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DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy
in the Graduate School of The Ohio State University
By
Anne Robinson Albers, B. A.
*****
The Ohio State University 1996
Dissertation Committee Approved by Georgia A. Bishop, Ph.D. * A
Philip R. Johnson, M.D. sJuJJL- QIJ ja/c y A* \ jlJUJ L X i P M. Sue O’Dorisio, M.D., Ph.D. ( Advisor Alan J. Yates, M.D., Ph.D. Neuroscience Graduate Program UMI Number: 9639178
UMI Microform 9639178 Copyright 1996, by UMI Company. All rights reserved. This microform edition b protected against unauthorized copying under Title 17, United States Code.
UMI 300 North Zeeb Road Ann Arbor, MI 48103 ABSTRACT
The hypothesis that the neuropeptide somatostatin and somatostatin receptors
play a role in the biology of neural crest development was tested by studying
neuroblastoma. A second hypothesis, that studies of somatostatin receptors in
neuroblastoma may lead to therapeutic advances in clinical neuroblastoma, was also tested. In vitro gene analysis, in vitro pharmacologic characterization, and in vivo growth
studies, pharmacologic characterization, and analysis of gene expression were pursued.
Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) was used to analyze
the expression of somatostatin and all five somatostatin receptor subtypes (ssti - sst5) in
34 tumor tissue samples from 26 patients with neuroblastoma. All 34 tumor specimens expressed a control gene, c-abl, and ssti ; 33/34 samples expressed SS; sst 2 was expressed in 29/34 samples, and sst 3 , ssU, and sstj variably expressed.
Pharmacologic analysis of binding affinities of somatostatin 14 (SS 14) and somatostatin congeners octreotide, CH275, and WOC3b, was studied in SKNSH neuroblastoma cell lines stably transfected with ssti and sst2. SSu bound with high affinity to the SKNSH/ssti and SKNSH/sst 2 neuroblastoma cell lines. Octreotide bound with high affinity only to SKNSH/sst 2 . CH275 bound with high affinity only to
SKNSH/ssti- WOC3b bound with high affinity to both SKNSH/ssti and SKNSH/sst 2 . No significant difference in tumorigenesis was observed between the cell lines
SKNSH, SKNSH/ssti, and SKNSH/sst 2 when used for a xenograft model of neuroblastoma. In vivo binding characteristics of the SKNSH, SKNSH/ssti, and
SKNSH/sst2 cell lines demonstrated variable binding by receptor specific analogues. In vivo gene expression of upregulated sst was preserved.
These studies present the first molecular analysis of the expression of the five sst in neuroblastoma; and demonstrate that neurpeptide-receptor interaction plays a role in neuroblastoma, and by inference, in neural crest development. Additionally, the application of receptor biology to detection of and/or therapy for neuroblastoma offers exciting prospects for improving the clinical management of neuroblastoma. Dedicated to M. Sue O’Dorisio ACKNOWLEDGMENTS
I would like to thank my family for their encouragement, support, and inspiration.
This dissertation is dedicated to MSO, whose confidence in me and enthusiasm for her work and life are the foundation for this work and inspiration for my career.
I am grateful to Dr. Debbie Martinez, Monica Summers, Dr. Gail Wenger, and
Dr. Anne Lewis for sharing techniques, styles, and ideas with me.
I am grateful for the efforts of Dr. Thomas M. O’Dorisio and Dawn Wray who helped me become a poised, engaging, public speaker.
I would like to acknowledge Dr. Steven Qualman for providing not only his own time and ideas but also for making available the resources of the Cooperative Human
Tissue Network and the help of the technicians in the department of anatomic pathology.
I also wish to thank Julia M. Kim, Steve Kirkby, Chris Elliott, Jeff Sail, Leigh
Sotos, Marsha Hauger, Dr. Sang Park, and Dr. Michael Fruehwald who have helped me immeasurably with this work.
I would finally tike to acknowledge Pat Davis, Sharon Palko, and Brad Baker for their help in preparing my dissertation's final form.
This work was supported by the Howard Hughes Medical Institute, by the
Neuroscience Graduate Program at The Ohio State University, and by NCI ROl Grant #
CAM 177 to MSO. VITAE
July 12, 1968 ...... Born - Columbus, Ohio
199 0 ...... B.A. English, Yale University, New Haven, Connecticut
1991 - present ...... Medical student, The Ohio State University College of Medicine Graduate Student, The Ohio State University Neuroscience Graduate Program
PUBLICATIONS
1. A.R. Albers, M.S. O’Dorisio, "Clinical Use of Somatostatin Analogues in Paediatric Oncology.” Digestion., 1996; 57(suppl 1):38-41.
2. A.R. Albers, M.S. O’Dorisio, and A.J. Yates, “Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis of somatostatin receptor expression (SST) in neuroblastoma, Soc. Neurosci. Abst., 21 (1995) 2133 (Abstract).
FIELDS OF STUDY
Major field: Neuroscience TABLE OF CONTENTS
Page
Dedication ...... iv
Acknowledgments ...... v
V itae...... vi
List of Tables ...... viii
List of Figures ...... ix
Chapters:
1. Introduction ...... 1
2. Background and Significance ...... 3
3. Materials and Methods ...... 42
4. R esults...... 72
5. Discussion ...... 113
References ...... 122 LIST OF TABLES
Table Page
2.1 Nomenclature for human somatostatin receptor subtypes (ssti - sst;) ...... 23
2.2 Expression of sst] through sst; in mouseM and ratR b rain ...... 24
2.3 Expression of ssti through sst; in rat peripheral tissues ...... 27
4.1 Patient prognostic factors and outcome ...... 78
4.2 Primer design for the c-abl, human p actin (HPA), somatostatin (SS) and somatostatin receptor genes 1-5 (ssti.j) ...... 80
4.3 Probe sequences for Southern confirmation of RT-PCR analysis ...... 81
4.4 Expression of ssti - sst5, somatostatin (SS), and c-abl in control tissues and neuroblastoma cell lines ...... 81
4.5 COS-7 expression of pcDNA3/ssti, pcDM 8 /sst2 . and pcDNA 3 /sst2 , evaluated by total binding to [l 2 5 I-tyrn ]-somatostatini 4 ...... 87
4.6 Affinity (K d ) of peptides for ssti and sst 2 ...... 97
viii LIST OF FIGURES
Figure Page
4.1 Total RNA isolation ...... 72
4.2 RT-PCR analysis of sst 2 and P actin expression in IMR32 and SKNSH total RNA ...... 74
4.3 Total RNA subjected to Ambion DNasel treatment ...... 75
4.4 PCR confirmation of sst primers ...... 76
4.5 Neuroblastoma total RNA analyzed by R T-PC R ...... 82
4.6 Southern confirmation of RT-PCR ...... 83
4.7 Neuroblastoma RT-PCR cDNA Southern analysis for ssti and sst 2 ...... 84
4.8 Gene expression in 34 specimens ...... 84
4.9 Expression vector pcDM 8 /sst2 with sst 2 cloned into polycloning region ...... 8 6
4.10 Transfection of mammalian cells with pcDNA3/sst| or pcDM 8 /sst2 -psV2Neo ...... 8 8
4.11 Binding analysis of stably transfected SKNSH cells ...... 89
4.12 RT-PCR confirmation of ssti and sst 2 upregulation in SKNSH neuroblastoma cells ...... 91
4.13 RT-PCR confirmation of sst 2 upregulation ...... 92
4.14 Competitive binding analysis on whole cells ...... 93 Figure Page
4.15 Competitive binding of l25I-SSu versus unlabeled SS 14 on SKNSH neuroblastoma cells, on SKNSH/pcDNA3/sst| , and on SKNSH/pcDM 8 /sst2 ... 94
4.16 Competitive binding of 1 2 5 I-SSu versus unlabeled peptides on SKNSH/ssti neuroblastoma cells ...... 95
I 7^ 4.17 Competitive binding of I-SS 14 versus unlabeled peptides on SKNSH/sst2 neuroblastoma cells ...... 96
4.18 Survival analysis for xenograft tumorigenesis ...... 98
4.19 SKNSH radioreceptor guided surgery time course ...... 99
4.20 SKNSH/ssti radioreceptor guided surgery time course ...... 100
4.21 SKNSH/sst2 radioreceptor guided surgery time course ...... 100
4.22 Autoradiographic analysis of in vivo b inding ...... 102
4.23 Autoradiographic analysis of in vivo bin ding ...... 105
4.24 Autoradiographic analysis of in vivo binding ...... 107
4.25 SKNSH xenograft neuroblastoma total RNA analyzed by RT-PCR ...... 110
4.26 SKNSH/ssti xenograft neuroblastoma total RNA analyzed by RT-PCR ...... 111
4.27 SKNSH/sst 2 xenograft neuroblastoma total RNA analyzed by RT-PCR ...... 1 12
x CHAPTER 1
INTRODUCTION
Neuroblastoma is one of a group of neuroblastic tumors that also includes ganglioneuroblastoma and ganglioneuroma; all of these tumors are derived from primordial neural crest cells ( 1 ). Neuroblastoma tumors have variable outcomes; stage
IV-S tumors may spontaneously regress, while Stage I tumors differentiate to ganglioneuroma, and most Stage III/IV tumors take an aggressive, devastating course (1).
Variations in tumor type may result from the pluripotent nature of neural crest cells which ultimately give rise to differentiated cells that include melanocytes, autonomic and sensory ganglia, glia (oligodendroglia), Schwann cells, pia and arachnoid, cranial nerve roots (V, VII, IX, X), medulla of the adrenal gland, cartilage, cells of the trunco-conal cushions of the heart, and bone (2-4). The variations of histologic presentation, location, and behavior of neuroblastic tumors might correlate with stages of neural crest development when oncogenesis occurs. Neuroblastoma in particular is the most common extracranial solid tumor of childhood with an annual incidence of 9 per million children in the US (1); yet the biology of neuroblastic tumors, such as neuroblastoma, is not understood.
1 The cells that make up neuroblastoma tumors derive from postganglionic
sympathetic neuroblasts, derived from the sympathoadrenal (SA) lineage ( 1 ). Two major and one minor types of cells arise from the SA lineage: the sympathetic ganglion neuron, chromaffin cells of the adrenal medulla, and the minor type of small intensely fluorescent
(SIF) cell (5,6). Neuroblastoma cells may be propagated in vitro and exploited for their plasticity (7-9). Addition of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), retinoic acid, vasoactive intestinal peptide (VIP), somatostatin (SS), dibutyryl cyclic AMP (dbcAMP), and ciliary neurotrophic factor (CNTF) all induce morphologic and biochemical changes in neuroblastoma in vitro (10-15). Studies of neuroblastoma cell lines in vitro offer insight 1 ) into the mechanisms that underly oncogenic transformation and progression of disease in neuroblastic tumors, as well as 2 ) to the role of neuropeptides and their receptors in the orchestration of neural crest differentiation. Recent studies have shown that proto-oncogenes and growth factors that are integral to development also play a role in the biology of neuroblastoma. An understanding of the development of the SA lineage and the biology of the neural crest is imperative in understanding the molecular oncology of neuroblastoma and developing effective means for detection, characterization, and treatment of this tumor. Accordingly, the purpose of the background and significance will be to introduce current knowledge of neural crest neuropoeisis and the roles of growth factors and neuropeptides, their receptors, and gene regulation using in vitro models of neural crest development which include in vitro neuroblastoma research.
2 CHAPTER 2
BACKGROUND AND SIGNIFICANCE
Development of the neural crest. The majority of tissues that make up the
nervous system derive from the neuroepithelium of the neural tube which forms at weeks
three to four in human development (16). The process of neurulation is the invagination
of neuroepithelium to form the neural tube; which subsequently gives rise to the brain and
spinal cord. Neurulation represents the third major phase in development for the
vertebrate embryo. The first phase consists of defining axes: anterior-posterior, inside-
outside, and dorsal-ventral; mesoderm induction between endo- and ectoderm must occur
during the second phase (17). It is the cells of the dorsal mesoderm which induce cells in
the ectoderm to become neural; the signaling that causes an ectoderm cell to become a
neuroepithelial cell thus initiates nervous system development ( 6 ).
Cells of the neuroepithelium give rise to a specialized group of migratory cells called the neural crest. Soon after the neural tube has closed, neural crest cells emerge
from the dorsal region. Their emergence follows a rostralcaudal pattern, with migration differing between axial segments of origin (18). Neural crest cells originating rostral in the neural tube “midbrain” region emerge as a monolayer of cells under the ectoderm,
3 while crest cells emerging from the hindbrain or trunk regions follow a segmental pattern
(18). The neural crest populations have been termed cranial, vagal, trunk, and
lumbosacral according to their origin on the rostrocaudal axis, their pathways of
migration, and final derivatives (18). The crest cells migrate away from the neural tube
and develop into a wide variety of tissues including the sensory and autonomic ganglia of
the peripheral nervous system, melanocytes of the skin, connective tissues of the face,
smooth muscle cells of the myocardium, neurons of the enteric nervous system, and
neuroendocrine tissue of the adrenal medulla (19). Clinical disorders that arise from
defects in neural crest development include pheochromocytoma, medullary carcinoma of
the thyroid, carcinoid tumors, melanomic progonoma, Hirschsprung’s disease, and neuroblastoma/ganglioneuroma ( 2 ,2 0 ,2 1 ).
Many studies have shown the plasticity of the neural crest progenitor phenotypes; their plasticity provided a model for environmental modulation of phenotype. Molecules such as growth factors, glucocorticoids, transcription factors, and cell surface proteins affect the phenotype of the neural crest cells. Cells of the sympathoadrenal lineage have been studied in detail for their plasticity and developmental characteristics (5,6). The S A progenitor cell has a plasticity between chromaffin character and SA progenitor, as well as a capacity to differentiate instead into a sympathetic neuron ( 6 ). Interestingly, the cells of the neural crest can arise from the same progenitor cells as the cells of the neural tube, indicating some signal must direct their differentiation and their migrating character, to make them specifically cells of the neural crest (18). The diverse products of neural crest progenitor cells makes them an appealing target for study of 1 ) development and 2 )
4 triggers for diversification; prompting the question, “how does an apparently
homogeneous population of nondifferentiated cells become channeled into different
developmental pathways?” (22). In the context of neuroblastoma, a tumor bom of neural
crest progenitors, the question equally applies to which of these mechanisms in the
developmental pathway/s have changed, yielding tumorigenesis.
Molecular evidence for neuropoiesis. How the nervous system develops from a
homogenous mass of cells has become more clear. Studies of defects in nervous system
development have pointed out normal developmental mechanisms. Also key to
understanding neural development, and the idea of neuropoiesis in particular, the
interaction of cell-extrinsic and cell-intrinsic factors have been studied (23).
Recent studies have described a stem cell for neurons and glia as a member of the
mammalian neural crest (22). Reflecting the studies that have established hematopoiesis,
Anderson and Stemple describe this stem cell, a single mammalian neural crest cell as 1)
multipotent, 2) able to generate multipotent progeny, and yet also 3) able to generate
clonal progeny. The theory of neuropoiesis applies to neuroblastoma, if the tumor cells
are considered as a physiologic expansion of precursor cells (24). Manipulation of
growth conditions, changing either culture substrate or media components, and analysis
using cell specific proteins with antibodies supports the above three neuropoietic characteristics of mammalian neural crest cells. Such manipulation has been applied to studies of in vitro neuroblastoma cell lines derived from human tumors. Antibodies to cell specific proteins facilitate separation of neural crest cells and determination of their final phenotype. For example, Anderson and Stemple used the antibody 192Ig, 217c
5 specific to extracellular epitopes of rat low affinity nerve growth factor receptor
(LNGFR) to isolate neural crest cells from 24 hr explants of E 10.5 rat trunk neural tubes
on fibronectin substrate (22). These crest cells also expressed nestin, an intermediate
filament marker common to all neuroepithelial precursors (23).
Antibodies to the extracellular portion of the protein coded by c-RET also have
aided in neural crest investigation; the orphan receptor tyrosine kinase c-RET has proven
to be a marker of postmigratory neural crest cells (3). Early migrating neural crest cells
lack c-RET expression while cells in primordia of autonomic ganglia or enteric
progenitors express c-RET (3,25).
Mutations in the RET gene have been associated with disorders of neural crest
derivatives (20). The protein is expressed in human tumors and cell lines derived from
the neural crest. Mutations that constitutively activate cRET play a role in thyroid
medullary carcinoma, and germ line point mutations in RET cause the dominantly
inherited syndrome of the Multiple Endocrine Neoplasia (MEN) types 2A and B (25).
Mutations of the RET proto oncogene also play a role in Hischsprung's disease (26,27).
Expression of RET has been explored regarding its role in differentiation of neuroblastoma (20). Circumventing the lack of an identified ligand, activated forms of
RET were transfected into SK-N-BE neuroblastoma cells, where the transfected activated
RET decreased proliferation and differentiation both in response to retinoic acid or by
RET transfection alone (20). The RET protein can be exploited as a marker for neural crest development and neural crest derivatives. It plays a role in both proliferation and
6 differentiation in neuroblastoma, as well as being expressed in pheochromocytoma and
thyroid cancer cell lines (28).
Further evidence for control of cell fate comes from genes expressed in
developing neural crest cells. Glial cells missings (gem) acts as a genetic switch in
drosophila that determines glial versus neuronal development (29,30). The novel nuclear
protein from gem was discovered in a mutant drosophila lacking glial cells (30). Studies
of transgenic gem gain-of-function mutants, presumptive neurons develop into glia, while
in loss-of-function mutants, presumptive glia develop into neurons (29,30).
In the drosophila, the achaete-scute complex (AS-C) is a complex of genes having characteristics of basic helix-loop-helix transcription factors. The AS-C has been shown to play a role in the development of sensory organ precursor cells in the peripheral nervous system (23). Because the basic helix-loop-helix family of transcription factors seems to have a major role in orchestrating development, mammalian analogues for the
AS-C were sought using degenerate primers from drosophila AS-C. PCR analysis was run on cDNA isolated from v-myc immortalized adrenal-derived HNK-1+ (MAH) cells. The
MAH cells derive from sympathoadrenal progenitors isolated from rat embryonic adrenal gland (23). With further analysis, including a knockout of MASH 1 in mice, the gene was found to be expressed in precursors of the parasympathetic and enteric neurons (23).
Its expression provides an early marker for autonomic progenitor cells in the embryo.
Interestingly, the function of the transcription factor Mash 1 remains unclear. Anderson notes in a review of neural crest development that “we also need to understand the biological function of Mash 1: does it regulate cell proliferation, migration, adhesion, or
7 aggregation; or does it directly activate a differentiation program?” (23). While the
upstream and downstream targets of the M ashl gene have not been identified, the two
studies of gem and M ash1 indicate 1) neurogenesis depends on molecular mechanisms to
drive diverse development and that 2 ) genes are conserved from invertebrate to
vertebrate neural development.
Homeotic genes, described first from drosophila genetics, play an integral role in choreographing development in organisms ranging from drosophila to humans. HOX homeogene expression has been reported in human neuroblastoma cell lines (24).
Because neuroblastoma cells in vitro may represent some frozen point of neural crest development, Pevarelli et at. examined expression of four HOX genes in five neuroblastoma cell lines; SK-N-BE, CHP-134, IMR-32, LAN-1, and SK-N-SH as untreated tumor cells and during/subsequent to retinoic acid exposure (24). Of the four
HOX genes studied, 3 were expressed in the five neuroblastoma lines. The result is interesting in that the expression of HOX genes was therefore stable, preserved in cultured cells passaged over time.
Oncogenes play a role in neuroblastoma. The proto-oncogene N-myc may be amplified in neuroblastoma, and that amplification correlates with prognosis. The N-Myc protein belongs to the Myc family of oncoproteins, a family of nuclear located proteins containing helix-loop-helix and leucine zipper DNA binding motifs common to transcription factors (31). Analysis of N-myc transcripts in the mouse indicate high levels of expression in embryonic development, with in situ analysis demonstrating N-myc expression at high levels restricted mainly to the CNS and neural crest derivatives (31).
8 In a study of N-Myc protein expression, cytoplasmic N-Myc was discovered. The
presence of cytoplasmic N-Myc protein was common to neurons with large cell bodies:
spinal ganglion neurons of the mouse at day 12.5 embryos, retinal and spinal ganglion
cells of the chicken at days 10 and older (31). These chick cerebellar Purkinje cells
demonstrated cytoplasmic but not nuclear N-Myc protein (31). Purkinje cells become
apparent by day 14 due to their large cell bodies, characteristic dendrites, and staining
profiles to anti-neurofllament stains. Cytoplasmic accumulation of N-Myc protein was
common to differentiated cells that possessed large cell bodies, and paralleled
morphological signs of differentiation and neurofilament expression.
