xenograft tumor in nude mice. CONCLUSIONS: ZD55-shMYCN provides a novel agent for treating MYCN-amplified and p53-inactive aggressive neuroblastoma, representing a promising approach for further clinical development.Journal of Cancer Research and Clinical Oncology 02/2013; · 2.91 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Neuroblastoma (NB) is one of the most common malignant solid tumors in childhood, which derives from the sympathoadrenal lineage of the neural crest and exhibits extremely heterogeneous biological and clinical behaviors. The infant patients frequently undergo spontaneous regression even with metastatic disease, whereas the patients of more than one year of age who suffer from disseminated disease have a poor outcome despite intensive multimodal treatment. Spontaneous regression in favorable NBs has been proposed to be triggered by nerve growth factor (NGF) deficiency in the tumor with NGF dependency for survival, while aggressive NBs have defective apoptotic machinery which enables the tumor cells to evade apoptosis and confers the resistance to treatment. This paper reviews the molecules and pathways that have been recently identified to be involved in apoptotic cell death in NB and discusses their potential prospects for developing more effective therapeutic strategies against aggressive NB.Cells. 06/2013; 2(2):432-59.
in a Transgenic Model of
Through p53 Induction1,2
Louis Chesler*,†,‡, David D. Goldenberg§,
Rodney Collins¶, Matt Grimmer*, Grace E. Kim¶,
Tarik Tihan¶, Kim Nguyen§, Slava Yakovenko§,
Katherine K. Matthay*and William A. Weiss*,†,§,#
*Department of Pediatrics, University of California,
San Francisco, San Francisco, CA 94143, USA;
†Diller Family Comprehensive Cancer Center,
University of California, San Francisco, San Francisco,
CA 94143, USA;‡Paediatric Oncology Group, The
Institute of Cancer Research, Sutton, Surrey, SM2 5NG,
UK;§Department of Neurology, University of California,
San Francisco, San Francisco, CA 94143, USA;
¶Department of Pathology, University of California,
San Francisco, San Francisco, CA 94143, USA;
#Department of Neurological Surgery and The Brain
Tumor Research Center, University of California,
San Francisco, San Francisco, CA 94143, USA
Chemoresistance in neuroblastoma is a significant issue complicating treatment of this common pediatric solid
tumor. MYCN-amplified neuroblastomas are infrequently mutated at p53 and are chemosensitive at diagnosis but
acquire p53 mutations and chemoresistance with relapse. Paradoxically, Myc-driven transformation is thought to
require apoptotic blockade. We used the TH-MYCN transgenic murine model to examine the role of p53-driven apop-
tosis on neuroblastoma tumorigenesis and the response to chemotherapy. Tumors formed with high penetrance and
low latency in p53-haploinsufficient TH-MYCN mice. Cyclophosphamide (CPM) induced a complete remission in
p53 wild type TH-MYCN tumors, mirroring the sensitivity of childhood neuroblastoma to this agent. Treated tumors
showed a prominent proliferation block, induction of p53 protein, and massive apoptosis proceeding through induc-
tion of the Bcl-2 homology domain-3–only proteins PUMA and Bim, leading to the activation of Bax and cleavage of
caspase-3 and -9. Apoptosis induced by CPM was reduced in p53-haploinsufficient tumors. Treatment of MYCN-
expressing human neuroblastoma cell lines with CPM induced apoptosis that was suppressible by siRNA to p53.
Taken together, the results indicate that the p53 pathway plays a significant role in opposing MYCN-driven oncogene-
sis in a mouse model of neuroblastoma and that basal inactivation of the pathway is achieved in progressing tumors.
This, in part, explains the striking sensitivity of such tumors to chemotoxic agents that induce p53-dependent apop-
tosis and is consistent with clinical observations that therapy-associated mutations in p53 are a likely contributor to
the biology of tumors at relapse and secondarily mediate resistance to therapy.
Neoplasia (2008) 10, 1268–1274
Address all correspondence to: Louis Chesler, MD, PhD, The Institute of Cancer Research, 15 Cotswold Rd. Belmont, Sutton, Surrey, SM2 5NG, UK.
1Grant numbers and sources of support: National Institutes of Health (grants R01CA102321 (W.A.W.), K08NS053530 (L.C.), the Thrasher Research Fund (W.A.W.) and the
Samuel Waxman Cancer Research Foundation (W.A.W.), The Katie Dougherty Foundation (W.A.W.), the Children’s Neuroblastoma Cancer Foundation (L.C.), the Campini
Family Foundation (L.C.), and the UK Neuroblastoma Society (L.C.).
2This article refers to supplementary materials, which are designated by Figures W1 to W4 and are available online at www.neoplasia.com.
Received 11 July 2008; Revised 13 August 2008; Accepted 14 August 2008
Copyright © 2008 Neoplasia Press, Inc. All rights reserved 1522-8002/08/$25.00
Volume 10 Number 11November 2008pp. 1268–1274
Neuroblastoma is the most common extracranial solid tumor of child-
hood . The proto-oncogene MYCN is amplified in one third of
neuroblastomas and is associated with high-risk disease and poor sur-
vival [1–5]. The ability of Myc proteins to induce malignant transfor-
mation is limited by coordinate induction of both proliferation and
apoptosis [6–8]. Consequently, mutations of p53 are common in sev-
eral Myc-driven malignancies at diagnosis and enhance oncogenesis.
In neuroblastoma, genetic defects in the p53 pathway function
(mutation in p53 or p14ARF, amplification of MDM2) are rare at di-
agnosis but are present in cell lines established from patients treated
with prolonged therapy [9–12]. In the absence of such mutations, am-
plification of MYCN in newly diagnosed neuroblastomas is typically
associated with epigenetic abnormalities that impair apoptosis: silenc-
ing of caspase-8 or overexpression of the antiapoptotic proteins Bcl-2
and survivin [13–15]. These events are independently associated with
poor outcome in neuroblastoma and represent potential mechanisms
contributing to malignant progression [13,15,16].
Despite such defects in apoptotic signaling, newly diagnosed neuro-
blastoma tumors typically respond to chemotherapeutics such as cyclo-
phosphamide (CPM) irrespective of risk group, suggesting that
epigenetic regulation of apoptotic pathways is either insufficient to
block apoptosis in response to cytotoxic chemotherapy or is not initially
present at diagnosis [17,18]. Because virtually all patients respond to
initial therapy, outcome in this disease is mainly determined by the like-
lihood of relapse, with high-risk patients (including tumors that show
amplification of MYCN) frequently relapsing with drug-resistant tu-
The TH-MYCN model shows that coordinate mutation of p53
is not required for MYCN-driven oncogenesis in neuroblastoma,
mirroring the situation in human high-risk tumors. It follows that
apoptosis is functionally suppressed by other means. However, this
presents a paradox in that MYCN-driven murine neuroblastomas
retain exquisite sensitivity to chemotherapeutics, which exert their
effects through induction of apoptosis. To examine this question,
we sought to determine whether the p53 pathway was active or in-
active in TH-MYCN and whether induction of the pathway occurred
in response to chemotherapy.
TH-MYCN is a native neuroblastoma model in which tumorigene-
sis is driven by targeted expression of a TH-MYCN transgene in the
neural crest of transgenic mice . These animals develop an aggres-
sive malignancy that is morphologically, genetically, and clinically sim-
ilar to human high-risk MYCN-amplified neuroblastoma [21,22]. We
show that p53 haploinsufficiency enhances the effect of MYCN-driven
transformation in neuroblastoma, that basal p53 pathway function
and apoptosis are suppressed in such tumors (in contrast to in vitro
observations), that haploinsufficiency amplifies the effect of MYCN
on transformation in neuroblastoma, and that the chemoresponse
of effective agents such as CPM requires intact p53-driven apopto-
sis. Mechanistically, CPM induces p53 and activates a variety of p53
targets critical to apoptosis, including PUMA, Bim, Bax, caspase-3,
caspase-9, and poly(ADP-ribose) polymerase (PARP). The in vivo re-
sponse of TH-MYCN–driven tumors to CPM is similar to that ob-
served in cultured human cell lines treated with CPM. In three cell
lines with equivalent levels of p53 protein but increasing levels of
Mycn, the apoptotic response to CPM correlated with the induction
of p53 and could be blocked by siRNA to p53.
Taken together, these data document the activity in murine tu-
mors of a frontline chemotherapeutic agent used in children with
newly diagnosed neuroblastoma and add to evidence validating mice
transgenic for TH-MYCN as a preclinical platform for developmental
therapeutics. Furthermore, these observations highlight the utility of
mice transgenic for TH-MYCN as a preclinical model in which to
delineate the kinetics and details of apoptotic signaling in response
to CPM. The induction and functional activation of p53 in these
tumors reinforce the importance of p53 signaling in the therapeutic
response to cytotoxic chemotherapy. These observations are also con-
sistent with clinical data that cytotoxic chemotherapy selects for loss
of p53 function, that recurrence in neuroblastoma is subsequently
driven by p53 mutation, and that p53 mutation in relapsed tumors
contributes secondarily to therapeutic resistance [10,23,24].