Retinoic acid (RA) has been shown to affect the expression of N-myc in
neuroblastoma SMS-KCNR cells in vitro (32). Incubation with RA caused a decrease in
N-myc message at 48 hours, with a parallel arrest of cell-cycle at that point. The cell line
SMS-KCNR has an amplified N-myc character, which was not affected by RA exposure,
while transcription of N-myc was depressed with RA incubation (32). Amplification of
N-myc in untreated human neuroblastomas correlated with poor outcome when analyzed
with Southern analysis for gene copy number (33). Retinoblastoma has been shown to
have amplified N-myc, however tumor cell lines from other tumor tissues have not been
shown to contain amplified regions of N-myc (33). N-myc amplification is one of the
current prognostic indicators for neuroblastoma because of the correlation of amplified N- myc with poor outcome.
Molecular components of neuroblastoma also include transcription factors. B- myb, a member of the Myb family of transcription factors, has been shown to play a role in the differentiation potential of human neuroblastoma cells (34). Using the retinoic acid
(RA) induced differentiation of neuroblastoma, the authors ablated B-myb expression and studied the effects on LAN-5 and SKNSH neuroblastoma cells (34). To study the regulation of B-myb in vitro, Northern analysis was run on RNA isolated from cells exposed to retinoic acid versus cells cultured without retinoic acid. Transcriptional down-regulation of B-myb was observed in cells incubated with retinoic acid.
Transfection of sense and antisense sequences of B-myb into LAN-5 neuroblastoma cells resulted in a decreased survival in cells transfected with the antisense B-myb, following
G418 selection (34). Transfection and stable expression of B-myb in LAN-5 neuroblastoma cells resulted in insensitivity to retinoic acid induced differentiation (34).
Constitutive expression of B-myb resulted in absence of outgrowing neurites, unchanging high levels of vimentin expression, lack of neurofllament production, and, typical of
Schwann like differentiation, a moderate increase of collagen Type IV expression (34).
Like N-myc, B-myb downregulation precedes morphological differentiation. Further experiments analyzing the effects of Trk receptor expression in neuroblastoma tie to transcriptional regulators such as B-myb, in particular in the effects transduced by TrkB, which are speculated by Rashcella et al. to perhaps involve B-myb (34).
Another Myb family member is c-myb. This transcription factor has been shown to be down-regulated with differentiation of both hematopoietic and neuroblastoma cells
(34). In neuroblastoma, the transcriptional downregulation of c-myb is an early event in differentiation. Furthermore, c-myb has been shown to inhibit proliferation of neuroblastoma and neuro-epithelioma cells (34).
10 The mammalian developing nervous system has long been observed to over
produce neurons. Widespread cell death occurs as development proceeds, with 50% of
the neurons created during neurogenesis dying by apoptosis (35). This restructuring and
refining of neuronal connections involves recently discovered orchestration of growth
factors, transcription factors, and receptors that integrate signals from the cell surface to
the nucleus of a developing neuron.
Molecular cues for apoptosis have been shown to be from the c-Jun and Fos
families of transcription factors. Using antibodies to c-Jun and Fos, Ham et al. studied expression of the transcription factors during apoptotic events in rat superior cervical ganglion cells (35). The superior cervical ganglion (SCG) model has yielded details about sympathetic development and dependence on NGF for viability. All SCG rat neurons are postmitotic at birth, with 35% of these dying between postnatal days 3 and 7.
Supplemented with NGF, SCG neurons harvested and cultured from postnatal day 1 can survive for a period of time. With removal of NGF, the neurons die by apoptosis within three days. Following the removal of NGF, an increase in c-Jun was observed. The protein was found to be more phosphorylated as well. The increased phosphorylation was hypothesized to be on the amino terminal transactivating domain which facilitates c-Jun mediated regulation of target gene transcription. Blocking this protein’s action with a dominant negative prevented apoptosis while overexpression of c-Jun induced apoptosis in the SCG system (35).
Cultured rat sympathetic neurons have been used to study the prevention of programmed cell death by the bcl-2 proto-oncogene (36). An expression vector
11 consisting of 1 . 8 kb of 5’ flanking DNA to the rat neuron specific enolase promoter
linked to DNA encoding human bcl-2 was microinjected into rat sympathetic neuron
nuclei (36). Species specific antibodies targeted expression of the human bcl-2. With
removal of NGF, the bcl-2 upregulated cells survived. Cells injected with control vector
lacking human bcl - 2 as well as un-injected cells demonstrated “phase-dark cell body and
neurite disintegration" within 48 hours of NGF deprivation (36). Cells injected with the
bcl-2 construct survived 7 days without NGF; decreasing by day 10 to 20% perhaps due
to lower expression of the bcl-2 construct with time (36). Rescue of these neurons with
NGF supplementation has been shown. The neurons responded to the NGF and survived
a further 12 days, indicating that receptors for NGF persisted in the absence of the growth
factor. The gene bcl-2 thus most likely plays a mediating role in trophic dependence of
sympathetic neurons and other dependent neurons.
Whether bcl-2 plays a role in unchecked growth and metastasis of neuroblastoma has been studied. The expression of protein coded by bcl-2 was determined by immunocytochemical localization using a monoclonal anti-bcl-2 antibody (37). Positive bcl-2 protein expression correlated with unfavorable histology (P — 0.002) and with N- myc gene amplification (P = 0.002) (37). A member of the bcl-2 gene family, bcl-x, has also been studied in neuroblastoma(38). bcl-x has been shown to be highly expressed in neural tissue; its gene products Bcl-xL and Bcl-xS as well as the Bcl-2 protein were screened for expression in 27 NB cell lines by quantitative immunoprecipitation (38).
Bcl-xL and Bcl-2 were expressed in 24 and 21 of the 27 cell lines, respectively; while none expressed Bcl-xS (38). Expression of Bcl-2 was restricted to neuroblastoma cells
12 of chromaffin lineage, while Bcl-xL was expressed in chromaffin and nonchromaffin
derived NB lines (38). Both Bcl-xL and Bcl-2 have been shown to protect NB cells from
chemotherapy induced apoptosis (38,39).
These molecular studies offer possible therapeutic interventions in neuroblastoma.
Downregulation of bcl-2, bcl-xL, or N-myc via antisense oligos or upregulation of c-Jun
could affect neuroblastoma cells' tumorigenic potential. Research studying the molecular
mechanisms of neural crest development offers insight into neuroblastoma; yet many
questions about molecular interactions between transcription factors, oncogenes, growth
factors/receptors, and neuropeptides/receptors remain unanswered.
Growth Factors, Neuropeptides, and Their Receptors. At the cellular level, growth factors such as neurotrophins, glial growth factors, neuropeptides, and cytokines have been implicated in uncommitted progenitor cell development. The interaction of these diverse peptides on neuropoiesis is becoming more clear. Using the PC 12 cell line, a cell line of adrenal medullary origin, Stemple et al. showed that addition of basic FGF induced neuronal differentiation and dependence on NGF (40). Shah et al. report that glial growth factor (GGF) strongly suppressed neuronal differentiation of rat neural crest stem cells while promoting or allowing glial differentiation (19). GGF has been defined as a Schwann cell mitogen, a growth factor for the supporting cells of the peripheral nervous system. GGF is a member of a family of ligands in the epidermal growth factor/transforming growth factor a (TGFa) superfamily for the receptor tyrosine kinases pl85eibB2/HER22/c-Neu and p!80erbB4/HER4 (41,42). GGF was found to affect lineage
13 determination of stem cells by suppressing neuronal differentiation while promoting glial
differentiation (19).
Shah et al. applied the in vitro clonogenic assay system described by Anderson
and Stemple (22) to a study of GGF effects on multipotent neural crest cells. An antibody
to the intermediate neuronal filament, peripherin, selectively marks neuronal derivatives
while an antibody to the intermediate glial specific filament, glial fibrillary acidic protein
(GFAP), labels glial derivatives. Differentiation of rat neural crest cells in vitro occurs
within two weeks of addition of GGF. Incubation of neural crest cultures in recombinant
hGGF2 (GGF2 is a soluble form of GGF) resulted in striking differences in the final cell
populations. GGF suppressed neuronal differentiation and facilitated glial differentiation;
thus GGF appeared to be acting as an environmental factor that plays an instructive role
on multipotent cell lineage (19). Interestingly, the studies did not define GGF as a
mitogenic factor in vitro. The cells incubated in the presence of hrGGF2 displayed
growth similar to the control cells. Preliminary studies of other Schwann cell mitogens
including TGFp I and J52, platelet-derived growth factor BB and fibroblast growth factor,
and members of the TGFa family did not show similar effects on neural crest populations
as GGF (19).
Growth factors have also been implicated later in sympathetic neuron
development. Following primitive ganglia formation of chick sympathetic neurons, the
growth factors IGF-1 and IGF-2 play a role in sympathetic neuron proliferation (43). The
cytokine ciliary neurotrophic factor (CNTF) has multiple effects on sympathetic neuron development; including induction of differentiation, inhibition of division in postmitotic
14 sympathetic neurons; induction of choline acetyltransferase (CHAT) expression, and
inhibition of tyrosine hydroxylase expression ( 1 0 ).
The group of four structurally related homodimeric molecules termed
neurotrophins include nerve growth factor (NGF), brain derived neurotrophic factor
(BDNF), neurotrophin 3 (NT-3), neurotrophin 4/5 (NT-4/5). All four play a role in neural
development, and act on two groups of cell surface receptors: 1) p75 binds all four
neurotrophins with a low affinity (K d of 10'9 nM) and 2) the tyrosine kinase group of
TrkA, TrkB, and TrkC which have varying affinities to the neurotrophins (44). Verdi and
Anderson studied the expression of two NGF receptor genes, trkA and p75 in thoracolumbar sympathetic ganglia neuroblasts (44). They linked expression of the receptors to the presence of both NGF and NT-3
Neurotrophins and their receptors have been studied in neuroblastoma. A study by Brodeur et al. of tumor samples from 77 patients showed a positive correlation between trk-A expression and favorable tumor stage (I, n, and IVS vs. Ill and IV), and an inverse correlation between amplification of the N-myc proto-oncogene (45).
Transfection of trk-A into human neuroblastoma cells induces sensitivity to NGF; the cells differentiate in response to NGF following trk-A upregulation (46). Brodeur 1 s study showed good prognosis neuroblastoma tumors express high levels of trk-A mRNA; while Nakagawara et al. report an inverse correlation between trk expression and N-myc amplification (47). Matsumoto et al. conducted experiments that showed activation of the pl45TrkB signal transduction pathway facilitated neuroblastoma cell survival, disaggregation, and invasion; three characteristics of metastatic cells ( 8 ). BDNF
15 promotes cel] survival and neurite outgrowth in SMS-KCN cells; a neuroblastoma cell line with N-myc amplification that expresses both pl45TrfcB and BDNF (12).
The functional coupling of nerve growth factors to their receptors is inconsistent in neuroblastoma cell lines. The SMS-KCN cell line offers a functional in vitro system for studying BDNF-pl45T,kB interaction. For example, Nakagawara et al. demonstrated that the downstream signaling for pl45TrkB is intact in SMS-KCN cells (12). After 1 minute of incubation with BDNF, PI-3K is phosphorylated. After 5 minutes PLC-yl,
ERK-1, and ERK2 are phosphorylated, and the immediate early genes c-FOS and NGFI-
A are induced (12). SMS-KCN cells express Trk-A, however the TRK-A/NGF pathways are not complete in SMS-KCN cells. NGF induced phosphorylation of pi40rjMM occurs however phosphorylation of ERK-1 and ERK-2, as well as induction of c-FOS and NGF-
1A do not occur (12). Such deficient NGF signaling has been demonstrated in neuroblastoma cell lines, and may explain the lack of morphological effects by NGF on incubation with neuroblastoma cells (48-51).
Expression of both BDNF and pl45TtkB in primary neuroblastomas has been analyzed. BDNF was detected by Northern analysis in 50 of 74 primary neuroblastomas with expression in 79% of stage IE/TV neuroblastomas, in five of five mature ganglioneuromas, and in 50% of stage I, II, or IV-S neuroblastomas; showing an increased frequency in more advanced stage neuroblastomas (12). Northern analysis of trk-B expression detected positive expression in 27 of 74 (36%) primary neuroblastomas; however due to different forms of mRNA, the expression proved complex (12). The full length messenger RNA for human trk-B, extrapolated from rat and mouse studies (52,53),
16 is encoded by 9.5 kb and 4.5 kb transcripts. The truncated message at 8.0 kb codes a
form of the receptor that lacks the tyrosine kinase domain (12). Full length Trk-B was
expressed more highly in immature neuroblastomas: with positive expression in 7 of 10
with N-myc amplification, but positive expression in only 2 of 64 with no N-myc
amplification. The truncated, 8.0 kb, message for trk-B was found in 18 of 64
neuroblastomas without N-myc amplification (12).
Neuropeptides and their receptors have been studied in neural crest development
and have been implicated in neuroblastoma. Neuropeptides, including neuropeptide Y
(NPY), vasoactive intestinal peptide (VIP) and somatostatin (SS) are thought to play a
role in neural crest differentiation. NPY, a 36 amino acid peptide originally isolated from
porcine brain, is expressed only in the peripheral and central nervous systems, and can be
detected in plasma of patients with tumors of neuroectodermal origin (54). Cohen et al.
studied by in situ hybridization the expression of NPY in normal human adrenal gland
through development, as well as in 38 neuroblastoma tumor tissues (54). Their studies
found a biphasic expression of NPY in the human adrenal, over an age span of 7.5 weeks
to 5 years. The earliest samples through 18 weeks gestation had positive expression of
NPY. However in 18 samples that were greater than 18 weeks gestation, no expression
was detectable in the adrenal medulla until 8 months following birth. Tyrrell and Landis
recently studied NPY and VIP in embryonic neurons, where restriction of these peptides’
expression plays a role in the fmal neuropeptide profile of sympathetic neurons (55).
Both peptides were first detected in dividing neuroblasts as well as in postmitotic neurons of the rat superior cervical and stellate ganglia (55). Neuropeptide expression has also
17 been shown to be affected by CNTF; the cytokine alters expression of the neuropeptides
in increasing VIP, SS, and substance P expression while decreasing NPY expression (10).
Vasoactive intestinal peptide (VIP) is a 28 amino acid peptide, originally isolated
from porcine intestine; its gene was cloned from a human neuroblastoma cell line (15).
The peptide exerts its effects by binding to high affinity cell surface receptors; activating
adenylate cyclase and protein kinase A (15). The role of VIP in neural crest development
and in neuroblastoma has been studied (14,15,56,57). VIP acts as a neurotransmitter or
neuromodulator throughout the central and peripheral nervous systems (14); it has been
shown to increase developing neurons’ survival through a survival promoting activity as
well as acting as an astroglial mitogen (56). VIP has also been shown to induce tyrosine hydroxylase in PCI2 cells, cells derived from a rat adrenal chromaffin cell tumor (58).
In the SKNSH and SHSY5Y neuroblastoma cell lines, VIP promotes morphological differentiation, inhibits growth, and potentiates RA induced transglutaminase activity which is associated with differentiation in neuroblastoma (14,57). Recent work by Dr.
O’Dorisio has shown that neuroblastoma tumors express receptors for VIP with variable synthesis of the peptide, further indicating that there is an autocrine regulation of VIP and its receptor in neuroblastoma (15).
The neuropeptide somatostatin (SS) and receptors for somatostatin (sst) may play a role in cerebellar development. They have been demonstrated in immature rat (59) and human cerebellum (60,61). Gonzalez et al. describe high levels of SS in the rat cerebellum for 2 weeks postnatally, while very low levels are detected in the adult cerebellum by radioimmunoassay (RIA) (59). Immunohistochemical studies of SS levels
18 demonstrate positive fibers from birth to postnatal day 5 (P5) in the rat cerebellum (59).
At the cellular level, the immunoreactivity shows no positivity in the cerebellar molecular layer. However, positive staining fibers innervate that layer. In situ studies indicate that cerebellar neurons express the SS gene from P5 to P10. The neurons expressing SS are most likely the Golgi and Purlcinje cells, located in the granule cell layer and the Purkinje cell layers. Experiments performed in vitro on cultured 8 -day-old rat cells demonstrated potent inhibition of forskolin-stimulated adenylate cyclase by somatostatin.
Granule cells of the cerebellum are neurons that are still dividing postnatally.
Studies indicate that receptors for the neuropeptide somatostatin are expressed on these neuroblasts (59-61). Little to no expression has been demonstrated in the adult rat (61).
A culture of immature granule cells from rat cerebellum was studied for binding to somatostatin, for cAMP formation, and for intracellular calcium concentration^ 1). The cells demonstrated saturation and competitive binding by a single class of high affinity binding sites (Kd=0.133 ± 0.013 nM, Bmax=3038 ±217 sites per cell). Forskolin- stimulated cAMP accumulation was inhibited by somatostatin in the granule cell cultures
(61). Somatostatin incubation caused a decrease in intracellular calcium concentration
(61). Further studies are necessary to determine the effects of somatostatin as a regulator of proliferation, migration, or differentiation in the immature granule cells.
A study of somatostatin receptor expression in the human cerebellum (60) supports the rat granule cell study. The concentration of sst in cerebellum tissue from fetuses and infants younger than 8 months was 2 - 1 0 fold higher than in adult cerebellum
(60). The external granule cell layer of the cerebellum (EGL) demonstrated a high
19 concentration of sst using brain slice binding of [l 2 3 I-tyr°, Dtrp 8 ]SS | 4 (5!). The internal
granule cell layer (IGL) and molecular layer (ML) expressed sst also. However the ML
demonstrated [l 2 5 I-tyr°, Dtrp*]SSi 4 binding only if the EGL remained. No labeling was
detected in the deep cerebellar nuclei.
Studies of neural crest development have provided clues for what to study in
neuroblastoma and vice versa. In the area of neuropeptide research in nervous system
development, the studies of somatostatin and its receptors have yielded not only
information about nervous system development but techniques for improved diagnostics
and therapy for neuroblastoma. High affinity analogues for the somatostatin peptide have
provided means for growth inhibition and peptide management of some tumors, as well
as effective means for imaging (by radiolabeling the tyrosinated analogue with various
isotopes of iodine) in a wide range of tumors, including neuroblastoma. These clinical advances and applications preceded intricate knowledge of receptor expression, function, and physiologic regulation. Further analysis of somatostatin receptor subtypes is essential to effective design and use of somatostatin analogues or receptor based gene therapy.
Somatostatin, or Somatotropin Release Inhibiting Factor (SRIF), was cloned in the form of preprosomatostatin I using a cDNA library constructed from human pancreatic somatostatinoma mRNA (62). The neuropeptide somatostatin regulates a wide range of cellular activities in the nervous system, gut, and skeletal system. Somatostatin inhibits the release of insulin, glucagon, gastrin, secretin, thyrotropin-stimulating hormone, and somatostatin itself from exocrine secretion (63). Two bioactive forms of somatostatin occur: SS | 4 and the NH 2 -terminally extended SS 2 g. Proteolytic, tissue-
20 specific processing of the 92 amino acid precursor prosomatostatin yields either the 14 or
28 amino acid forms of somatostatin (64).
The effects of somatostatin are translated through cell surface receptors; with somatostatin inhibiting hormone secretion and/or acting as a neurotransmitter. The cloning of human sst), sst 2 , and sstj was first reported as part of a project to characterize proteins expressed in the insulin producing P islet cells of the pancreas, particularly proteins such as somatostatin receptors that may be involved in regulation of insulin secretion (64). Five receptor subtypes for somatostatin have been cloned in the mouse and human (65-69). The cloned receptor subtypes have been studied for their binding characteristics to somatostatin and its analogs, as well as for the second messenger systems activated (70-73). Species specific tissue expression may reflect different pathways and functions of the five receptor subtypes in their transduction of somatostatin's effects. These effects include regulating neuronal firing, modulating transmitter release, inhibiting pancreatic endocrine/exocrine secretions, and inhibiting cell growth in tumor cells (70,74).
All five receptor genes are members of the G-protein linked receptor family, with
7 transmembrane domains, an extracellular NH 2 terminus and an intracellular COOH terminus. The five receptor genes have an overall 39-57% sequence identity; they diverge most in sequence identity in the NH 2 and COOH regions, with the most sequence identity in the transmembrane domains (55-70% identity) (75). Within the G-protein linked receptor family is a group of receptors called the DRY- family because of a common sequence of Asp-Arg-Tyr (DRY) that occurs at the boundary of the third
21 transmembrane domain and second intracellular loop (76). Somatostatin receptors are members of this DRY family, whose ligands include neurotransmitters, neuropeptides, glycoprotein hormones, and olfactory molecules (76). Using fluorescence in situ hybridization, human metaphase chromosomes have been probed to localize the hsst] , hsst 2 , and hsst 3 to chromosomes 14, 17, and 22 (77). The chromosomal locations of ssu and sstj are 20 and 16, respectively (75). The characteristics of the five human somatostatin receptors, including their binding affinities to the native SSu and SS 2 g are summarized in Table 2.1.
22 Subtype Chromosomal Amino Affinity Affinity GenBank™ References Location Acids s s „ SS, Accession (nM) (nM) #
set, 14 391 0.1 0.1 M81829 (64,70,77,78)
17 369 0.3 0.4 M81830 (64,70,77,78)
set, 22 418 0.1 0.1 M96738 (66,70,77,78)
L07061 •81, 20 366 1 2 L07833 (67-69,71,78,79) D16826 L14856
SSt, 16 364 2 0.05 D 16827 (69,71,78)
Table 2.1 Nomenclature for Human Somatostatin Receptor Subtypes - (sst/sst5 )
Nuclease protection assays, in situ experiments, and RT-FCR analysis of the sst expression have resulted in detailed expression maps of sst in mouse and rat. Patel et al. summarize the receptor expression characteristics in rat tissues in their review of the 5 sst
(75). Maps of sst expression in human are not as detailed as those for mouse and rat.
However, human neuroendocrine tumors express a high density of sst and have been studied in depth for use in receptor based imaging and therapy for these tumors.
23 SSti sst2 sst3 sst4 sst5 Cortex + M + M +R +H +H +R +R Amygdala + M + M +R +R +R +H +R Hippocampus + M +R +R +R + r (CA1) + R Hypothalamus + M + M +R +H +n +R +n Pretectum + M Substantia nigra + M + M Cerebellum + M +R +R
Table 2.2 Expression of sst] through ssts in Mousem and RatR Brain. Positive tissues are indicated, and data compiled from references (63,75,80-82).
The neuropeptide somatostatin modulates neuronal and neuroendocrine function, making the brain a target for studies of somatostatin receptor expression. Expression of sst in the brain is summarized in Table 2.2. In the brain somatostatin has been shown to act as a neurotransmitter or neuromodulator in affecting locomotor activity, cognitive functions, and behavioral processes. It regulates the release of dopamine, 5-HT, and excitatory amino acids (63). In situ hybridization was used for studies of mouse brain by
Breder et al., mapping sst) expression to the supra- and infragranular layers of the cortex, the amygdala, hippocampus, bed nucleus of the stria terminalis, substantia innominata, hypothalamus, pretectum, substantia nigra, parabrachial nucleus and nucleus of the solitary tract (80). The same group mapped sst 2 mRNA to mouse infragranular layers of
24 the cortex, the amygdala, claustrum, endopiriform nucleus, arcuate and paraventricular
nuclei of the hypothalamus, and medial habenular nucleus (80). Expression studies by
Kaupmann et al. on ssti-ssu demonstrated all four subtypes in mouse hippocampus and cortex; with sst 2 also demonstrated in the spinal cord, substantia nigra, and striatum (82).
Kaupmann et al. demonstrated that receptor subtype 3 was positive by Northern analysis in mouse olfactory bulb and cerebellum (82). Interestingly, in the cerebellum binding sites for somatostatin have been demonstrated only in immature cerebellar granular cells of the rat and human but not in mature cerebellar cells (61), yet mRNA for sst3 is abundant by in situ hybridization in mature mouse cerebellum (82). The sst 3 protein is unique among the 5 receptors in its intracellular carboxy terminus, with 13 of
16 amino acids as glutamic acid in mouse sst 3 and 9 of 12 amino acids as glutamic acid in human sst 3 (76). The discrepancy between in situ findings (+) for mRNA for sst 3 and binding analysis (-) for sst 3 may be explained by either translational inhibition of the mRNA or by a receptor function conferred by the intracellular carboxy terminus that does not facilitate or act via binding; or by a high turnover of the mRNA for this receptor subtype in cerebellum. However reports of expression corroborate the finding of predominantly sst 3 expression in cerebellum of mouse and rat (63,75,81,82).
Expression of sst in rat mesencephalon has also been demonstrated by Kaupmann et al.. Using oligo probes specific for ssti - ssU, in situ hybridization localized mRNA expression. High expression of sst] localized to layers V-VI of the cerebral cortex, in the granular layers of the dentate gyrus, and in the amygdala (82). Hybridization signal also demonstrated sst 2 in the frontal cerebral cortex (layers IV, V, and VI), in the CA1 area of
25 the hippocampus, in the central grey, in the granular cell layer of the dentate gyrus and in
the amygdala (82). Expression of sst 3 was found in the amygdala, the granular layer of the dentate gyrus, and in the red nucleus (82). Strongest expression of ssu was found in
the pyramidal layer and the CA1 area of the hippocampus (82).
Bruno et al. have studied sst mRNA expression in the rat, analyzing all 5 receptor subtypes by nuclease protection assay (81). Their studies considered both the central nervous system and peripheral tissues. Expression of ssti and ssU mRNA was highest in the amygdala, cortex, hypothalamus, and hippocampus; and measurable in striatum, midbrain, thalamus, cerebellum, and spinal cord. The highest expression of sst 3 was in the cerebellum, with low level expression of sst 3 in the remaining brain areas studied.
Highest levels of ssu expression were mapped to hippocampus, amygdala, olfactory bulb, cortex and preoptic area, with low levels in remaining areas, and no expression of ssu in the cerebellum. Bito et al. analyzed rat brain by in situ hybridization for expression of ssu and found expression in hippocampus (primarily CA1 region), dentate gyrus, lateral habenula, and neocortex (83). They demonstrated moderate expression of ssU in striatum, amygdala, and pyriform cortex, and no expression in cerebellum (83). Unique to the 5 receptor subtypes, sst 5 was expressed strongly in the hypothalamus and preoptic area, with low expression in the other areas studied, including striatum and olfactory bulb
(81).
Somatostatin receptor expression in the CNS and PNS reflects the wide range of effects that somatostatin has in the nervous system. For example, somatostatin as a neurotransmitter has been shown to facilitate release of serotonin from the hypothalamus,
26 (73). It has been shown to augment the K+ stimulated release of acetylcholine from rat
hippocampal slices (73). Widespread CNS and PNS sst expression supports the idea that
somatostatin plays a role in motor activity, cognition, pain transmission, and peripherally
mediated feeding behaviors (73).
SSti SSt2 SSt3 SSt4 ssts Pituitary + + + + + Lung + + Heart + + + Liver + + + Pancreas + + + + Adrenal + + + + Small + + + + intestine Cecum + Stomach + + + + Kidney + + + Spleen + + + + +
Table 2.3 Expression o f sst i through ssts in rat peripheral tissues. Summary of data of strongly positive tissues as reported in (63,75,81).
Peripheral expression of sst is summarized in Table 2.3. Applying their nuclease protection assays to peripheral tissues, Bruno et al. demonstrated that pituitary expressed sstt - sst5 in an order of sst 2 > ssti = sst3 > ssts > ssu. Expression in spleen was ordered as sst3 > ssti = ssU = ssts > sst2. Stomach was positive for sst] through ssu at equal intensity while demonstrating low expression of ssts. Small intestine was positive for ssti and sst 5 most strongly, with equal expression of sst 3 and ssU and no expression of sst2.
27 Heart demonstrated expression of ssu predominantly, in addition to expression of sstt and
sst3 . Kidney expressed sst 3 and ssU- Liver demonstrated expression of sst 3 only and
pancreas sst 2 only (81).
The messenger RNAs of all 5 sst have also been studied in adult rat CNS and
peripheral tissue by RT-PCR analysis combined with in situ hybridization histochemistry
(63). Raulf et al. described a distinct but overlapping expression of sst] through sst;
mRNA that to a degree is expected because of the diverse effects of somatostatin peptide
in different tissues (63). Their studies demonstrated all 5 genes in total brain, brain
cortex, and pituitary, sst] was expressed in liver, ovary, and in trace amounts lung,
pancreas, stomach, cecum, kidney, heart, muscle, and testes. sst 2 expression was limited
to adrenals and pancreas, and kidney with “barely detectable” expression in the retina,
stomach, small intestine, and cecum (63). sst 3 was expressed in stomach, pancreas, liver,
muscle, spleen, lymph nodes, and, faintly, in lung and prostate (63). Low expression of
ssu in the periphery demonstrated positivity in only lung, with little expression in liver.
Expression of sst; occurred in adrenals, liver, pancreas, cecum, and less so in retina,
spleen, kidney, stomach, small intestine, and testes (63). These results conflict with the
results from the nuclease protection analysis. More positive expression was detected in
reports from RT-PCR and in situ analysis, indicating that this is a more sensitive
technique for detecting sst gene expression. Strain differences between rats and transcriptional regulation may also be factors in expression analysis of sst.
Yamada et al. reported expression of sst] and sst 2 in the human by RNA blotting
(64). Human sst| mRNA was 4.8 Kb in size and was expressed in stomach, jejunum and
28 human islet cells of the pancreas while low levels were detected in colon, colon
carcinoma, and kidney and no levels detected in human brain (64). Two transcripts of
sst^ mRNA were detected at 8.5 kb and 2.5 kb with highest expression in human cerebrum and kidney. Jejunum, hepatoma, colon, colon carcinoma, and liver were found
to have low levels of ssl 2 expression (64). Their sst 2 probe also detected sst 2 expression
in rat pancreatic islet cells, indicating both ssti and sst 2 are expressed in pancreatic islet cells. A consideration in comparing species expression of sst is that human studies have been from abnormal samples, rather than from controlled samples such as rat and mouse tissues isolated specifically for research purposes. Alternatively, one must consider the possibility that there is tissue and species variability of sst expression (84).
Human expression of sst has been analyzed and shown predominantly in tumor tissues. Cell lines derived from human tumors as well as tumor samples have been analyzed for sst expression by membrane binding, RT-PCR analysis, in situ hybridization histochemistry, and in vivo and in vitro autoradiography.
Somatostatin receptors have been demonstrated in tumors of neural crest origin such as neuroblastoma (13,85,86). Somatostatin receptor (sst) expression has been demonstrated in neuroblastoma tumor tissue by several techniques, including scintigraphy (87), competitive binding on cell membranes ( 8 6 ), ex vivo binding and autoradiography (85) and in vivo radioreceptor guided surgery ( 8 8 ). In a study of l 2 5 I-
[tyr^J-octreotide-based autoradiographic analysis of 30 neuroblastoma tumors, Moertel et al. demonstrated a positive correlation between somatostatin receptor expression detected by ex vivo binding and autoradiography and a favorable prognosis, and an
29 inverse correlation between somatostatin receptors and N-myc amplification (85). Chen et al. performed competitive binding studies on preserved tumor tissues and demonstrated much higher somatostatin receptor expression in Stage I and II tumors than in Stage III or
Stage IV tumors (13). Neuroblastoma tumor samples were analyzed by extracting the tissues' membrane fraction and determining if high affinity binding sites for somatostatin were present. Competitive binding curves between l 2 1 I-tyr'-octreotide and 1 2 5 I-SSi4 and unlabeled SS | 4 were generated and the Kd determined for various membrane preparations. The binding data was correlated with clinical staging data to generate the observations that tumors with a favorable Evans stage classification demonstrated higher expression of somatostatin receptors.
Analyzing membrane preparations for binding affinity, O’Byme et al. demonstrated sst expression in six of nine small cell lung cancer (SCLC) cell lines as well as in all 5 of the following tumor samples: from 3 patients with non-small cell lung cancer (NSCLC), one patient with an atypical carcinoid, and one patient with a bronchial carcinoid (89). Reubi et al. analyzed multiple tumor types for sst protein using in vitro autoradiography and for sst gene expression using in situ hybridization histochemistry.
Their results correlated positive in vitro autoradiography primarily with expression of sst 2 by in situ hybridization; positive receptor expression was found in pituitary tumors, meningiomas, neuroblastoma, gastroenteropancreatic (GEP) tumors, breast tumors, lymphomas, medullary thyroid carcinoma, SCLC, ovarian, and renal cell cancer (90).
Feindt et al. studied the expression of ssti, sst 2 , sst3, ssL, and sst 3 on normal astrocytes, glial cell lines and gliomas using RT-PCR and membrane binding to a
30 somatostatin-gold conjugate (91). Overall, sst 2 had the most expression according to the reported data, with positive sst 2 cDNA in astrocytes from both rat and human, in 6 / 6 glioma cell lines (one rat and five human), and in 6 / 6 human gliomas (91). Also positive, though less than sst2, were ssti and ssu in both rat and human astrocytes, 5 of 6 glioma cell lines, and 3 of 6 human gliomas (91).
Somatostatin receptors have been demonstrated on pituitary tumors (92). Using
RNase protection assay the expression of ssti and sst 2 were correlated with tumor characteristics. Four of nine nonfunctioning pituitary tumors had neither ssti or sst 2 expression, four had expression of sst 2 , and one patient had expression of both ssti and sst2. Nine of ten acromegalic patients had positive expression of sst2. Three of seven
GH-secreting tumors were positive for ssti expression. Four of five prolactinomas were positive for sst| expression, while none were positive for sst2. One tumor was a CLL infiltrate into the pituitary and tested positive for sst 2 (92). This study considered only subtypes 1 and 2 , yet demonstrated a heterogeneity of receptor expression, reflecting the different effects somatostatin may have, acting as neurotransmitter or hormone.
RT-PCR was used by Kubota et al. to demonstrate sst in human endocrine tumors.
They examined sst|, sst2, sst 3 , ssU. and ssts in two glucagonomas and metastatic lymph nodes, four insulinomas, normal adrenal gland, three pheochromocytomas, and one carcinoid (84). All tumor samples analyzed expressed ssti mRNA. Only one insulinoma and the carcinoid tumor lacked sst 2 expression, making sst 2 positivity 85%. Expression of sst 3 was 38%, demonstrated in the glucagonoma and metastases, in one insulinoma
(the same tumor that expressed sst2), and in insulinoma # 4 (which was (-) for sst2).
31 Expression of ssu was 69%. Normal adrenal gland and 3 pheochromocytomas did not
express ssu. No tumor sample or normal adrenal gland was positive for ssu expression
(84). The analysis demonstrated the same expression pattern of sst between normal
adrenal gland and the 3 pheochromocytomas studied.
Human breast carcinoma, carcinoid tumor, and renal cell carcinoma have been
studied for ssti, sst 2 , sst3, and ssu expression using RT-PCR (93). These studies found
sst2 in all but one sample of 55 malignant and nonmalignant breast tissues. Positive
expression of sst| was 73% in the same tissues. Expression of sst 3 was 72% positive in
the breast tissues, however sst 3 expression was not analyzed in two of the breast samples.
Expression of ssu was 35% positive in the breast malignant and nonmalignant tissues. Of
28 human carcinoid and renal cell tumors, sst 2 was positive in 96% of tissues analyzed.
Expression of ssti was positive in 8 6 % of tumors studied, sst 3 positive in only 28%, and
ssu positive in 43% of tumors studied (93). No sst 3 was detected in renal cell carcinoma
only. The probability of sst expression (P) in the tumor types studied was ranked
uniquely for each tumor: for breast cancer P2 > P3 = PI > P4; for carcinoid tumor P2 >
PI > P3 = P4; and for renal cell carcinoma P2 > PI > P4 > P3 (93).
Semiquantitative RT-PCR has demonstrated sst expression in neuroendocrine
gastroenteropancreatic tumors. John et al. studied various tumors and found sst 2 at high levels in 13/13 tissues studied (94). Expression of ssti was found in 11/13 tissues, ssts expression in 7/13, ssu in 7/13, and sst 3 at very low levels in 6/13 samples (94). Any isolation of RNA for analysis from tumor also includes supporting tissue and blood in the tissue homogenate. Thus results of receptor expression analysis must consider the variety
32 of cells included in addition to tumor cells in the total RNA yield. Autoradiographic
analysis of tissue slices demonstrated receptor-ligand interaction predominantly on tumor
cells, indicating receptor expression reflects tumor cells and not contaminating cell
populations.
Three basic areas of research must be addressed to understand the biology of
somatostatin receptors: 1 ) mapping somatostatin receptor expression in normal and
tumor tissues: 2 ) identifying specific signal transduction mechanisms for each receptor
subtype; and 3) determining ligand binding characteristics for the sst. Through studies of mouse and rat normal tissue and extensive studies of human tumor tissue, sst expression maps are becoming more complete. The five cloned receptor subtypes are expressed in brain, and are also variably expressed in the gastrointestinal system, heart, lung, and tumors of neural crest and neuroendocrine origin. A second characteristic of these receptors is that they transduce ligand effects within the cell via second messenger systems that may be receptor and or cell specific. Third, the ligand binding characteristics of the five receptors varies, offering a means for receptor based imaging and/or modulation of physiologic response.
Defining the roles of the somatostatin receptor family members will guide the development of effective somatostatin analogue therapy (95). Somatostatin binding to its various receptors results in decreases in cAMP accumulation, regulation of ion channels and exchangers and stimulation of tyrosine phosphatase activity in cells. Second messenger systems affected by sst include inhibition of adenylyl cyclase, decreased conductance of voltage-dependent Ca++ channels, stimulation of K* channels, stimulation
33 of tyrosine phosphatase, inhibition of Na+/H+ exchanger, activation of cyclic GMP
dependent protein kinases, stimulation of inositol 1,4,5 triphosphate formation, and
activation of phospholipase C, among other actions (96).
Functional studies of G proteins have demonstrated that endogenous somatostatin
receptors couple to Gjai to decrease adenylyl cyclase activity (97), to G i a 3 to stimulate K+
channels, and G ^ to inhibit Ca+ channels (98). In pancreatic HIT-T15 cells,
somatostatin inhibits glucose-stimulated insulin release via a pertussis toxin sensitive G-
protein coupling. HIT-T15 cells are a clonal cell line transformed by SV40 and derived
from Syrian hamster pancreatic (3 cells. Using anti sera to specific COOH-termini of the
six G proteins expressed in HIT-T15 cells, Seaquist et al. demonstrated that the
somatostatin receptor/s were coupling to Goa proteins (99). The antisera were targeted to
the receptor-ligand-coupling sequence of the COOH-terminus of the G,ai, Gja 2 . G 1(13 ,
Goa 2 , and an additional two forms of Goa. Successful coupling of the receptor-Iigand
complex to G-proteins is reflected in GTPase activity, the index used to determine
antisera blocking of somatostatin induced G-protein activation (99). Maximal GTPase
stimulation by somatostatin occurred at somatostatin concentrations of 100 nM.
Preincubation of cell membrane preparations with antisera either directly or cross reactive
to Goa prevented the GTPase activity subsequent to somatostatin exposure. Because not
all GTPase activity was eliminated with the Goa antisera, some interaction between G,«
and somatostatin receptors must occur (99). Unfortunately the authors of these studies did not determine the sst profile of the HIT-T15 cells. The G-protein profile indicates
34 that somatostatin receptors couple to different G protein subunits as a ligand receptor
complex, however which receptor subtypes are involved remains to be determined.
All five receptor subtypes have been shown to effect a decrease in adenylyl
cyclase activity via a pertussis toxin sensitive coupling to G proteins (78). Initial reports
indicated that not all 5 could cause a decrease in cAMP accumulation, however cellular
components such as G-proteins play a role in this coupling. Characterization of the
receptor subtypes has been conducted by expressing the receptor genes both transiently
and stably in numerous cell lines such as COS-1, CHO-DG44, and CHO-K1 (78).