Materials and Methods
Cell Culture and Treatments
SHEP, SK-N-SH, SH-SY5Y, and Kelly neuroblastoma cell lines
were obtained from the University of California at San Francisco.
Cells were grown in Dulbecco’s modified Eagle’s medium with
10% FBS at 37°C. 4-Hydroxyperocyclophosphamide (4OH-CPM)
was obtained from Susan Ludeman (University of North Carolina)
and was dissolved at 20 mg/ml in water. For in vivo therapy, CPM
from the University of California at San Francisco Clinical Pharmacy
was dissolved at 1 mg/ml.
Proliferation and Apoptosis Assays
Cell growth and proliferation were assessed by soluble tetrazolium
(WST-1; Roche, Indianapolis, IN). Necrosis and apoptosis were
assessed using a Cell Death Detection ELISA (Roche) that detects
intracellular or released nucleosomal H2bDNA. Values were cor-
rected for vehicle (DMSO). Cells were plated and assayed at 6 to
24 hours using a Synergy HT-1 spectrophotometer (Biotek, Winooski,
VT). Percent toxicity was standardized using Triton X-100 as a
positive control (0-100 mM).
Immunoblot Analysis and Antibodies
Cells were suspended in nondenaturing lysis buffer (Cell Signaling
Technology, Danvers, MA) with 0.5% SDS. Lysates were sonicated
and cleared at 14,000g at 4°C. Protein content was assayed by the
BCA method (Pierce, Rockford, IL), and 20 to 40 μg of protein
was analyzed on 4% to 12% gradient denaturing gels (Invitrogen,
Carlsbad, CA). Membranes were incubated overnight at 4°C with pri-
mary antibodies [cleaved caspase-3 (9664; Cell Signaling Technology),
caspase-9 (9508; Cell Signaling Technology), p53 (9282; Cell Signal-
ing Technology), Bim (4582; Cell Signaling Technology), Bax (2772;
Cell Signaling Technology), PARP (9542; Cell Signaling Technology),
PUMA (4976; Cell Signaling Technology), and β-tubulin (05-661;
Upstate, Billerica, MA)] and visualized using HRP-conjugated second-
ary antibodies (Calbiochem, Gibbstown, NJ) and ECL-Plus reagents.
For frozen sections, mice were killed after therapy, and tumors
were embedded in Tissue-Tex O.C.T. compound (Sakura, Torrance,
CA), cooled at −80°C (24 hours), sectioned at 4-μm thickness,
mounted on Superfrost slides (Fisher Scientific, Pittsburgh, PA),
and placed in 70% ethanol (1 hour at 4°C). Slides were washed
three times with wash buffer (Dako, Carpinteria, CA), treated with
3% hydrogen peroxide (for 10 minutes), rewashed, blocked with
casein (for 10 minutes), and incubated with caspase-3 antibody
Neoplasia Vol. 10, No. 11, 2008Cyclophosphamide and p53 in NeuroblastomaChesler et al.
(CST 9664) at a dilution of 1:100 at room temperature in a humidi-
fied chamber (for 2 hours). Slides were rewashed three times and in-
cubated with rabbit Envision+secondary antibody (Dako) at room
temperature in a humidified chamber (for 30 minutes) treated with
Dako DAB+complex (for 4 minutes), rinsed with water, counter-
stained with Gills modified hematoxylin (American Master Tech,
Lodi, CA), dehydrated, and mounted for analysis. Paraffin-embedded
tissues were cut and mounted, deparaffinized with xylene through
graded alcohol to water, and then placed in PBS. Heat-induced epi-
tope retrieval was performed at 120°C in citrate buffer at pH 6.0, fol-
lowed by treatment in 3% peroxide (for 20 minutes). Washing and
antibody incubations were as previously mentioned. Ki-67 staining
in paraffinized sections was performed using a Ki-67 rat primary anti-
body (M7249; Dako) in a 3-hour incubation followed by process-
ing as previously mentioned. Development was with streptavidin
HRP (Amersham, Piscataway, NJ) at a dilution of 1:100 for 1 hour at
In Vivo Therapy in TH-MYCN Transgenic Mice
TH-MYCN mice were maintained in hemizygotic matings as pre-
viously described . Hemizygous TH-MYCN were serially crossed
to p53+/− mice (FVBN)  and E2F1-luciferase mice (FVBN) 
for more than four generations to re-establish tumor penetrance in the
129X1/SvJ strain. A similar approach was used in luciferase crosses.