Different G protein profiles inherent to the different cell lines masked some signal
transduction characteristics of the 5 sst. For example, ssti and sst 2 expressed in CHO,
NIH 3T3, COS-7, or COS-1 cells demonstrate no coupling to a decrease in adenylyl cyclase (72,95). CHO-DG44 cells lack expression of Giai and Gia2, G proteins that
couple with membrane associated adenylyl cyclase. CHO-K1 cells express G i a 2 and if
used for stable expression of all 5 sst, forskolin stimulated cAMP accumulation is inhibited by both SSu and SS2g in all 5 receptor expressing CHO-K1 cell lines; this effect is blocked with addition of pertussis toxin and GTPyS (78). Inhibition of forskolin induced cAMP accumulation was 80% for ssti expressing CHO-K1 cells, 70% for sst 2 expressing CHO-K1 cells, 40% for sst 3 expressing CHO-K1 cells, 60% for ssU expressing CHO-K1 cells, and 70% for sstj expressing CHO-K1 cells, with SS2g having a more potent effect than SSu only on ssts expressing CHO-K.1 cells (78). These experiments characterized the 5 cloned receptor subtypes in vitro, in stably expressing
CHO-K1 cells. Importantly, the G-protein expression profile in vivo for various sst
35 expressing cells has not been determined. Thus all the sst may be said to couple to G,ai
and Gio 2 to effect a decrease in membrane associated adenylyl cyclase activity; however which cells in the body express the various Ga proteins remains to be determined.
The specificity of the signal transduction pathways triggered by the five sst has been studied and is currently being analyzed. Multiple signal transduction pathways have been ascribed to the ssti receptor. Analysis of sst) demonstrated that it inhibits the ubiquitous Na+-H+ exchanger (NHE1) (98). Stable transfection of CHO cells with sst j demonstrated somatostatin or somatostatin analogue induced decrease in adenylyl cyclase as well as an increase in inositol 1 ,4,5-triphosphate formation; both effects were blocked by addition of pertussis toxin (100). Analysis of the expression of the three types of a
subunit of the Gj-protein in CHO cells demonstrated RNA for G i a 2 and G ^ but not Gioj
(100). Antiserum for G ^ blocked SSu inhibition of forskolin induced cAMP
accumulation, while antisera for Giai and Gjo 2 had no effect ( 1 0 0 ).
The molecular signaling of ssu has been studied in vitro. Bito et al. isolated rat hippocampal ssU cDNA and demonstrated that it couples to adenylate cyclase inhibition, arachidonate release and activation of the mitogen-activated protein kinase cascade
(MAP) (83). The cloned ssU cDNA was stably transfected in CHO-K1 cells, and the resulting cells subjected to analysis for cAMP reduction, arachidonate release, and MAP kinase activation. These second messenger effects of somatostatin on their CHO-Kl/ssU cells were blocked by pre-treatment with pertussis toxin (83). Florio et al. analyzed ssu expression in PC C 13 thyroid cells and determined it was the only receptor subtype expressed. Somatostatin inhibited the growth of the PC C13 cells in vitro, presumably
36 via the ssu receptor and its modulation of a phosphotyrosine phosphatase ( 1 0 1 ).
Somatostatin growth inhibition has been linked to tyrosine phosphatase in
pancreatic cancer cells (102). In the studies of pancreatic cancer, the somatostatin
octapeptide analogue RC-160 caused the greatest stimulation of phosphotyrosine
phosphatase activity, while the octapeptide analogue octreotide (SMS 201-995)
demonstrated no growth inhibition of the pancreatic cancer cells, nor any stimulation of
tyrosine phosphatase activity (102). Interestingly, the peptide RC-160 binds with high
affinity to ssu with a KD of 45 nM, while octreotide has little affinity for ssu with a KD of
> 1000 nM for the receptor (103). These data, coupled with data from thyroid cell growth
inhibition, indicate that some of somatostatin’s growth inhibiting characteristics stem
from receptor specific signal transduction.
Peptide-receptor based imaging of tumor tissue offers sensitive, specific means for
detection of occult primary and metastatic neuroendocrine and other human tumors.
Peptide-receptor based imaging complements conventional radiologic imaging such as
MRI and CT because of the physiologic component inherent to peptide-receptor
interaction and receptor expression. Tissue enlargement as seen on MRI or CT does not
indicate whether the enlargement is due to acute or chronic inflammation, or neoplastic growth (104). Diminished or absent tissue abnormalities detected by MRI or CT also do not indicate whether disease foci are indeed eradicated. Peptide-receptor imaging offers a means for more specific analysis of such areas. Receptor expression may be used to analyze progression of disease (13)(unpubiished observations). With further research on receptor subtype expression and development of subtype specific analogues, peptide-
37 receptor based imaging will also direct potential therapy (104). Finally, analogues
coupled to high energy emitting compounds such as or a- emitting radionuclides offer
means for radiotherapy such as has been used in pheochromocytoma by l 3 'l-
metaiodobenzylguanidine (MIBG) (105).
The physiology of peptide-receptor scintigraphy has been applied to the
neuropeptide somatostatin and its receptors (104). Two factors resulted in the
development of somatostatin and its receptors as an initial system for peptide-receptor
based imaging. First, long acting analogues for somatostatin were developed that
preserved the receptor binding amino acids and could contain a tyrosine substituted for a
phenylalanine for iodination (104). Second, receptors for somatostatin are highly
expressed on a wide range of tumors such as gastrinomas, lymphomas, neuroblastomas,
prostate cancer, small cell lung cancer, pheochromocytoma, breast cancer (87,106,107).
The development of long acting analogues for the neuropeptide somatostatin yielded
small compounds that when tyrosinated could be radiolabeled with l25I or other isotopes.
These radiolabeled peptides bind with high affinity to their receptors, which are highly expressed by various tumors. Subsequent scintigraphy or intraoperative gamma probe detection has become a standard means for imaging for tumors such as gastrinoma (107).
Somatostatin also has antiproliferative effects via inhibition of growth hormone release, inhibition of angiogenesis (104), and stimulation of antiproliferative tyrosine phosphatases (95,101,102,108).
Neuroblastoma as a Model of Neural Crest Differentiation. The malignant cells in neuroblastoma may be either neuroblasts or Schwaan cells, both of which are
38 derivatives of the neural crest (5). The molecular signaling pathways which regulate
proliferation and differentiation to guide normal neural crest development or presumably,
malfunction to give rise to neuroblastoma, remain unclear. Neuroblastoma presents a
clinical manifestation of transformed neural crest development (21,109). This hypothesis
has been tested in neuroblastoma by analyzing molecular markers (20,24,34,110), by
demonstrating phenotypic plasticity (9,111,112), and by analyzing dependence of
differentiated neuroblastoma cells in vitro on factors that are key to normal neural crest
maturation (46,113).
Several laboratories have recently investigated the possible use of neuropeptide
levels and neuropeptide receptor expression as markers of differentiation as well as
prognostic factors in neuroblastoma (114), Increased NPY in plasma has been
demonstrated to correlate with rapidly growing tumors (Stage IVS and Stage IV) and to
predict early relapse (54). High plasma or tumor levels of VIP and somatostatin correlate
with Stage I and Stage II tumors and tumor differentiation by Shimada classification; thus
VIP and somatostatin peptides are good prognostic indicators (114).
Expression of somatostatin receptors correlates with tumor differentiation and has
been identified as a good prognostic indicator in neuroblastoma (13,85). Three
independent groups have utilized octreotide scintigraphy to image tumor in patients with
neuroblastoma. These combined studies of 27 patients have demonstrated that octreotide
scintigraphy fails to image neuroblastoma in 15-30% of children with documented disease (87,115,116), but the reasons for these negative results are presently unknown. I hypothesize that lack of sst 2 may explain these results and that somatostatin receptor
39 subtype expression may be a marker of neural crest differentiation as well as a clinical
prognostic indicator in neuroblastoma.
The interactions of molecular signals, neurotrophins/receptors, neuropeptides/receptors, and glucocorticoids are interlinked and determine phenotype for both normal multipotent neural crest or neuroblastoma cells (5,6,117). Whether neural crest stem cells express receptors for somatostatin has not been studied. However, studies of multipotent sympathoadrenal cells as well as superior cervical ganglion cells indicate that neurotrophic factors and neuropeptides interact in directing these cells’ development (10,55). In sympathetic neuroblasts, the neuropeptides VIP and NPY regulate mitosis, differentiation, and survival, having early induction/expression in a wide cell population, with later development of a restricted, heterogeneous expression
(55,118). Somatostatin modulates second messenger systems that are also affected by
VIP and NPY such as adenylate cyclase, and has been shown to enhance NGF-induced neurite outgrowth in PC 12 cells, a cell line derived from adrenal medulla (73,119). The assumption is made that the neuropeptides act via specific cell surface receptors, and expression of these receptors is essential to transmission of neuropeptide signal (76).
My hypothesis for these experiments is that a specific somatostatin receptor subtype is expressed in neuroblastoma. By inference, this receptor will play a role in neural crest development. A second hypothesis is that delineation of somatostatin receptor subtype expression in neuroblastoma and identification of subtype specific ligands will provide the basis for improved receptor based scintigraphy in neuroblastoma.
The following studies were designed to 1) analyze somatostatin receptor gene expression
40 in neuroblastoma tumor specimens; 2 ) to study receptor subtypes and identify subtype specific analogues; 3) exploit sst in tumor detection in vivo. These studies were pursued both in vitro and in vivo, analyzing gene expression as well as studying pharmacologic characteristics of receptor-peptide interaction between analogues of somatostatin and the receptor subtypes 1 and 2 .
41 CHAPTER 3
MATERIALS AND METHODS
Solutions:
1) Solution D(4M guanidinium thiocyanate, 7.04 ml 0.75 M sodium citrate pH 7.0,
10.56 ml 10% sarcosyl and 0.36 ml fl mercaptoethanol per 50 mis solution D)
2) 10 X TAE buffer (400 nM Tris base, 20 mM disodiumdihydro-EDTA, 200 mM
sodium acetate anhydrous, 296 mM glacial acetic acid , deionized water to 1 L, at pH
7.8 @ 25°C) (120).
3) Formamide dye (80% deionized formamide, 10 mM EDTA pH 8.0, 1 mg/ml xylene
cyanol FF, I mg/ml bromophenol blue (120))
4) TRlzol™ reagent (Gibco BRL)
5) Guanidinium solution (4 M guanidinium isothiocyanate, 20 mM sodium acetate, pH
5.2,0.1 mM dithiothreitol (DTT), 0.5% AMaurolylsarcosine (Sarkosyl) all pH 5.5)
42 6 ) TES (10 mM Tris-Cl, pH 7.4, 5 mM EDTA, 1 % sodium dodecyl sulfate)
7) 10 X PCR Buffer II (Perkin-Elmer-Cetus; 500 mM KC1, 100 mM Tris-HCl, pH 8.3)
8 ) Electrophoresis Sample Buffer (freshly prepared prior to loading as 0.75 ml deionized
formamide, 0.15 ml 10 X MOPS (0.2 M of [3-(N-morpholino) propanesulfonic
acid]))
9) 10 X MOPS/EDTA buffer (0.2M MOPS, 50 mM sodium acetate, 10 mM EDTA
adjusted to pH 7.0 and autoclaved)
10) Northern electrophoresis buffer X(I MOPS/EDTA buffer diluted from 10X with
autoclaved, DEPC treated water)
11) 20 X SSC (3 M NaCl, 0.3 M sodium citrate, 800 mis ddHiO, pH to 7.4 using 10 N
NaOH, qs to 1 L with H 2 O) (120)
12) Northern prehybridization/hybridization solution-. 50% v/v deionized formamide,
0.47 X Denhardts solution, 4.7 X SSPE, 0.1% w/v SDS, 0.18 mg/ml denatured
salmon sperm carrier DNA, and 10% dextran sulfate
43 13) J M Tris (120): 121.1 gm Tris base, 800 mis ddH 2 0 , allow to cool to room temperature, qs to 1 L pH by adding concentrated HC1 ( 1 1.4 M/L) as follows: pH HC1 7.4 70 ml 7.6 60 ml 8.0 42 ml
14) TE (120): pH 7.4, 10 nM Tris-Cl (pH 7.4), 1 mM EDTA (pH 8.0) pH 7.6, 10 mM Tris-Cl (pH 7.6), 1 mM EDTA (pH 8.0) pH 8.0, 10 mM Tris-Cl (pH 8.0), 1 mM EDTA (pH 8.0)
15) 1 OX PBS: 1.37 M NaCl, 27 mM KC1, 43 mM Na2HP04, and 14 mM KH2P04, 800
mis of ddH20 , adjust pH to 7.4 with HC1, qs to 1 L with ddH20 (120).
16) Luria-Bertani Medium or "LB" broth (for 1 liter: 950 mis of deionized water, 10 gm
bacto-tryptone, 5 gm bacto-yeast extract for 1 X or 7.5 gms for 1.5 X, 0.17 M NaCl,
stir until dissolved, pH to 7.0 with approximately 0.2 ml 5 N NaOH, volume adjusted
to 1 L with deionized water, autoclaved to sterilize) (120).
17) TET (50 mM Tris-HCl pH 8.0, 50 mM EDTA, 0.1% Triton X-100) lysing solution
18) 7 X TNE (pH 8.0) 10 mM Tris-Cl (pH 8.0), 1 mM EDTA (pH 8.0), 100 mM NaCl
( 120)
19) TEG (25 mMTris pH 8.0, 50 mM EDTA, 50 mM glucose)
44 20) Neutralizing Solution: 3 M Potassium acetate, 1 . 8 M Formic Acid
21) Phenol:Chloroform:isoamyl (120)
22) 50 X Denhardt’s reagent: 5g Ficoll, 5 g polyvinylpyrrolidone, 5 g bovine serum
albumin, ddH 2 0 to 500 ml (120)
23) 10% SDS: Sodium dodecyl sulfate (sodium lauryl sulfate), 100 gm electrophoresis-
buffer grade SDS, 900 mis ddH 2 0 , pH to 7.2 using concentrated HC1 (120)
24) Alkaline/SDS lysis solution: 0.2 N NaOH, 1% SDS.
25) 1 M Dithiothreitol (DTT): 3.09 gm of DTT, 20 ml 0.01 M sodium acetate (pH 5.2)
( 120).
26) B ufferS (50 mM HEPES (A/-2-hydroxyethylpiperazine-jVT-2-ethanesulfonic acid), 10
mM CaCl2 , 5 mM MgCli. 50 ug/ml bacitracin, 200 KlU/ml Aprotinin, 0.5% BSA,
0.02 jig/ml PMSF (phenylmethylsulfonylfluoride), pH 7.5)
27) S.O.C. Medium (97 ml ddH 2 0 , 2 g bactotiyptone, 0.5 g yeast extract, 10 mM NaCl,
2.5 mM KC1, 20 mM Mg""\ 20 mM glucose, filtered at 0.2 pm, pH 7.0 ± 0.1)
45 Culture of Neuroblastoma and COS-7 Cell Lines. Cell culture reagents were
purchased from Gibco BRL (Grand Island, NY). The cells were incubated in a
humidified atmosphere of 95% air, 5% CO 2 at 37°C. The COS-7 cell line and the human
neuroblastoma cell lines IMR32 and SKNSH were purchased from American Type
Culture Collection (ATCC, Rockville, MD). IMR32 cells were derived from an abdominal primary and express multiple copies of N-myc (7), while SKNSH cells were derived from bone marrow metastases and express a single copy of N-myc (121). The neuroblastoma cell lines were grown in monolayers in Minimal Essential Medium
(MEM) supplemented with 15% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, 100 {ig/ml streptomycin and nonessential amino acids. The COS-7 cells were grown in monolayers in Dulbecco’s Modified Eagle Media (DMEM) supplemented with
10% FBS, 100 U/ml penicillin, 100 |ig/ml streptomycin and nonessential amino acids.
Tumor Procurement. Human neuroblastoma tumor specimens were obtained through the Cooperative Human Tissue Network (CHTN). Tissues had been obtained at the time of biopsy and frozen to -80°C within 60 minutes of extraction. All available patient data, including age at diagnosis, sex, site of primary tumor, stage, Shimada classification, DNA analysis for N-myc content, and survival (Table 4.1) were provided by CHTN in accordance with guidelines established by the National Cancer Institute and the Children’s Hospital Institutional Review Board (IRB).
RNA Isolation. Total RNA was isolated from cultured cells following the methods of Chomczynski et al. and Chirgwin et al. (122,123) and by CsCl gradient separation. The Trizol reagent (Gibco BRL) was used for RNA isolation from cultured
46 cells and frozen tissue samples. Cell culture is described above. For all manipulations
of RNA, preventive measures to avoid RNase contamination were as follows. All
glassware used for preparation of RNA was baked 4 hours or overnight at 210° C to
inactivate RNase enzyme. All solutions (except those containing Tris) used for RNA
isolation were treated for 12 hours with diethyl pyrocarbonate (DEFC) and autoclaved to
inactivate the DEPC. Solutions containing Tris were prepared using baked, RNA- designated bottles, DEPC treated water and RNA utensils. All utensils were baked if
feasible, autoclaved, or designated solely for RNA isolation.
Isolation from cultured cells was conducted as follows. Briefly, T75 or T 150 culture flasks with confluent cells were placed on ice in a tray. The culture media was aspirated, the monolayers covered with solution D (100 gm guanidinium thiocyanate,
117.2 ml DEPC treated distilled water, 7.04 ml 0.75 M sodium citrate pH 7.0, 10.56 ml
10% sarcosyl, and 0.72% (5 mercaptoethanol), scraped using a sterile cell scraper, and the solution D/celi mixture collected in a 50 cc sterile centrifuge tube. A serial wash of the flasks with solution D was added to the collection tube. The solution D/cell mixture was either frozen at -80°C or immediately used for RNA isolation. A polytron, setting "7,1' was used to homogenize the solution D/celt mixture. The mixture was homogenized twice, 15 seconds each, with 30 seconds on ice in between. The homogenate was transferred to Corex tubes for the remaining steps. Following homogenization, 0. IX (X refers to the volume of cell/solution D mixture) 2 M sodium acetate pH 4.0 was added, with mixing. DEPC treated water-saturated phenol at 1 X volume was added, with mixing. Chloroform:isoamyl alcohol at 49:1 was added at 0.2 X volume, with mixing,
47 and placed on ice 15 minutes. After chilling on ice, the tubes were centrifuged at 10,000
x g for 20 minutes at 4°C. The aqueous phase (top) was recovered and placed in a new
tube. An equal volume of isopropanol was added and the mixture kept at -20°C for at
least one hour to precipitate the RNA. Centrifuging at 10,000 x g 20 minutes at 4°C
recovered the RNA pellet. The supernatant was discarded and the pellet drained well.
The RNA pellet was resuspended in 0.3 X volume of solution D and precipitated with an
equal volume of isopropanol as before. The pellet was recovered as above, rinsed with
75% EtOH, drained well and vacuumed dry. The pellet was resuspended in DEPC
treated water, quantified by reading a 1:100 dilution at OD 2 6 0 and OD 2 8 0 on the
spectrophotometer (Beckman), and analyzed for quality by an agarose gel visualized with ethidium bromide staining. The 1 pg sample (as determined from the spectrophotometer
reading at OD 2 W/ 2 8 0 where 40 pg RNA gives a reading of 1.0 at an OD 2 6 0 (120)) was
denatured by boiling 10 minutes, chilled on ice 5 minutes, and added with formamide dye
(80% deionized formamide, 10 mM EDTA pH 8.0, 1 mg/ml xylene cyanol FF, 1 mg/ml bromophenol blue (120)) and ethidium bromide (1 mg/ml) to the well of a 1% agarose gel and the gel electrophoresed 30 minutes at 90 Volts in 1 X TAE buffer (400 nM Tris base, 20 mM disodiumdihydro-EDTA, 200 mM sodium acetate anhydrous, 296 mM glacial acetic acid, deionized water to 1 L for 10 X TAE, pH 7.8 @ 25°C).
Total RNA was also isolated by ultracentrifugation over a cesium chloride gradient (124). The CsCl method lyses cells using guanidinium, releasing RNA and cell contents. First the culture media was aspirated and the cells washed with 1 X PBS.
Guanidinium solution (4 M guanidinium isothiocyanate, 20 mM sodium acetate, pH 5.2,
48 0.1 mM dithiothreitol (DTT), 0.5% AMaurolylsarcosine (Sarkosyl) all pH 5.5) was added
in volume of 3.5 mis per < 108 cells. Cells were scraped directly into the guanidinium
solution. The viscous lysate was collected using a sterile cell scraper and the lysates combined. Using a 20 gauge needle, the lysate was drawn up and down four times to shear the chromosomal DNA and reduce viscosity thus allowing for complete removal of
DNA in the centrifugation step. Ultracentrifuge tubes were filled with 2 ml of 5.7 M
CsCl per tube. The cell lysate was layered on top of the CsCl and the tubes placed in ultracentrifuge buckets (SW60 rotor (Beckman) with swinging buckets). An overnight centrifuge spin was run at 150,000 x g, 18°C. RNA was pelleted at the bottom of the ultracentrifuge tube; DNA and proteins remained at higher levels in the tube, facilitating
RNA purification/separation.