For the analysis of the in vivo effects of CPM on tumor growth in mice
transgenic for TH-MYCN (in strain 129X1/SvJ), a tumor intervention
design was implemented. The assay end point was tumor mass. In par-
allel experiments, tumor response and progression were confirmed
using luciferase bioimaging of TH-MYCN/E2F1-Luc doubly trans-
genic animals, in which a luciferase signal is emitted from proliferating
tumor tissue and is a surrogate indicator of Mycn activity. Animals
(seven per arm) with palpable tumors (approximately 60 days of life)
were treated three times per week with intraperitoneal injections of
CPM (150 mg/kg in 100 μl of saline) or vehicle (saline) and were
then killed. In parallel, animals (three per arm) were followed by
noninvasive imaging, using the Xenogen IVIS Lumina system (Caliper
Life Sciences, Hopkinton, MA) with a background bioluminescence
setting of 38,000 photons/sec per square centimeters. At sacrifice,
tumors were imaged, excised, measured, weighed, and snap-frozen for
further analysis. Lysates were prepared for immunoblot analysis as pre-
viously mentioned, except that tumors were initially homogenized
in TBS with protease inhibitors (Compleat; Roche, Inc.) before lysis.
Significance analysis was performed using the Student’s t test. All ani-
mals were handled in accordance with institutional guidelines for safe
and ethical treatment of mice.
Effect of p53 Heterozygosity on Neuroblastoma Tumor
Formation in Mice Transgenic for TH-MYCN
Given the critical role of p53 in regulating myc-driven onco-
genesis, we sought to evaluate induction of apoptosis by p53 in
spontaneously growing neuroblastoma tumors originating within a
native murine background. We therefore used the well-characterized
TH-MYCN neuroblastoma model in which tyrosine hydroxylase
(TH)–mediated overexpression of MYCN is targeted to the periph-
eral neural crest . This model faithfully recapitulates high-risk
human neuroblastoma tumorigenesis by all major clinicopathologic
criteria. To evaluate the effect of p53 dysfunction on TH-MYCN–
driven neuroblastoma tumor formation, we introduced TH-MYCN
into a p53-deficient background by serial crosses against animals with
an inactivated germ line p53 allele . Tumors arose in doubly
transgenic animals with higher penetrance and reduced latency
(Figure 1A). The initial anatomic appearance of these tumors was
similar to that of p53 wild type TH-MYCN tumors (Figure 1B),
but end-stage tumors were larger and were characterized by a higher
density of proliferating neuroblasts and lower levels of apoptosis and
necrosis on histologic analysis (Figure 1, C and D). Taken together,
the data imply that, in neuroblastoma tumor progression, inactiva-
tion of p53 could enhance MYCN oncogenicity, at least in part by
decoupling the growth-stimulatory activity of MYCN from its ability
to concurrently drive apoptosis.
Cyclophosphamide Induces Durable Remissions in Mice
Transgenic for TH-MYCN
Because intact p53 function inhibits growth and progression of TH-
MYCN neuroblastoma tumors, we used CPM to assess whether estab-
lished murine TH-MYCN tumors are sensitive to chemotherapeutics
that exert their activity at least in part through p53 up-regulation.
Cyclophosphamide induced complete regression of established tumors,
with a dramatic effect on survival intervals (cumulative tumor free days
Figure 1. p53 heterozygosity enhances tumorigenesis in TH-
MYCN neuroblastoma. TH-MYCN mice were introduced into a
p53+/− background by sequential breeding against p53+/− FVBN
animals, and cohorts of mice were followed for tumor formation.
(A) Kaplan-Meier survival analysis of animal cohorts showing in-
creased tumor penetrance (85% in p53+/−, 60% in p53+/+ ani-
mals) and shorter time to tumor onset (70 days of life in p53+/+,
50 days of life in p53+/−). (B) Early-stage focal tumor (T) of adre-
nal origin (a) on the superior pole of the kidney (K). (C, D) Histo-
logic appearance of representative, large, late-stage p53+/+ and
p53+/− TH-MYCN tumors showing a reduction of apoptotic cells
with hyperchromatic nuclei on pale backgrounds and necrotic cells
with pyknotic nuclei with hyaline staining (white arrows) in p53+/−
tumor (D) versus p53+/+ tumor (C), white arrows. The p53+/−
tumor is also relatively more monomorphous with densely packed
neuroblasts (white arrows).
Cyclophosphamide and p53 in NeuroblastomaChesler et al.Neoplasia Vol. 10, No. 11, 2008
of life; Figure 2A) and time to death from overwhelming tumor burden
(Figure 2B). Tumors in the control cohort were readily apparent on nec-
ropsy and by luciferase bioimaging of parallel animals doubly transgenic
for TH-MYCN and E2F1-luciferase (representative animal shown in
Figure 2C). Seven CPM-treated animals were killed at 100 days of life,
with no evidence of tumor on necropsy and negative luciferase signals
(representative animal shown in Figure 2D). These data show that tu-
mors in TH-MYCN transgenic mice are sensitive to CPM, mirroring
similar observations in MYCN-amplified human neuroblastoma.
Cyclophosphamide Inhibits Tumor Proliferation and
Stimulates Apoptosis In Vivo
To analyze the effect of CPM on apoptosis, necrosis, and prolifera-
tion, we treated a cohort of transgenic animals with early, established
tumors, sacrificing animals at several time points within 24 hours
of CPM treatment. Tumors showed no changes in vascularity (data
not shown) and exhibited no obvious cellular or structural changes
(observable apoptosis or necrosis) in response to CPM treatment (Fig-
ure W1, A–C). In vehicle-treated tumors, immunoreactivity for the
proliferation marker Ki-67 was positive in most nuclei (Figure 3, A
and B). In response to CPM, Ki-67 labeling decreased dramatically
even by 24 hours (Figure 3C). Whereas control tumors had negligible
staining for the apoptosis marker cleaved caspase-3 (Figure 3D), high
levels of cleaved caspase-3 were observed 3 and 6 hours after treatment
with CPM (Figure 3, E and F). A wave of apoptosis throughout the
tumor was demonstrated at low power (Figure 3, H–J), with maximal
staining visible at 6 hours after treatment. At 24 hours, apoptotic stain-
ing was associated with foci of necrosis (Figure 3J, inset). Significantly,
levels of apoptosis induced by similar treatment of p53-haplodeficient
tumors with CPM were minimal, as assessed by immunostaining for
cleaved caspase-3 (Figure W2). No significant differences in apoptosis
were seen by immunohistologic analysis in vehicle-treated p53+/− versus
p53+/+ tumors (data not shown).
Apoptosis in Tumors Treated with Cytotoxic Chemotherapy
Is Driven by p53
To evaluate apoptotic signaling in response to CPM, we treated a
cohort of early tumor-bearing transgenic animals and assessed levels
of key apoptotic proteins by immunoblot (Figure 4A). In contrast to
basal apoptotic activity detectable by immunoblot analysis of cul-
tured neuroblastoma cell lines, activation of the p53 pathway and
caspase-3 cleavage were minimal in vehicle-treated tumors, consistent
with the low-level caspase-3 staining seen in Figure 4. Rapid induc-
tion of p53 was observed at 3 hours after treatment with CPM, with
a peak at 6 hours after treatment (Figure 4A). Cleaved caspase-3 and
-9 were maximal at 6 hours after treatment and were sustained dur-
ing a 12-hour period. These data indicate that basal apoptotic activity
in spontaneously progressing TH-MYCN neuroblastomas is minimal
but that p53-driven apoptosis is intact and can be strongly induced
in response to cytotoxic chemotherapy.
CPM Treatment of Murine Neuroblastoma
PUMA and Bim are Bcl-2 homology 3 (BH3)–only proteins,
proapoptotic components of the mitochondrial cell death pathway,
and are critical effectors of p53-induced apoptosis in Myc protein–
driven tumors . In response to CPM treatment, PUMA was
strongly induced in vivo, peaking at 3 hours and dissipating by
12 hours (Figure 4A). Bax, a downstream target of PUMA and a criti-
cal effector of myc-induced mitochondrial apoptosis, was strongly
expressed (Figure 4B). Bim, a BH3-only protein necessary for apop-
tosis in myc-driven lymphoma, was induced prominently. Cleavage
of caspases-3 and -9 (Figure 4A) and PARP (Figure 4B) occurred
concurrently, indicating high levels of apoptosis. Taken together,
these findings indicate that the proapoptotic effect of CPM proceeds
through the intrinsic p53 regulatory pathway of BH3 proteins, im-
plicating PUMA, Bax, and Bim as key effectors of p53 function in
primary murine neuroblastoma tumors.