After the centrifugation, the tubes were removed from the buckets and the DNA band and RNA pellet visualized. The supernatant was removed in a serial fashion, changing pipettes to avoid DNA contamination of the RNA pellet. The tube was inverted to drain when a final 100 pi of supernatant remained. The tube bottom and RNA pellet were cut from the top portion using a sterile scalpel blade. A volume of 0.36 ml TES (10 mM Tris-Cl, pH 7.4, 5 mM EDTA, 1 % sodium dodecyl sulfate) was added to each pellet and the pellet resuspended with up and down pipetting. The pellet/TES was totally resuspended by standing 5 to 10 minutes before continuing. A volume of 40 pi of 3 M sodium acetate, pH 5.2 and 1 ml of 100% EtOH were added to precipitate the RNA for thirty minutes @ -80°C. The pellet was recovered by spinning in a microcentrifuge 30 minutes, discarding the supernatant. The pellet was resuspended in 0.36 ml DEPC treated distilled water and the precipitation/recovery repeated. The pellet was drained and
dissolved in approximately 0.2 ml DEPC treated distilled water. Quantitation was run by
diluting 5 pi sample into 0.5 ml water and the dilution read on the spectrophotometer at
OD 26W280 -
The TRIzol™ method described by GIBCO was used to isolate RNA from both
cultured cells and from tissue. The TRIzol™ protocol is a modification of the
guanidinium/phenol extraction (123,125). Briefly, the cultured cell media was aspirated
and the cells washed with 1 X PBS. The TRIzol TM reagent was added and cells lysed
with exposure to the guanidinium (122). The cell lysate mix was immediately exposed to
the phenol component of the TRIzol™ reagent. This mixture was left at room
temperature for 10 minutes. Chloroform was added to the TRIzol™/cell lysate mixture,
the mixture left to stand for 2-3 minutes, and then centrifuged 12,000 x g for 15 minutes.
The aqueous layer was removed from the centrifuged mixture. Isopropanol was added to
precipitate the RNA, the pellet collected, washed with 75% EtOH and dried on a sterile air flow hood grate.
The TRIzol™ method was applied to the frozen tumor tissues. The frozen tissues were weighed into tared eppendorfs, kept frozen on dry ice while minced on a sterile culture dish, and added to a sterile tube. Alternatively, a mortar and pestle (chilled with liquid nitrogen) were used to grind the tumor tissue into a powder. The TRIzol™ reagent was added to the tissue/powder to a volume of 1 ml per 100 mg tissue. The mixture was homogenized with an Omni pH Handheld Rechargeable Micro-Homogenizer (Omni
50 International, Inc., Gainesville, VA) for two 15 second bursts, at room temperature. The
TRIzol ™/homogenized-tissue mixture was further treated as above for cultured cells.
Total RNA was resuspended in diethylpyrocarbonate-treated (DEPC) water and
quantified using UV spectrophotometry at 260 nm. The Message Clean kit of GenHunter
Corporation (Nashville, TN) was used to DNAse the isolated total RNA. Following
DNAse treatment, the total RNA was run on an agarose gel with ethidium bromide for
visualization of the 28 and 18 S RNA bands, and an mRNA smear, as well as the 5 S transfer RNA band running smaller (tRNA visualized only on some gels). Once the quality of the RNA prep was confirmed, RT-PCR analysis was run to determine gene expression. RNA kept for storage was EtOH precipitated by adding 3 M sodium acetate pH 7.4 at 0.1 times the product volume and 100% EtOH at 2.5 times the volume of the
RNA product.
RT-PCR Analysis of Total RNA. The GeneAmp RNA PCR kit from Perkin
Elmer Cetus (Norwalk, CT) was used to analyze mRNA expression from total RNA isolated from tumors or cultured cells. Total RNA templates were first denatured 5 minutes at 70°C and chilled to 4°C. The master mix for reverse transcription of the total
RNA to cDNA was as follows: 25 mM MgCl2, 10 X PCR Buffer H (500 mM KC1, 100 mM Tris-HCl, pH 8.3), DEPC-treated distilled water, dGTP, dCTP, dATP, dTTP, MuLV
Reverse Transcriptase, RNase Inhibitor, Random Hexamers, and 1 pg total RNA template. The 20 pi reaction volume was placed at 23°C for 10 minutes, 42°C for 15 minutes, 99°C for 5 minutes and cooled at 4°C. The primers for PCR amplification of c- abl, somatostatin (SS), somatostatin receptor types one through five (ssti.s) (Table 4.2)
51 were designed using the published sequences for the genes. The master mix for the PCR
reaction follows: 25 mM MgCh solution, 10X PCR Buffer II {as above), sterile
(autoclaved) distilled water, and AmpUTaq DNA Polymerase. The program for PCR
amplification was 5 minutes to reach 94°C, then 33 cycles of 1 minute at 94°C, 1 minute at 63.8°C, and 1 minute at 72°C, and finally dwell at 4°C. The PCR products were electrophoresed on an agarose gel and visualized using ethidium bromide staining and uv
illumination.
Southern Confirmation of PCR Analysis, A 10 p.1 sample of each PCR reaction was run on a 1% agarose gel, depurinated in 3 N HC1 for 15 minutes, denatured with 0.4
N NaOH-0.6M NaCl for 30 minutes, neutralized in 1.5M NaCl-0.5M Tris-HCl, pH 7.5, for 30 minutes all with gentle agitation and at room temperature. The products were transferred from the gel to a Hybond membrane (Amersham, Arlington Heights, IL) by capillary action overnight. A stratalinker (Stratagene, La Jolla, CA) was used to autocrosslink the transferred products to the membrane. Membranes were prehybridized for 3 hours at 60°C in hybridization buffer supplemented with 100 |ig/ml Herring sperm
DNA (Gibco BRL). Probes were designed nested to the PCR primers. Five pmol of dephosphorylated DNA oligo were incubated 10 minutes at 37°C with IX Forward
Reaction Buffer, 10 Units T4 Polynucleotide Kinase (Gibco BRL) and 2.5 (ll [V2P] ATP
(10 pCi/pl, 3000 Ci/mmol Amersham). Labeling reactions were stopped by adding
EDTA to 5mM per reaction. Labeled probes were purified using the Bio-Spin 6 prefilled column (Bio-Rad, Hercules, CA). The end-labeled oligos were then boiled 10 minutes to denature, chilled on ice, and added to the hybridization buffer/membrane, and hybridization run overnight at 60°C. Following hybridization, the membranes were washed twice with 2X SSC (saline sodium citrate) at room temperature with agitation for
5 minutes each, twice with 2X SSC-1%SDS (sodium dodecyl sulfate) at 65°C with agitation for 30 minutes each, and finally twice with 0.1X SSC at room temperature with agitation for 30 minutes each. The membranes were placed in autoradiography cassettes and exposed to x-ray film for 1 and 3 hour exposures before developing the film. Probe sequences are listed in Table 4.3.
Northern Analysis of Total RNA. Total RNA was isolated as above. A 25 jig sample was recovered as a pellet from ethanol precipitation and resuspended in 25 mM
EDTA, 0.1 % SDS. Twenty five (ll of electrophoresis sample buffer (freshly prepared prior to loading as 0.75 ml deionized formamide, 0.15 ml 10 X MOPS (0.2 M of [3-(N- morpholino) propanesulfonic acid]), 0.24 ml of 37% formaldehyde, 0.1 ml deionized
RNase-free H 2 O, 0.1 ml glycerol, 0.08 ml 10% w/v bromophenol blue) was added and the sample heated to 65°C for 15 minutes, then placed on ice. One jil of ethidium bromide at
1 mg/ml was added to the sample and the solution mixed thoroughly. The sample was loaded on the gel. A 0.24 to 9.5 Kb RNA ladder purchased from Gibco BRL was prepared as a sample and loaded onto the gel. Gel preparation follows: a 1 % agarose gel of 10 X MOPS/EDTA buffer (0.2M MOPS, 50 mM sodium acetate, 10 mM EDTA adjusted to pH 7.0 and autoclaved) and autoclaved DEPC treated water was heated and then cooled to 50°C. In a fume hood, 37% formaldehyde at 5% of gel volume was introduced into the agarose and gently mixed. The gel was poured into an RNase free gel frame, the comb added, and all left to stand in the hood for 1 hour before use.
53 Prior to loading the gel, the wells were flushed with electrophoresis buffer (1 X
MOPS/EDTA buffer diluted from 10X with autoclaved, DEPC treated water). The wells were loaded with prepared samples (see above) and the gel electrophoresed at 30 V constant voltage at room temperature for 18 hours. The gel was visualized on a UV light box to determine extent of migration. A fluorescent ruler was photographed aligned next to the migrated RNA for reference following probe hybridization and autoradiography.
The gel was prepared for transfer by soaking it for two 20 minute periods in 10 X SSC (at room temperature with gentle shaking). During the washing procedure, the membrane was pre-wet in distilled water for 5 minutes followed by a 5 minute soak in 10 X SSC.
The RNA was transferred from gel to membrane by capillary action using paper towels and 10 X SSC on a blotting apparatus overnight. The RNA was fixed to the membrane using a stratalinker (Stratagene). The membrane was prehybridized for 1-4 hours and hybridized for 12-16 hours at 42°C with rotation. The solution for both prehybridization and hybridization was 50% v/v deionized formamide, 0.47 X Denhardt’s solution, 4.7 X
SSPE, 0.1% w/v SDS, 0.18 mg/ml denatured salmon sperm carrier DNA, and 10% dextran sulfate. The probe was a PCR product labeled with [oc32P]dCTP (6000 Ci/mmol)
(Amersham) using the Pharmacia Oligolabeling Kit (Pharmacia Biotech, Piscataway. NJ)
(126,127). Briefly, a PCR product was diluted with TE pH 7.6 and run through a column to remove nucleotides and enzyme from the product. The PCR product was precipitated using 3 M sodium acetate and 100% EtOH. The purified product was resuspended in TE pH 7.6 and 50 ng used to make the probe. The 50 ng template was brought to a volume
54 of 34 ill and denatured by heating to 95-100° 2-3 minutes, placing on ice for 2 minutes
and centrifuging the tube briefly.
To a clean microcentrifuge tube, the following were added: 34 pi denatured
DNA, 10 pi of the Reagent Mix (buffered aqueous solution containing dATP, dGTP,
dTTP, and random hexadeoxyribonucleotides, provided with kit), 5 pJ (50 pCi) of
[a 3 2 P]dCTP (6000 Ci/mmol), and 1 pi of Klenow Fragment (provided with kit). The tube
contents were mixed gently and centrifuged briefly. The tube was incubated at 37°C in a
water bath/heat block overnight. The labeled probe was prepared for hybridization by
heating at 95-I00°C for 2 minutes, cooled immediately on ice, then added to the
hybridization tube.
Following hybridization, the membrane was washed under moderate stringency using two 20 minute washes in 1 X SSC, 0.1 w/v SDS at room temperature followed by two 20-minute washes in 0.1 X SSC, 0.1% SDS at 50-55°C. The membrane was finally placed in a film cassette with Kodak XAR film (Eastman Kodak Company, New Haven,
CT) and the cassette placed at -70°C for 2-3 days before developing.
TA Cloning of cDNA Products. Primers designed to amplify bp 82-1192 of sst 2
Tli (GenBank accession # M81830) were used for PCR to amplify an sst 2 cDNA from
RNA isolated from the IMR32 neuroblastoma cell line. The PCR product was ligated into a PCRII cloning vector (Invitrogen, San Diego, CA) and subcloned to the pcDM 8 plasmid (Invitrogen) using EeoRI. Briefly, the insert containing bp 82-1192 of sst 2 was gel purified from PCRII using Geneclean (Bio 101, Vista, CA). The plasmid pcDM 8 was digested with EcoRI and the insert ligated using DNA ligase (Gibco BRL). Ligation
55 reaction products were used to transform DH10B/p3® bacteria (Gibco BRL). The
transformed DH10B/p3® bacteria were then plated on LB/AMP/Tet plates and positive
colonies miniprepped and analyzed for inserts using restriction enzyme digestion with
EcoRI.
Subcloning of sst 2 into pcDNA3. The vector pcDNA3 (Invitrogen) was opened using the Xba-I enzyme targeting a site in the plasmid’s polycloning region. Following the digest, the restriction enzyme was inactivated by heating to 75° C for 10 minutes.
The pcDNA3 was then treated with Calf Intestinal Alkaline Phosphatase (C.I.A.P.,
Boehringer Mannheim) (120) to digest the 5' protruding ends left by the Xba-I digest and therefore prevent self ligation during the ligation reaction with sst 2 - C.I.A.P. was used at a concentration of 0.01 units per pmol of DNA 5' overhang ends at 37°C for 30 minutes.
C.I.A.P. was inactivated by heating the mixture to 75°C for 10 minutes following the 30 minute reaction.
Linear pcDNA3 was digested with Xba-I, treated with C.I.A.P., and purified by electrophoresing it on a LMP gel. Purified pcDNA3 was removed from the gel using the
GELase method (Epicentre Technologies, Madison, WI). Because of its size (5.5 Kb) the pcDNA3 vector was purified from an agarose gel using the GELase protocol. The
GELase protocol exploits a unique enzyme preparation for recovery of DNA from low- melting-point agarose gels (128). The high activity protocol was followed for pcDNA3 isolation, as follows. The desired band was cut out of a LMP agarose and the gel slice weighed in a tared tube. The gel slice was soaked in three volumes of 1 X GELase buffer for one hour. After one hour, the excess GELase buffer was carefully removed with a
56 pipette and the gel slice melted at 70cC. The molten agarose was equilibrated to 45°C
and one unit GELase per 600 mg of 1 % LMP gel was added. The agarose/enzyme
mixture was incubated for one hour at 45°C. Following the incubation, one volume of
fresh 5 M ammonium acetate was added to the reaction. Two volumes of room
temperature ethanol were added. The pellet of precipitated DNA was recovered by
centrifugation for thirty minutes at room temperature at 12,000 x g. The supernatant was
aspirated and the pellet washed gently with 70% EtOH. A brief centrifugation at 12,000
x g to re-pellet was followed by aspiration of the wash and resuspension of the pellet in
water or TE.
Subcloning of ssti into pcDNA3. The construct of pcDNA3/sst] was provided by Dr. Feng Chen, a post doctoral fellow in Dr. O’Dorisio’s laboratory. Briefly, a cassette containing bp 7-1498 of ssti (GenBank™ accession # M81829) was provided by
Graeme Bell. The cassette was cloned into the EcoRV site of pcDNA3 (Invitrogen) by
Dr. Chen.
Transformation of Bacteria. Max Efficiency DH10B/p3® competent cells
(Gibco BRL) were transformed with pcDM 8 /sst2. The cells were thawed on wet ice, and
100 pi aliquoted to each transformation tube (Falcon 2059 tubes). Between 5 and 50 ng of ligation reaction (from above) was added to the chilled cells, and the mixture placed on ice for 30 minutes. The cells were then heat shocked for 45 seconds in a 42°C water bath, and placed on ice for 2 minutes. Room temperature S.O.C. media was added to the cells
(0.9 ml). The mixture was placed in an orbital shaker at 37°C 225 rpm for 1 hour. The cells were plated on LB/Amp/Tet plates and incubated overnight. Colonies were picked
57 and grown as minipreps for initial screenings. Max. efficiency DH5a cells (Gibco BRL)
were used for pcDNA3/ssti transformation and propagation, using the same methods used
for the DH10B/p3® cells.
Bacterial Plasmid Preps. Wizard Mini Preps (Promega, Madison, WI) were
used to confirm colonies from Ecoli transformation. Briefly, the colony was picked using
a pipette tip, and cultured overnight in a 2 ml culture of LB + 100 pg/pJ Ampicillin
(Sigma, St. Louis, MO).
Two methods were used in developing large preparations of plasmid. Plasmid
containing the gene of interest, sst 2 , were used to transform DH5a E.coli bacteria. A 250-
500 ml volume of E.coli thus transformed was cultured and served as template for the plasmid isolation. One method is a purification over a cesium chloride gradient (120).
The plasmid pcDM 8 /sst2 was prepared using the CsCl gradient isolation. The second method is an isolation not requiring ultracentrifugation (120). The second method was used to isolate all pcDNA 3 /sst2 and pcDNA3 plasmids.
Briefly the CsCl method was as follows. Day one of the preparation: four mis of
Luria-Bertani Medium or "LB" broth (for 1 liter: 950 mis of deionized water, 10 gm bacto-tryptone, 5 gm bacto-yeast extract for 1 X or 7.5 gms for 1.5 X, NaCl 10 gm, stir until dissolved, pH to 7.0 with approximately 0.2 ml 5 N NaOH, volume adjusted to 1 L with deionized water, autoclaved to sterilize) were inoculated with 2 0 ul of glycerol stock of a miniprep of bacteria containing desired plasmid/gene construct. The small culture was grown in a QUEUER (Parkersburg, West Virginia) orbital shaker (37°C, 225 rpm) for
58 4 hours at which point it was used to inoculate a 500 ml volume of LB broth. The large culture was grown overnight at the same conditions as the small culture.
Day two: the overnight culture was poured into two 500 ml sterile plastic bottles and spun in a JA10 rotor (Beckman) at 4,420 x g for 15 minutes at 4°C. The supernatant was completely decanted from the pellet. The pellet could have at -20°C for several days at this point. Adding 5 mis (2.5 mis per pellet) of cold 25% sucrose in 50 mM tris-HCl pH 8.0 (sterile) loosened the pellet. Vortexing and pipetting facilitated complete resuspension of the pellet. The bacteria were transferred to a 30 ml sterile polypropylene centrifuge tube. Another 2.5 mis each of the sucrose solution was used to wash the pellet bottle, and added to the 30 ml tube. Lysozyme (Sigma, 15 mg/ml in 0.25 Tris-HCl pH
8 .0 ), freshly made, was added to the bacteria, the contents mixed gently and left on ice.
After 5 minutes, 0.5 ml 0.25 M EDTA (pH 8 ) was added with gentle mixing, and left on ice. After 5 minutes, 5 mis cold TET (50 mM Tris-HCl pH 8.0, 50 mM EDTA, 0.1%
Triton X-100) lysing solution was added and the tube gently inverted to mix the contents.
The contents were centrifuged in a JA-20 rotor (Beckman) at 27,200 x g for 60 minutes at
4°C. The clear supernatant was transferred to a sterile 50 ml conical tube. One gram solid CsCl per ml of clear supernatant was added. The solution was mixed to dissolve the
CsCl. The supematant/CsCl solution was transferred to a 30 ml quick seal ultracentrifuge tube (Beckman) through a syringe fitted with an 18 gauge needle. Ethidium bromide at
10 mg/ml and 0.4 ml volume was added through the syringe/needle. Tubes were balanced with 1 g/CsCl/ml TNE solution (10 mM Tris pH 8.0, 10 mM NaCl, 25 mM
EDTA). The balanced tubes were rilled with mineral oil and the balance checked again.
59 A tube sealer (Beckman) was used to seal the tubes, which were checked for leaks. The tubes were fitted with metal ultracentrifuge hats, the balance checked again to be within
1%.
The rotor (Ti70, Beckman) was prepared for ultracentrifugation by applying
Spinkote (Beckman) around the grooves of the lid and vacuum grease on the o-rings of the lid. The samples were placed in the rotor and the centrifuge run for 24 hours at
150,000 x g, 20°C.
Day three: a large UV lamp was placed near a ring stand positioned with a beaker, a mount for a tube, and absorbent paper. The tubes were removed from the ultracentrifuge and one tube placed in the tube mount on the ring stand. The UV light illuminated the isolated plasmid band in the ultracentrifuge tube. Three bands and a pellet were visible. The top band indicated protein, the second highest, nicked or linear
DNA. The lower band represented supercoiled plasmid DNA, and the pellet RNA. An
18 gauge needle was inserted into the top of the ultracentrifuge tube to release pressure.