CPM Induces p53-Dependent Apoptosis in Human
MYCN-Amplified Neuroblastoma Cell Lines
To determine whether levels of Mycn influence response to CPM
in human tumors, we examined a panel of human neuroblastoma
cell lines in which levels of Mycn were undetectable (SH-EP), inter-
mediate (SH-SY5Y), or high (Kelly). All lines expressed roughly com-
parable levels of p53 at baseline (Figure 5A). Cells were treated with
CPM, and proliferation and apoptosis were assessed using soluble
MTT and histone DNA-cleavage assays, respectively (Figure 5, B and
C). Treatment with 4OH-CPM (the active metabolite of CPM) re-
sulted in effective growth inhibition in 10% serum (Figure 5B) or
Figure 2. CPM induces durable remission in mice transgenic for
TH-MYCN. Cohorts of transgene-positive animals (seven per arm)
were randomly assigned to receive treatment with either control
(saline) or CPM (150 mg/kg per day injected intraperitoneally three
times per week). Animals were treated at approximately 60 days of
life, when tumors were palpable. (A) Survival after treatment (day of
life, 60). All treated animals survived to >100 days of life. Saline-
treated animals required euthanasia owing to signs of advanced
disease at or before 90 days of life. (B) Tumor-free survival after
treatment on day of life 60. All CPM-treated animals remained
tumor-free, whereas saline-treated animals developed tumors
10 days after treatment (70 days of life). The proportion of saline-
treated animals remaining tumor-free is shown on the Y axis, plot-
ted against tumor-free days after treatment on the X axis. (C) At
treatment initiation, animals harbored focal tumors (white arrow),
with strong E2F1-driven luciferase bioluminescence of up to 6 ×
105photons/sec per square centimeters. At autopsy, animals in
or luciferase bioluminescence were detected in treated animals at
100 days of life (white dashed outline).
Neoplasia Vol. 10, No. 11, 2008Cyclophosphamide and p53 in NeuroblastomaChesler et al.
in low serum (data not shown). 4-Hydroxyperocyclophosphamide
induced apoptosis in all cell lines, with increased apoptosis noted
in the MYCN-amplified Kelly cell line (Figure 5C). These data are
consistent with published findings that neuroblastoma cells ampli-
fied for MYCN are sensitized to the effects of cytotoxic chemo-
therapeutics . Coincident with the induction of apoptosis by
4OH-CPM, activation of p53 was still observed in such cells. We
treated MYCN-amplified Kelly cells with 4OH-CPM and cisplatin
[cis-diamminedichloridoplatinum(II), CDDP], a bifunctional alkylator
also used in frontline therapy for human neuroblastoma. In con-
trast to a lack of apoptosis observed in vehicle-treated spontaneous
in vivo tumors, the vehicle-treated cultured cell lines displayed de-
tectable basal apoptosis by immunoblot analysis. Analogous to the
response observed in TH-MYCN tumors growing in vivo, p53 was
strongly induced and phosphorylated in response to treatment with
4OH-CPM, and maximal activity correlated with strong induction of
caspase-3 cleavage at 6 hours (Figure 5D). Comparable activation was
observed in response to CDDP, although with a slightly longer time
course of p53 activation (Figure W3). Treatment of these cells with
siRNA against p53 led to decreased levels of p53 total and phos-
phorylated proteins 6 hours after treatment with 4OH-CPM, with
a concomitant decrease in levels of the apoptotic marker cleaved
caspase-3 (Figure W4). These results are consistent with previous
findings indicating that the induction of p53 observed in murine
Figure 3. Proliferation blockade and apoptosis induction in CPM-treated murine neuroblastoma. Tumor-bearing animals were treated
with CPM and killed, and tumors were fixed in paraffin. Immunoreactivity of Ki-67 (myb-1) in tumors (three per time point) treated for 0, 6,
or 24 hours, respectively. BV indicates blood vessel; K, kidney; T, tumor. (A) Vehicle-treated tumors under low power (4×) show ex-
tensive Ki-67 immunoreactivity, reflecting the high proliferative index of such lesions. (B, C) Ki-67 staining is maximal at 6 hours and
is notably diminished at 24 hours after CPM, highlighting the responsiveness of this tumor to the drug. (D–G) Cleaved caspase-3 im-
munostaining in formalin-fixed paraffin-embedded tumors is minimal in vehicle-treated tumors (D), but is rapidly induced at 3 hours (E),
is maximal at 6 hours (F), and largely resolves by 24 hours (G) after CPM. Caspase-3 immunoreactivity at 24 hours is greatly reduced and
is colocalized to regions of cellular breakdown and nuclear atypia (G). Insets: original magnification, ×20. BV indicates blood vessel; K,
kidney; T, tumor. (H–I) Quantitation of Ki-67 staining (H) and cleaved caspase-3 staining (I) in the tumor sections shown in D–G at time
points after CPM in five high-power fields per section from three separate tumor samples.
Cyclophosphamide and p53 in Neuroblastoma Chesler et al.Neoplasia Vol. 10, No. 11, 2008
neuroblastoma is also evident in human tumors and that MYCN-
amplified human neuroblastoma likely requires p53 for induction of
apoptosis in response to cytotoxic chemotherapy.
To our knowledge, this study is the first in vivo demonstration of
the importance of intact p53 function to neuroblastoma tumor pro-
gression and chemoresponsiveness. In this MYCN-driven malignancy,
the proliferative and apoptotic functions of MYCN are effectively
uncoupled by functional suppression of p53-induced apoptosis (in
the absence of p53 mutation). Furthermore, any coexistent apoptotic
defects required for transformation by MYCN are insufficient to
block the apoptotic effect induced by cytotoxic chemotherapy. Mu-
tation at p53 is not generally observed in neuroblastoma tumors
at diagnosis; however, epigenetic alterations (including silencing of
caspase-8, increased levels of survivin and Bcl2, and cytoplasmic
sequestration of p53) likely contribute to malignant progression
[13,15,29]. Although these epigenetic abnormalities may underlie
some relative chemoresistance, our data are consistent with observa-
tions in other tumor types suggesting that these abnormalities spare
intrinsic mitochondrial apoptotic signaling, which can drive apopto-
sis in response to cytotoxic chemotherapy.
Cyclophosphamide, a first-line agent in children with newly di-
agnosed neuroblastoma, is active in murine neuroblastoma. Baseline
apoptosis in spontaneously progressing TH-MYCN tumors is sup-
pressed, and activation of such proteins is not detectable by immuno-
blot analysis, in contrast to observations in cultured neuroblastoma
tumor cell lines in vitro. Cyclophosphamide treatment induces mas-
sive apoptosis proceeding through activation of p53 and the down-
stream Bcl-2-homology domain-3 (BH3) proteins, PUMA and Bim,
indicating that apoptosis proceeds through mitochondrial-dependent
pathways. Bax, a known effector of apoptosis in murine lymphoma,
is also strongly induced . PUMA, Bim, and Bax are all key effec-
tors of p53-induced apoptosis, and alterations in PUMA expression
modulate the phenotype of c-myc–driven tumors in genetically engi-
neered mouse models of hematopoietic malignances . PUMA
complexes directly with p53, linking nuclear and cytosolic p53 frac-
tions, and is required for induction of apoptosis by nuclear p53 .
Our observations argue that p53-driven apoptosis is functional but
suppressed in spontaneous TH-MYCN tumors growing in vivo,
that p53 and its effector PUMA are potential therapeutic targets in
Figure 5. CPM induces apoptosis in MYCN-amplified neuroblas-
toma cell lines are sensitized to CPM-induced apoptosis and acti-
vate p53. Tumor cells were cultured in 10% FBS and treated with
4OH-CPM at indicated doses for 24 hours. (A) Expression levels of
Mycn and p53 protein in cultured cell lines SH-EP (MYCN diploid,
no Mycn protein), SH-SY5Y (MYCN diploid, intermediate Mycn
protein), and Kelly (MYCN-amplified, high Mycn protein) treated
with 4OH-CPM for 6 hours. (B) To assess the effect of CPM on pro-
liferation, cells were analyzed at 24 hours by water-soluble tetra-
zolium (WST-1) assay. Kelly cells showed enhanced inhibition
across the range of doses tested. Values represent means of trip-
licate measurements. (C) CPM induces apoptosis in human neuro-
blastoma cell lines. The same cells were treated for 24 hours,
and apoptosis was assessed by nucleosomal DNA ELISA. Apopto-
sis was more readily evident in the MYCN-amplified Kelly cells.
These data suggest that amplification of MYCN sensitizes human
neuroblastoma cells to apoptosis induced by CPM and are consis-
tent with observations that amplification of MYCN generally in-
creases the susceptibility of cells to cytotoxic chemotherapy. (D)
MYCN-amplified Kelly cells were treated with 20 μM 4OH-CPM for
24 hours and analyzed by immunoblot analysis for levels of p53,
phospho-p53 (S15), and caspase-3 proteins. Induction of caspase-3
cleavage occurs 6 hours after treatment with CPM coincident with
maximal p53 induction and phosphorylation. This is consistent
with the observations in vivo using TH-MYCN tumors.