Another 18 gauge needle was inserted at the bottom of the tube, with slight rotation to avoid the RNA pellet at the tube bottom. The lower needle was removed to allow the tube contents to flow from the tube. Only the bottom band was collected in an orange capped 15 ml centrifuge tube. A second CsCl gradient separation was conducted because the plasmid construct was to be used for sequencing and transfection. The second run was prepared and conducted as above.
Day four: The band from the second gradient spin was collected in the same manner as the first gradient spin’s band. The ethidium bromide was extracted from the
60 sample by adding an equal volume of CsCl-saturated isopropanol. The solution was diluted 3-5 fold with 1 X TNE (pH 8.0). An equal volume phenol:chloroform:isoamyl alcohol (25:24:1) was added to extract protein. The mixture was centrifuged at 1000 x g,
10 minutes at 4°C. The upper aqueous layer was transferred to a clear chloroform- resistant (polypropylene) tube and mixed with an equal volume of chloroform: isoamyl alcohol (24:1). The mixture was centrifuged as above. The upper aqueous layer was transferred into a 30 ml polypropylene centrifuge tube. Sodium chloride to a final concentration of 0.1 M, and 2 volumes of cold 100% EtOH were added to precipitate the
DNA for > 1 hour. The precipitate was recovered by centrifugation for 30-45 minutes at
20,400 x g. The supernatant was decanted and the tube drained by inversion. Five mis of
70% EtOH were added to wash the pellet before drying in a speed vacuum.
The pellets were resuspended in 0.4 ml 1 X TNE pH 8.0 and transferred to a sterile eppendorf tube. The 30 ml tube was washed with another 0.4 ml of TNE and the wash pooled in the eppendorf tube. The concentration was measured by reading a 1:100 dilution of the product on a spectrophotometer at OD 2 6 0 /2 8 0 - The plasmid DNA quality was verified by PCR amplification, restriction enzyme digest, and by sequence analysis as described.
The second plasmid preparation spanned one day and did not require special equipment (ultracentrifuge) (120). The protocol follows: 250-500 mis of LB broth was inoculated with a glycerol stock of bacteria containing the desired plasmid. The large culture was grown overnight as above. After overnight incubation, the culture was divided into two centrifuge tubes and the bugs pelleted by spinning at 3,000 x g 20
61 minutes at room temperature. Following centrifugation, the supernatant was decanted
and the pellets resuspended in 20 mis of cold TEG (25 mMTris pH 8.0, 50 mM EDTA,
50 mM glucose) per 250 ml culture at 4°C. Forty mis of alkaline/SDS solution made
fresh for each experiment was added and the solutions gently mixed until a precipitate
formed. The mixture was left on ice for 10 minutes. After the incubation 30 mis of
Neutralizing solution (3 M potassium acetate, 1.8 M formic acid) were added with gentle
mixing and returned to ice for 5 minutes. The mixture was centrifuged at 3000 x g for twenty minutes at 4°C. The resulting supernatant was poured through sterile gauze into a new 50 ml tube. An equal volume of cold isopropanol was added to the supernatant and mixed gently by inversion. The supematant/isopropanol mixture was centrifuged at 3000 x g, twenty minutes at 4°C.
The resulting supernatant was decanted and discarded. The pellet was resuspended in 3 mis total of sterile distilled water. An equal volume of 5 M LiCl, 50 mM MOPS pH 8.0 was added to the resuspended pellet with mixing and the solution placed on ice for 10 minutes to precipitate most of the RNA. The solution was centrifuged as above, 3000 x g for 20 minutes @ 4°C. Following the centrifugation, the supernatant was transferred to a new 50 ml tube. The supernatant contained the plasmid.
The supernatant was extracted once with an equal volume of phenol/chloroform.
Centrifugation at 1000 x g, 22°C for 5 minutes separated the phases. Returning to 4° for the isolation, the upper phase was transferred to a new 50 ml tube and an equal volume of cold isopropanol added to precipitate the DNA. The precipitate was recovered by spinning at 3,000 x g, 4°C for 20 minutes. Following centrifugation, the supernatant was
62 decanted and discarded and the pellet allowed to air dry for 5 minutes. The dry pellet was
resuspended in 1 ml of TE containing RNase A (100 fxg/ml, from bovine pancreas (120)).
The solution was incubated at 37°C for 15 minutes. Following incubation, the 1 ml was
divided into two 0.5 ml samples; thus each 250 ml culture yields two 0.5 ml samples.
Each sample was extracted with an equal volume phenol:chloroform:isoamyl alcohol
(25:24:1) and centrifuged at 1,000 x g, for 2 minutes to separate the phases. The aqueous
upper phase was transferred to a new microfuge tube and the extraction repeated until the
interface between the phases was clear. At that point a final extraction with chloroform:isoamyl (24:1) was conducted.
The aqueous upper phase was removed and one half volume of 7.5 M ammonium acetate added with mixing. One volume of cold isopropanol was added with mixing.
The precipitated DNA was recovered by a 15 minute microcentrifugation at 12,000 x g, at
4°C. The pellet was washed twice with 70% cold EtOH and dried in a speed-vacuum.
The final pellets were resuspended in 0.3-0.4 ml of TE, quantified on a spectrophotometer, and analyzed on an agarose gel. The maxi prep plasmids were used as templates for dideoxy sequencing, restriction enzyme digest and polymerase chain reaction analyses. They were also used as vectors for transient and stable transfection of mammalian cells.
Dideoxy Sequencing Analysis. Dideoxy sequencing analysis was used to confirm successful ligation of the complete sst 2 gene into the pcDM 8 or pcDNA3 vector.
The reactions were run using the Sequenase version 2.0 enzyme, a genetic variant of the
T7 DNA polymerase (129). Briefly, 6 pg of the plasmid template was subjected to
63 alkaline lysis and precipitated with 50 ng of primer using 3 M sodium acetate pH 7.4 and
100% EtOH for 1 hour or overnight. The denatured DN A/primer complex was recovered
by centrifugation at 14,000 x g, 4°C for 45 minutes. The pellet was washed with 70%
EtOH, centrifuged at 14,000 x g for 15 minutes and dried in a speed-vacuum. The pellet
was resuspended in 8 pi of sterile distilled water. A 96 well round bottom plate (Falcon)
was used for the reactions. While isolating the primer/DNA complex, the 96 well plate
was warmed to 37°C. Aliquots of 2.5 pi of G,A,T and C termination mixtures (ddG
termination mix: 80 pM dGTP, 80 pM dATP, 80 pM dCTP, 80 pM dTTP, 8 pM ddGTP,
50 mM NaCl; other mixes vary respectively with which ddNTP is present) were added to
the warmed reaction plate with four wells per each sequencing DNA/primer pair.
A labeling reaction master mix was prepared containing 1.0 pi of 0.1 M DTT, 2.0
pi of diluted labeling mix (5 X concentrate: 7.5 pM dGTP, 7.5 pM dCTP, 7.5 pM dTTP),
0.5 pi of [ 3 5 S] dATP, and 2 pi of 5 X reaction buffer were assembled in an eppendorf.
The Sequenase enzyme (United States Biochemical Corporation, Cleveland, OH) (13
units/pl in 20 mM KPO 4 , pH 7.4, 1 mM DTT, 0.1 M EDTA, 50% glycerol) was diluted in its reaction buffer (10 mM Tris.HCI, pH 7.5, 5 mM DTT, 0.5 mg/ml BSA). A volume of
15.5 pi of labeling reaction mix was added to each DNA/primer pair. Then 2 pi of
Sequenase dilution was added to the mixtures. The reaction was run for 5 minutes at room temperature. Following the incubation, 3.5 pi of each reaction mixture were aliquoted into each of four respective termination mixtures in the warm 96 well plate.
The reactions were run for 5 minutes at 37°C, and terminated by adding 4 pi of stop
64 solution (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% xylene
cyanol) to each well. The reactions were stored at -20°C until heated to 80°C and loaded
on a denaturing acrylamide gel. The denaturing acrylamide gel used was 6 %
concentration, with 5.7 gm/0.3 gm acrylamide/bis-acrylamide, 42-50 gm urea (7-8.3 M),
10 ml 10X TBE buffer and 40 ml H 2 O. When ready to pour, 1 ml of 10% ammonium
persulfate, and 25 pJ TEMED were added to the gel mix. The gel was run in a model
STS 45 Standard Thermoplate Sequencer, (International Biotechnologies Incorporated
(IBI), New Haven, CT) sequencing apparatus for a 20 minute pre-run slow increase to
80W, then with loaded samples, ran for 2 hours at 80W, followed by a second load of samples run 1 additional hour at 80W. Following the ran, the plates and gel were removed and the gel washed with 10% methanol, 10% acetic acid in H 2 0. The gel was dried, exposed to Fuji 35 x 43 cm medical x-ray film overnight, and developed on a
Kodak RP X-OMAT model M7B processor. Sequences were read using an IBI film reader and the DNAStar/Lasergene software program (DNASTAR Inc., Madison, WI) for sequence analysis.
Restriction Enzyme Analysis of Plasmid Constructs.Plasmid preparations were further subjected to restriction enzyme analysis to confirm subcloning ( 1 2 0 ).
Because sst2 was cloned bidirectionally into the Xba-I site of the pcDNA3 polycloning region, digests using the enzymes Kpn-I and Xmn-I were used to determine the gene’s orientation in the pcDNA3 vector. Kpn-I cuts sst 2 at bp 740 and cuts uniquely at pcDNA3 bp 900. The expected products for 5 ’3 ’ orientation of sst 2 in pcDNA3 by Kpn-I digest is
722 bp, for 3’-5’ orientation, 407 bp. The expected products for digestion with Xmn-I,
65 which has a sites at 1741 and 5123 in pcDNA3 and at 1046 in sst 2 , are 3382 bp, 2265 bp,
and 818 bp for an insert in the 5’-3’ orientation. For 3’-5’ orientation digested with Xmn-
I, the expected product sizes are 3382 bp, 1745 bp, and 1368 bp.
Expression/analysis of sstj & sstj constructs. Two methods of transient
transfection were applied to determine protein expression of ssti and sst 2 in plasmid
vectors: the Lipofectamine (Gibco BRL) agent and DEAE-Dextran (120) chemical
methods were both used. The plasmid constructs pcDNA3/sstj, pcDM 8 /sst2 and
pcDNA 3 /sst2 were transiently expressed in low passage COS-7 cells (passage 14 or less)
for 24 hours at which point total binding was determined by whole cell plate binding with
[ 1 2 5 I-tyrM] Somatostatin - 1 4 (30 pmoles/35 mm well) (Amersham). The cells were plated
at 1.5 x 10 5 cells/15.5 mm diameter well 24 hours prior to transfection. At transfection,
the cells were washed with serum free media, followed by addition of 0.8 ml Optimem
(Gibco BRL) media. Two pg of plasmid DNA was added to 100 pi Optimem per well
and mixed with 4 pi Lipofectamine/100 pi Optimem per well. The DNA/Lipofectamine
mixture incubated at 25°C for 45 minutes, at which point it was added to the monolayer cells. After 4 hours incubation at 37°, 1 ml of DMEM/20% FCS was added to each well.
The cells were incubated for 48 hours at which point total binding analysis was conducted.
Alternatively, DEAE-Dextran was used for transient transfection of COS-7 cells.
Low passage (less than passage 14) COS-7 cells were plated at a density of 7.5 x 10 4 cells/well of Coming (Coming, New York) 6 well culture plates. DEAE-Dextran (3.2 mg/ml; final concentration = 100 pg/ml) was used (120). Cells were plated and placed in
66 a 37°C, 5% COi, humidified culture incubator for 24 hours with a media containing
Dulbecco’s Minimum Essential Media/10% Fetal Calf Serum (FCS), 1% 1-glutamine,
1% Nonessential amino acids, and 2% penicillin/streptomycin as above. They were then
washed with 1.5 ml IX PBS, and returned to the incubator with 1.5 mis DMEM/10%
NuSerum (Collaborative Biomedical Products, Bedford, MA). DNA, approximately 1
Hg. was added to warm DEAE-Dextran solution. DN A/DEAE Dextran was added
dropwise to each cell well, with swirling, followed by addition of chloroquine (4.127
mg/ml; final concentration 100 pM) in similar manner to each well. Four hours
incubation at 37°C, 5% CO2 followed. After four hours, DNA/DEAE-Dextran was
aspirated and 10% DMSO in I X PBS added to each well. Cells were incubated for 1
minute with DMSO, followed by aspiration, and addition of 2 ml 1 X PBS to wash the
monolayers. The cells were incubated for 48 hours at 37°C, 5% CO 2 , followed by plate
binding and analysis by autoradiography, AMBIS (AMBIS, Inc, San Diego, CA), or
trypsinization and gamma counter analysis. Total binding analysis indicated which
constructs to use for stable transfection experiments.
For stable transfections the pcDNA3/ssti construct and the pcDM 8 /sst2 plus psV2Neo (Invitrogen) constructs were transfected into SKNSH neuroblastoma cells using
Transfectam (Promega, Madison, WI) for the pcDM 8 -sst2 + psV2Neo constructs or
Lipofectamine (Gibco BRL) for pcDNA3-sst]. Selection with geneticin (G4I8, Gibco
BRL) 500 fXg/jjJ yielded SKNSH cells expressing either sst| or sst 2 . These cells were subjected to limiting dilution and subsequent clonal expansion from a single cell.
67 Binding analysis of ssti and sst2. The resulting cell lines expressing either sstt
or sst 2 were then subjected to competitive binding analysis using [l 2 5 I-tyrn ] Somatostatin-
1 4 (Amersham) and unlabeled SS 1 4 (Bachem, Torrance, CA) as well as three long acting
analogues of SS)4; CH275 (130), Octreotide (Sandoz, Basel, Switzerland), and WOC3B
(Woltering, O’Dorisio, O’Dorisio, Coy).
Receptor expressing cell lines were plated in seven wells of a 24 well plate.
Serum free MEM (Gibco BRL) was used to wash the cells, and 0.4 ml Buffer S (50 mM
HEPES, 10 mM CaCh, 5 mM MgCb, 50 pg/ml bacitracin, 200 KlU/ml Aprotinin, 0.5%
BSA, 0.02 pg/ml PMSF, pH 7.5) was added to each well. [I 2 5 I-tyrn ] Somatostatin- 14 was
added at 50,000 cpm/well in 50 pi Buffer S/0.5% human serum albumin per well.
Dilutions of respective unlabeled peptides in Buffer S were made and 50 pi added per
well at 0,0.01 nM, 0.1 nM, 1 nM, 0.01 pM, 0.1 pM, and 1 pM final concentrations of
unlabeled peptide. The binding reactions were incubated at 37°C, 5% CO 2 for 30
minutes, at which point the reaction mixture was aspirated, the cell monolayer washed by
adding 1 ml warm MEM and aspirating, and 0.5 ml of 0.25% trypsin- 1 mM EDTA
(Gibco BRL) added to release the monolayer. The cells were collected in biovials and the mixtures counted on a gamma counter (Beckman). Radioactivity recorded in cpm was converted to dpm and subjected to analysis by the LIGAND program (131,132) to generate affinity constants (Kd) for the respective unlabeled peptides.
Xenograft animal experiments. Mice were acquired within two weeks of birth from Harlan Sprague Dawley Inc. (Indianapolis, IN). Athymic mice were used for these studies, following one week vivarium quarantine. Cells cultured as above were harvested
68 at 80-90% confluency to ensure exponential growth phase. Briefly, 10 T-175 flasks
(Falcon) were plated at 1:5 with either SKNSH, SKNSH/ssti, or SKNSH/sst 2 neuroblastoma cells in MEM (Gibco BRL) supplemented with 1 5% fetal bovine serum
(FBS), 1% 1-glutamine, 2% penieillin-streptomycin, 1% non-essential amino acids, 500
|ig/ml G418 (Geneticin, Gibco BRL). On the day of injection, the culture media was aspirated, the monolayers washed with 4 mis trypsin-EDTA, and incubated with 4 mis
0.05% trypsin with 0.53 mM EDTA to disrupt the monolayer. The cells were resuspended (and trypsin subsequently diluted) in 6 mis warm sterile MEM with no additives/flask, pelleted and resuspended in 20 mis MEM per 5 flasks for cell count. The cells were counted on a Coulter counter (Coulter Corporation, Hialeah, FL), pelleted, and resuspended to a concentration of 3 X 10 7 cells/0.2 ml for injection. All manipulations preserved the sterility of the cells.
The mice were tagged using ear punch and identified as numbers 1-6 by position of holes in the ear. Right ear anterior was noted “ 1 mid right ear “2,” posterior right ear
“3,” and left ear for #4-6. The cells were drawn into a 1 cc syringe and a 22 g, needle affixed to the syringe. Restraining the mice by neck scruff and tail in the left hand, the injection was aimed sub cutaneously from left lateral thorax to distal left leg. The long sub-cutaneous path for injection reduced cell leakage. Mice were observed 24 hours following injection for signs of complications, and thereafter were observed Monday,
Wednesday, and Friday for tumor development and subsequent measurement.
Calipers were used for measuring development of tumors in injected neuroblastoma xenografts. One investigator measured tumor diameters for consistency.
69 Radiolabeling studies of the xenograft tumors were conducted when the tumor
dimensions exceeded 1 cm X 1 cm. The isotope 125I was used for all radiolabeling.
Radiolabeled compounds were produced either by Amersham (Arlington Heights, IL),
([l2 5 I-tyr‘'] Somatostatin- 1 4 ), or by the peptide laboratories at The Ohio State University
Hospitals ( 1 2 5 I-tyr3-octreotide and ,25I-CH288). The pediatric or small size Neoprobe
1001 (Neoprobe Corporation, Columbus, OH) gamma-detecting instrument was used to
monitor the mice ( 8 8 ). The Neoprobe 1001, equipped with a radiosensitive 7- or 12-mm
cadmium telluride crystal and enclosed in a chromium plated copper housing, detected
low-level gamma radiation of ,25I with an efficiency of 60% ( 8 8 ). A collimator, or lead
shield-cap for the probe, was also used for the in vivo studies. Gamma radiation detected
in the probe was relayed through a preamplifier and a signal processor and displayed as a
digital count of radioactivity.
The mice to be studied were weighed and their tumor dimensions measured. The
radiolabeled peptides were injected intra peritoneally with 0.9 % NaCl as 20 pCi
radiolabeled peptide in 0.2 ml volume and 0.3 ml NaCl for a total injection volume of 0.5
ml. Triplicate count readings were recorded for the heart, abdomen, right leg
(background), tumor, and thyroid at 5 minutes, 15 minutes, 30 minutes, and 60 minutes after injection. The animals were sacrificed with 0.075 ml telazol/rompum anesthetic following the 60 minute time point. The thorax was opened and 1 cc of blood removed from the left ventricle. Tumor was then harvested and divided for analysis by total RNA isolation, formalin fixation/parafm embedding/autoradiography, and biovial/gamma counter radioactivity per gram of tissue determination. Finally, the large intestine and
70 submandibular gland area were isolated and divided for formalin fixation/parafin
embedding/positive control of autoradiography, and biovial/gamma counter radioactivity
per gram of tissue determination.
Autoradiography of radiolabeled studies was performed. The resources of Dr.
Steven J. Qualman and the technicians of the pathology histologic laboratory were
invaluable in the autoradiographic analysis. Parafln-embedded tissue was cut to 20 pM
thickness and mounted wet to a microscope slide, 2 sections or more per slide, 2 slides
per tissue. The slides were deparafinized using xylenes and rehydrated in a series of 6
washings from 100 % EtOH to distilled deionized water. The slides were dried and dipped in photoraphic emulsion (Kodak), dried 3 hours and packed with desiccant for 3 and 6 week exposure times. After allotted exposure times, the slides were developed and fixed using the Kodak Dektol developer and fixer. The developed slides were stained with hematoxylin and eosin and coverslipped using permaslip. Observations were made at 100-X under oil for determination of background, and overall grain presence.
71 CHAPTER 4
RESULTS
To examine the molecular expression of sst 2 in the neuroblastoma cell lines
IMR32 and SKNSH, total RNA was isolated from confluent cultured cells using the
method of Chomczynski et al. and Chirgwin et al. (122,123), shown in Figure 4.1.
28 S 18 S
5 S
1 2 3 4 5 6
Figure 4.1: Total RNA Isolation. One p.g total RNA was denatured by boiling for 10 minutes in the presence of formamide loading dye, chilled on ice, and loaded with ethidium bromide on a 1% agarose-1 X TAE gel. The products were electrophoresed at 70 volts for 25 minutes and visualized with UV illumination. The gel shows the 28 S and 18 S ribosomal RNA bands, as well as the 5 S transfer RNA band, with the mRNA smear appearing between the 28 S and 18 S bands. Lane 1: Adrenal; Lane 2: Pituitary; Lane 3: Neuroblastoma; Lane 4\ Neuroblastoma; Lane 5: IMR32; Lane 6: SKNSH.
72 Analysis of ssto expression and human (3 actin expression in SKNSH and IMR32 total RNA was run using Northern analysis (data not shown). Three preparations of RNA from the two cell lines were subjected to RT-PCR analysis for sst 2 expression. The primers for p actin were used as a control for RNA quality for each preparation of total
RNA. Reverse transcription was carried out using random hexamer priming and reverse transcriptase enzyme as described in methods. Results from analysis of sst 2 expression demonstrated relatively higher expression in IMR32 cells than in SKNSH cells, while levels of human pactin in both cell lines were similar as shown in Figure 4.2.
73 <1107 bp
1 2 3 4 5 6 MW MW 9 10 11 12 13 14
Figure 4.2: RT-PCR Analysis of sst; and P Actin Expression in IMR32 and SKNSH Total RNA. Total RNA from three independent preparations of each cell line was transcribed to cDNA using reverse transcriptase and random hexamers. PCR amplification of cDNA was run using Amplitaq DNA polymerase, primers specific for sst; and P actin at 33 cycles of 1 minute at 94°C, 1 minute at 63.8°C, and I minute at 72°C, and finally dwell at 4°C. Ten pi of reaction product was loaded with bromophenol blue loading dye and run for one hour on a 1% agarose/lX TAE gel @ 80 V. Ethidium bromide added to the agarose gel for visualization of the DNA with UV illumination. Lane I: sst; product from IMR32 total RNA; Lane 2: sst; product from IMR32 total RNA; Lane 3: sst; product from IMR32 total RNA; Lane 4: sst; product from SKNSH total RNA; Lane 5: sst; product from SKNSH total RNA; Lane 6: sst; product from SKNSH total RNA; Lane 7: Molecular weight marker, Boehringer-Mannheim Number III; Lane 8: Molecular weight marker, Boehringer-Mannheim Number VI; Lanes 9-11: Human P actin product from IMR32 total RNA: Lanes 12-14: Human P actin product from SKNSH total RNA.
74 The ethidium bromide stained \% agarose/TAE gel depicts the expected band size for primers targeting sst; and human P actin. However, because the somatostatin receptor genes are intronless, the lack of control for genomic contamination persisted. DNase treatment of total RNA prior to RT-PCR analysis was necessitated and carried out using
DNase. DNase treatment using DNasel from Ambion, however resulted in RNA degradation (Figure 4,3). The MessageClean DNasel eliminated DNA contamination without RNase activity and was applied to all tumor RNA analyzed. Furthermore, primers designed to span an intron in the somatostatin peptide and c-abl genes were synthesized and run in parallel RT-PCR reactions.
< 28 S < 18 S
1 2 3 4 5
Figure 4.3: Total RNA Subjected to Ambion DNasel treatment - After and before the reaction. RNA was electrophoresed on a \% agarose-1 X TAE gel and stained with ethidium bromide for UV visualization. Lane 1. RNA isolated from cultured patient neuroblastoma cells (PGA) following DNasel treatment; Lane 2: RNA isolated from cultured patient neuroblastoma cells (PML) following DNasel treatment; Lane 3: PGA RNA prior to Ambion DNasel reaction; Lane 4 : PML RNA prior to Ambion DNasel reaction; Lane 5: SKNSH/sst; total RNA prior to any DNasel treatment.
75 Primer and Probe Design. Primers and oligo probes were designed as described in Methods. Primers to the five sst were confirmed using PCR of a SKNSH genomic
DNA template (Figure 4.4). Probes for Southern confirmation were demonstrated to be specific for each of the five sst by probing the transferred PCR products (data not shown).
< 600 bp
< 200 bp
1 2 3 4 5 6 7
Figure 4.4: PCR Confirmation of sst Primers. PCR amplification of cDNA was run using Amplitaq DNA polymerase and primers specific for the 5 receptors at 33 cycles of 1 minute at 94°C, 1 minute at 63.8°C, and 1 minute at 72°C, and finally dwell at 4°C. Ten fil of reaction product was loaded with bromophenol blue loading dye and run for one hour on a 1% agarose/lX TAE gel @ 80 V. Ethidium bromide was added to the agarose gel for UV illumination of the DNA. Lane 1: 100 bp ladder (Gibco BRL); Lane 2: ssti 481 bp product; Lane 3: sst2 1107 bp product; Lane 4: sstj 517 bp product; Lane 5: ssL 516 bp product; Lane 6 : ssts 625 bp product; Lane 7: 100 bp ladder (Gibco BRL).
76 Somatostatin Receptor Expression in Hum an Neuroblastoma Tumors. Thirty four tissue samples from 26 cases of neuroblastoma diagnosed between 4/91 and 3/95 were provided by the CHTN (Table 4.1).
77 Patient # Age @Dx Sex Primary Stage Shimada9 N- Survival (months) Location Myc A/D** *
t 1 2 Male R IV U U D 2 32 Female R IV U A 3 1 0 Female A IF - A 4 2 1 Male A IV U - D 5 2 2 Female R IV - u D 6 26 Male Abdomen m u FD 7 40 Female A/R IV - U D 8 84 Female M rv - F D 9 4 Female A IV-S - F A 1 0 24 Female A IV u U D 1 1 24 Female N n F F A 1 2 1 day Female M IV-S - - A 13 96 Female A m - - A 14 12 Female A IV High MKI UA 15 12 Male A IV U FA 16 42 Male A IV U FA 17 8 Female Abdomen m F FA 18 48 - A m U FD 19 6 - M i u FA 2 0 15 Female A in u FA 2 1 - - - rv F FA 2 2 1 2 Male A i - - A 23 40 * M n - FA 24 8 Male M i F FA 25 36 Female A IV U UA 26 4 Male M m F F A
Table 4.1 Patient Prognostic Factors, and Outcome
'A, Adrenal; M, Mediastinum; R, Retroperitoneal; N, neck; F, favorable (unamplified); U, unfavorable (amplified) **A, Alive; D, Dead “Histopathologic classification based on stroma rich/poor, mitosis-karyorrhexis index, ferritin (133,134) bInformation not available from CHTN
78 The age of patients at diagnosis ranged from 1 day to 8 years. Eight male and 14 female
patients’ tumors were studied. Primary location of the neuroblastoma was 50% adrenal,
27% mediastinal, 15% retroperitoneal, and 8 % abdominal. Clinical staging is also
indicated in Table I: 46% were Stage IV, 23% were Stage III, 23% were Stages MI, and 2
patients were Stage IV-S. Shimada classification of the 26 patients was 42% unfavorable,
23% favorable, and 30% not classified. N-myc analysis of the 26 cases indicated 53%
with a single copy of N-myc, while 23% had amplified N-myc by Southern blot DNA analysis. N-myc data were not provided for 23% of the cases. Survival at most recent follow up remained at 69% overall for the 26 cases of neuroblastoma, and 50% for patients with Stage IV; none of the patients are > 5 years from diagnosis.
Two neuroblastoma cell lines and three human tissues were subjected to RT-PCR analysis and Southern confirmation as control samples. RNA was isolated from IMR32 and SKNSH neuroblastoma cultured cells, as well as from human adrenal, brain, and pituitary tissue. The control gene c-abl cDNA was expressed in both cell lines and each of the three normal human tissues. No SS cDNA was detected in either the cell lines or the normal human tissues. The receptor subtype analysis demonstrated sst) in both cell lines, adrenal, brain, and pituitary; sst 2 expression in brain and IMR32 cells; sst 3 in none of these samples; ssU expression in both neuroblastoma cell lines; and sst 5 expression only in IMR32 cells (Table 4.4).
79 _ 5' Primer 3' Primer gDNA cDNA
Product Product
(bp) (bp)
c-abl TTC AOCGGCCAGTAGCATCTQ ACTT TOT OAT TAT AOC CTA AGA OCC GGA G 764 bp 201 bp
HpA CAC CTCACC ATG GAT OAT OAT CTC GOC COT OGT GOT GAA GOT 1199 bp 625 bp
SS TATOCTCTCCTGCCGCCTCCAG GAAOACAGGATGTOAAAGICTTCCA 1234 bp 356 bp 1
SSt) COC TOG CTO OTC GOC TIC OTO TTC COC COC COG ACT CCA GGT TCT CAG 481 bp 481 bp
SSt; CAT OOA CAT OOC OGA TOA O CTC AGA TAC TOO TTT OGA G 1107 bp 1107 bp
sst3 GOG AOC COG CTT CAT CAT CTA CAC OAC CCG GCC GTT CAT CTC CTT C 517 bp 517 bp
SSL) TGC TCG GCA OTC TTC CTG OTC TAC CTT OCO OCC GGG TTC TOOT 316 bp 516 bp
SStj OCC GCC TOG OTC CTO TCT CT CCC CCO CCT OCA CTC TCA C 627 bp 627 bp
Table 4.2 Primer Design for the c-abl, human p actin (H$A), Somatostatin (SS) and Somatostatin Receptor Genes 1-5 1(sst.5).
80 Gene Target Probe Sequence
c -a b l CCT TGG AGT TCC AAC GAG CGG CTT CAC TCA GAC CCT GAG GCT
SS CTG GGA CAG ATC TTC AGO TTC CAG GCC ATC ATT CTC CGT CTG
SSt) ACA OCT GAC TCA CCC TGG CGT CGT CCT GCT CAG CAA ACG CGT
SSt; GAG GTC AAA TGG AAT GGA TAG CCA TGT GTG GCT TCC ATT GAG
SStj GOC AGT GGG CAC ACC ACG TTG ACG ATG TTG AGO ACG TAG AAG
ssL> CGC AGC TGT TGG CAT AGC TGA GGA TAA GGG ACA CGT GGT TGA |
SStj CTG GGG CAG CGC CAC GOC CAG GTT GAC GAT GTT GAC GGT GAA
T able 4.3 Probe Sequences for Southern Confirmation of RT-PCR Analysis.
asti itti sat, s*t4 u t, SS cabl Adrenal Brain Pituitary IMR32 SKNSH
T able 4.4 Expression of sstis, Somatostatin (SS), and c-abl (cabl) in control tissues and NB cell lines
81 The thirty four tumor tissue samples from 26 patients with neuroblastoma were
subjected to RNA isolation and RT-PCR analysis (Figure 4.5) with confirmation by
Southern blot (Figure 4.6, Figure 4.7).
< 600 bp
< 200 bp
aat, sst, sst, ast4 sat, SS c-abl bp
Figure 4.5: Neuroblastoma total RNA analyzed by RT-PCR.Total RNA was transcribed to cDNA using reverse transcriptase and random hexamers. PCR amplification of cDNA was run using Amplitaq DNA polymerase, primers specific for the 5 receptors, SS and c-abl at 33 cycles of 1 minute at 94°C, 1 minute at 63.8°C, and 1 minute at 72°C, and finally dwell at 4°C, Ten pi of reaction product was loaded with bromophenol blue loading dye and mn for one hour on a 1% agarose/lX TAE gel @ 80V. Ethidium bromide added to the agarose gel for visualization of the DNA. Lane 1: sst] 481 bp product; Lane 2: sst2 1107 bp product; Lane 3: sstj no product; Lane 4\ sst4 516 bp product; Lane 5: sst5 no product; Lane 6: SS 355 bp cDNA product only; Lane 7: c-abl 201 bp cDNA product only; Lane 8: 100 bp ladder, Gibco BRL.
Expression of the constitutively transcribed c-abl was included as a positive control for
RNA quality (135). All thirty four samples were positive for the c-abl gene 201 bp cDNA product (Figure 4.6); the 764 bp gDNA product was not seen in any of the 34 samples, demonstrating RNA free of DNA contamination. SS cDNA of the expected
356 bp size was positive in 33/34 tumor tissue RNA samples for 97% expression, with
0/34 tumors showing the 1234 bp product for SS gDNA, again verifying the purity of the
82 RNA. These 5 receptor genes are intronless: that the product generated is the resuit of
amplification of cDNA transcribed from mRNA is substantiated by the lack of gDNA
products, for either c-abl or SS. and the positive Southern blot analysis using nested
probes. Representative Southern blots of ssti and sst; are shown in Figure 4.7. Receptor
expression was 100% for ssti, 85% for sst;, 26% for sst;, 85% for sst4, and 2 1% for sst?
(Figure 4.8).
Figure 4.6: Southern Confirmation of RT-PCR.Neuroblastoma RT-PCR cDNA Southern analysis for c-abl (A) and SS (B), control gene cDNA expression demonstrating lack of gDNA product for c-abl and SS. PCR products of amplified cDNA from twelve representative neuroblastoma tumor samples were transferred to a Hybond membrane and uv crosslinked to the membrane using a Stratalinker. Internal 44-mer c-abl or SS oligo endlabeled with [ 7—P*1-]dATP using T4 Polynucleotide kinase were used to probe the membranes by hybridization at 42°C overnight. The membranes were washed @ 65°C following hybridization. A: Amplified cDNA products in lanes 1-12 are positive for 201 bp c-abl cDNA PCR product and negative for the 764 bp c-abl gDNA product. B: Amplified cDNA products are positive for 355 bp cDNA SS PCR product in lanes 1-12. and negative for 1233 bp SS gDNA PCR product.
83 1 2 3 4 S 6 7 S 9101112 1 2 3 4 5 G 7 9 9 10 1112 A B
Figure 4.7: Neuroblastoma RT-PCR cDNA Southern analysis for ssti (A) and sst2 (B). Neuroblastoma RT-PCR cDNA Southern analysis for c-abl (A) and SS (B), control gene cDNA expression demonstrating lack of gDNA product for c-abl and SS. PCR products of amplified cDNA from twelve representative neuroblastoma tumor samples were transferred to a Hybond membrane and uv crosslinked to the membrane using a Stratalinker. Internal 44-mer ssti or ssti oligo endlabeled with [Y-P33]dATP using T4 Polynucleotide kinase were used to probe the membranes by hybridization at 42°C overnight. The membranes were washed @ 65°C following hybridization. A: Amplified cDNA products in lanes 1-12 are positive for 481 bp ssti PCR product. B: Am plified cDNA products are positive for 1107 bp PCR product in lanes 1-3, 6, 8-10. and 12, and negative for 1107 bp PCR product in lanes 4,5,7,11.
Figure 4.8: Gene Expression in 34 Specimens. Summary of expression of sst( somatostatin (SS), and c-abl genes in human neuroblastoma tumors.
84 Multiple tissue samples were analyzed from four of the 26 patients included in our
study. All four patients were positive for both ssti and sst 2 at diagnosis. The sst 2 receptor
was downregulated with progression of disease in two patients. In one of these two
patients, tumor samples were analyzed at diagnosis, at second look surgery, and at
relapse. Positive sst 2 expression was observed at diagnosis and at a second surgery, but
no sst 2 expression was seen at relapse. In the second patient with downregulation of sst 2 ,
the tumor sample from diagnosis was positive for sst 2 while the tumor sample obtained at
relapse was negative for sst 2 expression. In a third patient with multiple tumor samples, 2
samples from 2 foci at diagnosis were examined and identical patterns of expression of
ssti *■ ssts were observed in the two samples. The fourth patient from whom multiple
samples were analyzed had positive sst 2 expression in tumor samples obtained at initial diagnosis and at relapse.
Because 100% of the neuroblastoma tumor specimens studied had sst] expression and because the clinically exploited compound octreotide has affinity to predominantly the sst 2 receptor, the genes for sst 2 and ssti were cloned into expression vectors in order to study the pharmacologic characteristics of both receptors and ligands (i.e. somatostatin analogues).
The product of RT-PCR analysis of IMR32 neuroblastoma cells total RNA using primers for sst 2 (Table 4.2) was ligated into PCRII (Invitrogen) using DNA ligase. The cDNA was cut from PCRII using BstXI, subcloned into pcDM 8 (Fig. 6 ), and sequenced for verification of the coding sequence integrity (data not shown). A cassette containing
85 bp 7-1498 of ssti (GenBank™ accession # M81829) was provided by Graeme Bell. The cassette was cloned into the EcoRV site of pcDNA3 (Invitrogen) by the addition of linkers to blunt end the ssti-containing cassette.
CMV
Figure 4.9; Expression vector pcDM8/sst2 with sst2 cloned into polycloning region.
The constructs pcDM 8 /sst2 and pcDNA3/ssti were transiently expressed in COS-7 cells using either DEAE-Dextran or Lipofectamine transfection. Expression of receptor protein was confirmed by total and nonspecific binding analysis on transfected COS-7 cells, with cell wells analyzed by autoradiography or whole cells by gamma counter.
86 Gene Plasmid Construct 1iiaf-SSi4 Bound (cpm)a
ssti bp 6-1498 pcDNA3 18138 ± 1574
sst2 bp 83-1192 pcDMS 1674 ± 103
sst2 bp 83-1192 pcDNA3 536 ± 30
sst2 bp 13-1253 pcDM8 360
sst2 bp 13-1253 pcDNA3 500
- pcDNA3 209 ± 69
Table 4.5 COS-7 Expression of pcDNA3/sstj, pcDM8 /sst 2 , and pcDNA3/ssti, evaluated by total binding[ ito 2 I-tyrn] Somatostatinj4 . “Values are cpm for whole cells bound in serum-free MEM 30 minutes at 37°C, 5% C 0 2 , 100,000 cpm/50 p 1/15.5 mm well. Binding is 48 hours following Lipofectamine (Gibco BRL) transfection of 2 pg plasmid DNA per well of 1.5 x 105cells plated in triplicate 24 hours prior to transfection.
Stable Expression of ssti and sst2 in Neuroblastoma Cell Lines. Transient transfection of the plasmid constructs in COS-7 cells identified the pcDNA3/ssti and pcDM 8 /sst2 constructs which expressed high affinity binding of SS 14 (Table 4.5). Stable transfection of mammalian cells was carried out as shown in Figure 4.10.
87 M utton CtofMl Expansion
Figure 10: Transfection of mammalian cells with pcDNA3/sst| or pcDMS/sst^* psV2Neo, followed by limiting dilution and clonal expansion from single cells. Cells plated in a 6 well culture plate (Coming, Coming, NY) were transfected, selected with geneticin, plated to one cell per well of a 96 well culture plate (Coming, Coming, NY), and expanded from the one cell to a culture flask of a continuous cell line.
The cell line SKNSH was selected for stable transfection with the receptor-expressing plasmids because 1) SKNSH is a human neuroblastoma cell line, 2) SKNSH have low levels of ssti and no levels of sst 2 mRNA expression, and 3) SKNSH cells demonstrate little binding to l 2 5 I-SS | 4 (Figure 4.11). SKNSH cells stably transfected with pcDM 8 /sst2 were compared to SKNSH cells stably transfected with pcDNA 3 /sst2 for total and nonspecific binding to 125I-SSi4 with or without SS | 4 (Figure 4.11).
Stable transfections of pcDM 8 /sst2 were created using a co-transfection of the construct with the resistance vector psv2neo. A ratio of 20:1 of receptor vector to resistance vector was used for transfection to ensure expression of the sst 2 gene and not solely Neo. Stable transfections with pcDNA3/ssti were carried out directly for G418 selection, because pcDNA3 contains the Neo gene in the construct. The transfected cells were selected under concentrations of 500 [ig/ml G418 (Gibco BRL).
88 Figure 4.11: Binding analysis of stably transfected SKNSH cells.Transfected SKNSH were plated in 35 mm culture wells at 2 x 10s cells/well and analyzed by total binding (TB) with , 2 3 I-SS |4 or by nonspecific binding (NSB) with 1 2 5 I-SS |4 and 0.1 |iM SS,4 - The binding results were analyzed by autoradiographic analysis of the wells in the pictured 5 x 5 well matrix. A l: pcDM 8 /sst2 NSB A2-A3: pcDM 8 /sst2 TB A4-A5: clonal dilution cell line of pcDM 8 /sst2 NSB. B1-B4: clonal dilution cell line of pcDM 8 /sst2 TB B5: SKNSH cells transfected with only pcDNA3 NSB. Ct: pcDNA3 only NSB C2-C3: pcDNA3 only TB C4-C5: pcDNA3/sst 2 NSB D1-D2: pcDNA3/sst 2 TB
D3: pcDNA3/sst 2 NSB D4-D5: pcDNA3/sst 2 TB E1-E2: SKNSH cells only NSB E3-E4: SKNSH cells only TB E5: No well present.