Figure 4. In vivo CPM treatment of murine neuroblastoma tumors
induces p53 and p53 downstream targets. Tumors were harvested
from transgene-positive animals at 0, 3, 6, 12, and 24 hours after
therapy with CPM. Lysates were analyzed by immunoblot analy-
sis for levels of apoptosis proteins. (A) Activation of the intrinsic
(mitochondrial) apoptotic pathway occurs at 3 hours, with induc-
tion of p53 peaking at 6 hours after treatment. Activation of p53
was associated with cleavage of caspases-3 and -9, also peaking
at 6 hours. PUMA was induced strongly, with a peak at 3 hours
after treatment. (B) To assess the effect of CPM treatment on
BH3-family proteins, lysates were probed for the activation of
Bim and Bax. Rapid activations of Bax and Bim were coincident.
These proteins are downstream targets of PUMA and c-myc, re-
spectively, subject to p53 regulation of the chemotherapy-induced
DNA damage response. Poly(ADP-ribose) polymerase cleavage
indicates potent induction of apoptosis.
Neoplasia Vol. 10, No. 11, 2008Cyclophosphamide and p53 in NeuroblastomaChesler et al.
neuroblastoma, and that agents that enhance the activity of p53 or
PUMA may show efficacy in this disease.
Impaired p53 signaling likely enhances the oncogenic activity of
MYCN in neuroblastoma [10–12,33,34] and probably contributes
to relapse as additional (mutational) defects in this pathway are ac-
quired with genotoxic therapy. These studies help to clarify observa-
tions in cell lines from human neuroblastoma, in which acquisition
of mutations in p53 occurred in cell lines established from relapsed
tumors [11,24,33]. Collectively, our murine data are in agreement
with human cell line data generated here and published by others,
suggesting that cytotoxic drugs drive relapse by selecting for muta-
tions in p53 or in the p53 pathway. We are currently generating mice
transgenic for TH-MYCN that are inducibly dysfunctional at p53
and are backcrossing them into the penetrant 129/SvJ strain to de-
termine the utility of these animals as a model for drug-resistant
high-risk neuroblastoma at relapse.
The authors thank Scott Lowe, Gerard Evan, Jason Shohet, and mem-
bers of the Weiss laboratory for useful discussions. The authors also
thank Larry Donehower for supplying p53+/− mice, Eric Holland
for supplying E2F1-luciferase mice, and Susan Ludeman for supplying
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Cyclophosphamide and p53 in NeuroblastomaChesler et al. Neoplasia Vol. 10, No. 11, 2008
Figure W1. Morphology of TH-MYCN neuroblastoma tumors treated with CPM. (A–C) Animals were treated intraperitoneally with
150 mg/kg CPM for 0, 3, and 6 hours, and tumors were harvested, fixed in formalin, embedded in paraffin, and sectioned for hema-
toxylin and eosin staining. No major structural changes were observed, and cellular morphology of neuroblasts (arrows) was unaltered.
BV indicates blood vessel; K, kidney; T, tumor.
Figure W2. Apoptosis in p53+/− TH-MYCN tumors in response to CPM administered in vivo. In contrast to p53 +/+ TH-MYCN tumors,
which show potent activation of caspase-3 cleavage 6 hours after treatment with CPM (150 mg/kg given intraperitoneally), p53+/−
tumors fail to show significant induction of caspase-3 staining after treatment (representative staining from a cohort of three replicate
tumors per group). Caspase-3 cleavage is quantitated in the right panel. No significant changes in caspase-3 staining were seen be-
tween vehicle-treated p53+/− and p53+/+ tumors (data not shown).
Figure W4. Inhibition of p53 using RNA interference. MYCN-
amplified Kelly cells were transiently transfected with p53 siRNA.
Inhibition of p53 reduces p53 activation and phosphorylation of
p53 (Ser 15) at 6 hours, with consequent reduction of caspase-3
cleavage, implying a requirement for p53 function in the apoptotic
response of these cells to chemotherapy.
Figure W3. Response of human MYCN-amplified neuroblastoma
cells to cisplatin (CDDP). Kelly cells were cultured and treated as
in Figure 4, using 15 μM cisplatin, and were immunoblotted for
p53, phospho-p53 (Ser 15), and caspase-3. Induction and activa-
tion of p53 is observed commencing at 3 hours after CDDP, with
prolonged activation until 12 hours corresponding to the activation
of caspase-3 cleavage beginning at 6 hours.