89 Figure 4.11 (continued)
90 Transcriptional upregulation of both receptor genes was demonstrated by RT-PCR analysis of ssti (Figure 4.12) and sst 2 (4.12 & 4.13) in transfected SKNSH and the sst 2 expressing neuroblastoma cell line, IMR32 (Figure 4.13) and confirmed by binding studies (Figure 4.14). RT-PCR on total RNA isolated from clonal cell lines demonstrated upregulated sst 2 (Figures 4.12 & 4.13).
< 600 bp < 200 bp
1 23456789
Figure 4.12: RT-PCR confirmation of ssti and sst2 upregulation in SKNSH neuroblastoma cells. Respective volumes of 100 pi RT-PCR reactions electrophoresed on a 1% agarose-IX TAE gel, stained with ethidium bromide, and photographed under UV illumination. Confirmation of RNA quality by positive expression of c-abl cDNA product. Lane 1: 100 bp DNA ladder (Gibco BRL); Lane 2: 20 pi SKNSH cDNA product, 481 bp ssti product; Lane 3: 10 pi SKNSH cDNA product, no 1107 bp sst 2 product; Lane 4: 10 pi SKNSH cDNA product, 201 bp c-abl cDNA product only; Lane 5: 5 pi SKNSH/pcDNA3/ssti cDNA product, 481 bp ssti product; Lane 6: 10 pi SKNSH/pcDNA3/ssti cDNA product, 201 bp c-abl cDNA product only; Lane 7: 5 pi
SKNSR/pcDM 8 /sst2 cDNA product, 1107 bp sst 2 product; Lane 8; 10 pi SKNSH/pcDM 8 /sst2 cDNA product, 201 bp c-abl cDNA product only primers; Lane 9: 100 bp ladder (Gibco BRL).
91 Figure 4.13: RT-PCR confirmation of sst2 upregulation. Ethidium bromide stained 1% agarose/1 X TAE gel with 10 pi of RT-PCR reaction samples electrophoresed with the 100 bp ladder (Gibco BRL). Confirmation of RNA quality by positive expression of c-abl primer product. Lane L. Positive IMR32 cDNA, sst 2 primers; Lane 2: Negative SKNSH cDNA, sst2 primers; Lane 3: Positive SKNSH/pcDM 8 /sst2 , ssti primers; Lane 4: 100 bp ladder; Lane 5: Positive IMR32 cDNA, c-abl primers; Lane 6: Positive SKNSH cDNA, c-abl primers; Lane 7; Positive SKNSH/pcDM 8 /sst2 , c-abl primers; Lane 8: 100 bp ladder (Gibco BRL). Competitive binding analysis confirmed protein expression of the two receptor
subtypes in SKNSH neuroblastoma cells (Figure 4.14). Competitive binding analysis of
the SKNSH cell line in whole cell binding demonstrates little total binding; while
upregulation of either ssti or sst 2 increases the total binding of SKNSH 8 fold (Figure
4.15). Expression of the genes for ssti and sst 2 in the neuroblastoma cell line SKNSH
facilitated a study of the pharmacologic characteristics of the two receptors, and of three
analogs for SS)4.
Incnulnfl eone. of poptldo
Figure 4.14: Competitive binding analysis on whole celts.Constant amount of Buffer S/0.5 % human serum albumin versus varying amounts of unlabeled peptide, incubated for 30 minutes at 37°C, washed, trypsinized and results counted on a gamma counter.
93 Binding: SKNSH Neuroblastoma Cells
■ SKNSI-VpSV2neo 2000 - a SKNShVsstl o, *-SKNSH/sst2 ■oc 1500- 3 O m 1000- cn CO ■n CM 500-
_r— |— |— |— r 12 11 10 9 6 7 6 -log [SS14], M
Figure 4.15: Competitive Binding of Somatostatin-[tyru ] 3-[l25IJ iodotyrosyl 11 versus unlabeled SS 14 on SKNSH neuroblastoma cells, on SKNSH/pcDNA3ssti, and on SKNSH/pcDM 8 sst2 neuroblastoma cells. Cultured SKNSH/psv2neo (filled
squares), SKNSH/pcDNA3ssti (filled triangles), and SKNSH/pcDM 8 sst2 (filled circles) cells plated in 7 wells each in a Coming 24-well culture plate. Cells were incubated for 30 minutes at 37°C, 5% C 0 2 in buffer S, 15 pmoles 1 2 3 I-SSi4t and indicated concentration of unlabeled peptide after which the reaction mixture was aspirated and cells were washed with serum free MEM (Gibco BRL). The cells were trypsinized and counted on a gamma counter (Beckman).
94 Pharmacology of ssti and sst2 in Neuroblastoma. Analysis of binding in the
SKNSH/ssti cell line demonstrated high affinity binding of SS|4. CH275 (130), and
WOC3b, but little affinity to octreotide (Figure 4.16).
125f-SS14 Binding: SKNSH/sst-i
SS14 a - -O C T ++z 100 - --W O C -3 b O CH275 3?
50-
12 11 10 9 e 7 6 -log [unlabeled peptide], M
Figure 4.16: Competitive Binding of Somatostatin-ftyr11] 3-[,25I] iodotyrosyl11 versus unlabeled SSj4 on SKNSH/pcDNA3ssti neuroblastoma cells. Cultured SKNSH/pcDNA3sstj cells plated in 7 wells each in a Coming 24-well culture plate. Cells were incubated for 30 minutes at 37°C, 5% CO2 in buffer S( 15 pmoles 5 I-SSi4, and indicated concentration of unlabeled SS14, octreotide (OCT), WOC3b( or CH275 after which the reaction mixture was aspirated and cells were washed with serum free MEM (Gibco BRL). The cells were trypsinized arid counted on a gamma counter (Beckman).
95 Analysis of binding in the SKNSH/sst 2 cell line demonstrated high affinity binding of
SS14, octreotide, and WOC3b, but little affinity to CH275 (130) (Figure 4.17).
125,f-SS14 Binding: SKNSH/sst2
SS14 OCT
100- /— WOC-3b CH275
.8 50- E Q. O
12 11 10 9 8 7 6 -log [unlabeled peptide], M
Figure 4.17: Competitive Binding of Somatostatin-flyr11] 3-[125I] iodotyrosyl11 versus unlabeled SS14 on SKNSH/pcDM8sst2 neuroblastoma cells. Cultured SKNSH/pcDM 8 sst2 cells plated in 7 wells each in a Coming 24-well culture plate. Cells were incubated for 30 minutes at 37°C, 5% C 0 2 in buffer S, 15 pmoles 1 2 5 I-SSm, and indicated concentration of unlabeled SS|4( octreotide (OCT), WOC3b, or CH275 after which the reaction mixture was aspirated and cells were washed with serum free MEM (Gibco BRL). The cells were trypsinized and counted on a gamma counter (Beckman).
96 The results of the binding analyses of these two cell lines and their affinities (K d , nM) to
somatostatin and three somatostatin analogues are summarized in Table 4.6, The two cell
lines demonstrate high affinity to both SS | 4 and a recently developed analogue WOC3b
(Woltering, O’Dorisio, and Coy).
Peptide SKNSH/ssti SKNSH/sst2
KD(nM) Kd (nM)
S S 14 1.2 ± 0 .7 7.8 ± 4.3
Octreotide >200 12.0 ± 7.8
CH275 5.6 ± 2.8 >200
WOC3b 49.2 ±41.0 19.7 ± 5.6
Table 4.6 Affinity(Kd) of Peptides for sstf and sst2
The pharmacologic analysis of sst| and sst 2 in SKNSH with various analogues for somatostatin indicate that WOC3B is the first long acting analogue to somatostatin with affinity to the two families of sst: ssti and sst2. The compound CH275 demonstrates affinity to ssti but not to sst2. The clinically exploited long acting analogue octreotide demonstrates high affinity to sst 2 but not to sst|.
97 Neuroblastoma xenografts of SKNSH, SKNSH/ssti, and SKNSH/sst 2 were
analyzed to study the in viva growth rate of upregulated sst neuroblastoma cells, and in
vivo binding of , 2 5 I-labeled somatostatin analogues as detected by radioreceptor guided
surgery (RRGS) and autoradiography.
Growth of Xenografts. Fifty six athymic mice were injected with neuroblastoma
cells as follows: 19 SKNSH, 16 SKNSH/sst|, and 21 SKNSH/sst2. Tumor development
was recorded when a visible mass appeared on gross examination of the mouse; results of
tumor development are recorded in Figure 4.18.
Neuroblastoma Xenograft T umorigenesis
1 — SKNSH JS — SKNShVsst 3 —- SKNSH/sst:
1-Tumor Free, 0-Tumor
o 0 25 50 75 100 Days to Tumor Appearance
Figure 4.18 Survival Analysis for Xenograft Tumorigenesis.
98 In vivo RRGS binding studies demonstrated tumor to background ratios ranging from 1.4 to 10. Tumor binding was determined on the left leg location of the tumor, and background was the counts measured on the right leg of the same animal, where no tumor occurred. Figures 4.19,4.20, and 4.21 demonstrate the time course and radioactivity counts using ,25I-CH288 (130), the tyrosinated form of CH275 or 123I-tyr3-octreotide and
RRGS to study on SKNSH, SKNSH/ssti , and SKNSH/sst 2 tumors.
SKNSH RRGS 7.5n □ 125I-CH288 125 l-octreotide S. s 8 50
i s 2.5- O O
0.0 Time (minutes)
Figure 4.19: SKNSH RadioReceptor Guided Surgery Time Course
99 SKNSH/sst, RRGS
10.CH « □ 125I-CH288 H U 1251-octreotide 5 1 7.5- - £ QC ® 5.0- I s I H O 2.5-I I
0 . 0 - 30 60i Time (minutes)
Figure 4.20: SKNSH/sst] RadioReceptor Guided Surgery Time Course
SKNSH/ssfe RRGS 7.5-1 □ 125I-CH288 H 125l-octreotide
Time (minutes)
Figure 4.21: SKNSH/sst2 RadioReceptor Guided Surgery Time Course
100 Autoradiography of xenograft tumor tissue and mouse tissue following the radioreceptor study demonstrated uptake of 123I-tyr3-octreotide in the ganglion cells within the muscular layers of the colon (Figure 4.22). I25I-CH288 uptake was not demonstrated in colon ganglion cells (Figure 4.22). Radioreceptor analysis using injected
1 2 5 I-SSu was run on 1 SKNSH mouse and two SKNSH/sst 2 mice. , 25 l-SSi4 uptake by the colonic ganglia was markedly positive in these animals’ colons (Figure 4.22).
Autoradiographic analysis of the xenograft neuroblastoma tumor tissues has not demonstrated silver grain positivity.
101 A.
Figure 4.22: Autoradiographic analysis of in vivo binding. Photograph of formalin fixed, parafin-embedded tissue section exposed for 21 days to Kodak photographic emulsion and developed using Kodak Dektol developer and fixer, at 40 X magnification demonstrating positive in vivo binding in radioreceptor-studied mice. A: l 2 5 I-SS ]4 studied animal, silver grains cover ganglion cells; B: I2 3 I-tyr3-octreotide studied animal, silver grains cover ganglion cells; C: 125I-CH288 studied animal, no silver grains cover the ganglion.
102 Figure 4.22 (continued) Figure 4.22 (continued)
C. The binding o f 123I-tyr3-octreotide to colonic ganglion cells could be inhibited in vivo. A
10,000 fold excess of unlabeled octreotide inhibited colonic binding as demonstrated in
Figure 4.23.
A.
Figure 4.23: Autoradiographic analysis of in vivo binding. Photograph of formalin fixed, parafin-embedded tissue section exposed for 21 days to Kodak photographic emulsion and developed using Kodak Dektol developer and fixer, at 40 X magnification autoradiography demonstrating positive in vivo and negative in vivo binding from radioreceptor studied mice injected with l25I-tyr3-octreotide or 125I-tyr3-octreotide and a 10,000 fold excess of unlabeled octreotide five minutes prior to 125I-tyr3-octreotide injection. A: I-tyr -octreotide injected animal, silver grains cover colonic ganglion cells; B; 123I-tyr3-octreotide studied animal with 0.1 jiM octreotide added five minutes prior to radioactive label injection, no silver grains cover colonic ganglion cells.
105 Figure 4.23 (continued)
B.
Functional binding of 125I-CH288 was demonstrated in the interstitial tissue surrounding the exocrine submandibular gland tissue. I25I-tyr3-octreotide did not demonstrate binding on the neuronal cells in the same area. The binding in the submandibular gland region is shown in Figure 24.
106 A. p w ? *' %
Figure 4.24: Autoradiographic analysis of in vivo binding. Photograph of formalin fixed, parafin-embedded tissue section exposed for 21 days to Kodak photographic emulsion and developed using Kodak Dektol developer and fixer, at 40 X magnification autoradiography demonstrating positive in vivo and negative in vivo binding from radioreceptor studied mice injected with A: 123I-CH288 injected animal, silver grains cover interstitial neuronal cells; B: I-tyr -octreotide studied animal, no silver grains cover interstitial neuronal cells.
107 Figure 4.24 (continued)
B. Xenograft tumors were analyzed for ssti and sst; expression using RT-PCR and the primers described in Table 4.3. Three different mouse tumors from each neuroblastoma cell line were processed for total RNA isolation as described for the patient neuroblastoma tumors above. RT-PCR analysis demonstrated that the
SKNSH/sst) and SKNSH/sst; xenograft tumors express upregulated sst| and sst 2 respectively, in vivo (Figure 4.26 and Figure 4.27). Each are more highly transcribed than ssti and sst 2 in SKNSH xenograft tumor RNA, which demonstrated only c-abl expression by RT-PCR analysis (Figure 4.25).
109 mm W-f <600 bp <200 bp
1 2 3 4 5 6 7
Figure 4.25: SKNSH Xenograft Neuroblastoma total RNA analyzed by RT-PCR. Total RNA was transcribed to cDNA using reverse transcriptase and random hexamers. PCR amplification of cDNA was run using Amplitaq DNA polymerase, primers specific for ssti, sst 2 , SS and c-abl at 30 cycles of 1 minute at 94°C, 1 minute at 63.8°C, and 1 minute at 72°C, and finally dwell at 4°C. Ten (±1 of reaction product was loaded with bromophenol blue loading dye and run for one hour on a 1% agarose/IX TAE gel @ 80 V. Ethidium bromide added to the agarose gel for visualization of the DNA with UV illumination. Lane I: 100 bp ladder (Gibco BRL); Lane 2: sstj no product; Lane 3: ssts 1107 bp product; Lane 4: SS no product; Lane 5: c-abl 201 bp cDNA product only; Lane 6: c-abl, No Reverse Transcriptase Added, no product; Lane 7: 100 bp ladder (Gibco BRL)
110 < 600 bp
1 2 3 4 5 6 7
Figure 4.26: SKNSH/ssti Xenograft Neuroblastoma total RNA analyzed by RT- PCR. Total RNA was transcribed to cDNA using reverse transcriptase and random hexamers. PCR amplification of cDNA was run using Amplitaq DNA polymerase, primers specific for ssti, sst2, SS and c-abl at 30 cycles of 1 minute at 94°C, 1 minute at 63.8°C, and 1 minute at 72“C, and finally dwell at 4°C. Ten pi of reaction product was loaded with bromophenol blue loading dye and run for one hour on a \% agarose/IX TAE gel @ 80 V. Ethidium bromide added to the agarose gel for visualization of the DNA with UV illumination. Lane 1: 100 bp ladder (Gibco BRL); Lane 2: ssti 481 bp product; Lane 3: sst2 no product; Lane 4: SS no product; Lane 5: c-abl 201 bp cDNA product only; Lane 6: c-abl, No Reverse Transcriptase Added, no product; Lane 7: 100 bp ladder (Gibco BRL)
111 <1100 bp <600 bp
1 2 3 4 5 6 7
Figure 4.27: SKNSH/sst 2 Xenograft Neuroblastoma total RNA analyzed by RT- PCR. Total RNA was transcribed to cDNA using reverse transcriptase and random hexamers. PCR amplification of cDNA was run using Amplitaq DNA polymerase, primers specific for ssti, sst 2 , SS and c-abl at 30 cycles of l minute at 94°C, 1 minute at 63.8°C, and 1 minute at 72°C, and finally dwell at 4°C. Ten fil of reaction product was loaded with bromophenol blue loading dye and run for one hour on a 1% agarose/1 X TAE gel @ 80 V. Ethidium bromide added to the agarose gel for visualization of the DNA with UV illumination. Lane I: 100 bp ladder (Gibco BRL); Lane 2: ssti no product; Lane 3: sst2 1107 bp product; Lane 4\ SS no product; Lane 5: c-abl 201 bp cDNA product only; Lane 6: c-abl, No Reverse Transcriptase Added, no product; Lane 7: 100 bp ladder (Gibco BRL).
112 CHAPTER 5
DISCUSSION
The molecular signaling pathways which regulate proliferation and differentiation
to guide neural crest development remain unclear. Neuroblastoma presents a clinical
manifestation of transformed neural crest development. Studies of neuroblastoma in vitro
and in vivo have helped expand what is known about neural crest progenitors and their derivatives. While increasing our understanding of neurogenesis, these studies have also yielded therapeutic opportunities for a devastating tumor of childhood.
The idea of cell dependence on external signals activating internal signaling pathway has increased our understanding of neuronal development and survival.
Neurotrophic factors have been implicated in neuroblastoma in correlation with the N- myc oncogene, as well as in correlation with survival and tumor stage. Neuropeptides also play a role in neural crest development and neuroblastoma.
My hypothesis for these experiments is that a specific somatostatin receptor subtype is expressed in neuroblastoma. By inference, this receptor will play a role in neural crest development. A second hypothesis is that delineation of somatostatin receptor subtype expression in neuroblastoma and identification of subtype specific
113 ligands will provide the basis for improved receptor based scintigraphy in neuroblastoma.
The hypotheses were tested in three areas: 1) in vitro analysis of gene expression; 2) in vitro pharmacologic characterization; and 3) in vivo growth studies, pharmacologic characterization, and analysis of gene expression.
The first studies analyzed the expression of the somatostatin peptide gene as well as expression of five somatostatin receptors in 34 tumors from 26 patients with neuroblastoma. Somatostatin peptide gene expression was positive in 33/34 tumor samples analyzed. Expression of somatostatin receptor type 5 had the fewest number of positive tumor samples among the genes analyzed, with expression in only 9/34 tumor samples. Receptor subtype 4 was expressed in 29 of 34 samples. Because sst4 has been shown to inhibit proliferation via activation of tyrosine phosphatase activity (101), this widespread expression of sst4 in neuroblastoma offers an exciting area for further study.
All tumor specimens expressed ssti while 29/34 expressed ssti.
The long acting analogue currently exploited in somatostatin receptor based imaging is octreotide, which binds with high affinity to ssti, with intermediate affinity to sst3 and sst;, but with low affinity to ssti and ssu (103). The lack of expression of ssti in
15% of the tumor samples analyzed parallels the 15% false negative rate of somatostatin receptor based scintigraphy observed in neuroblastoma patients (87,106,115).
In Dr. O’Dorisio’s* study of 8 patients » who underwent “ I-tyr-3-octreotideI ^ "I scintigraphy, two had false negative results (115). Both of the patients who had false negative results by scintigraphy were scanned at relapse. The analysis of sst; expression by RT-PCR confirmed this downregulation of sst; at relapse in two patients. The other
114 two independent studies have confirmed a 15-20% false negative rate in octreotide based
scintigraphy of neuroblastoma (87,106). Downregulation of sst 2 with progression of
disease may contribute to false negative scintigraphy. A larger prospective study will be
required to determine whether downregulation of sst 2 during tumor progression has
clinical significance.
The three neuroblastoma cell lines SKNSH, SKNSH/ssti, and SKNSH/sstj were
studied in vitro to characterize the pharmacologic profiles of somatostatin and the
somatostatin analogues octreotide, CH275, and WOC3b. These studies confirm the
finding of Liapakis et al. that CH275 binds to human ssti with high affinity (130). The
studies described in this dissertation are the first to examine affinity of CH275 for human
ssti. Thus, des-A A125[DT rp8,IAmp9] SRIF (CH275) displays high affinity to human ssti
but low affinity (K d > 200 nM) to human sst 2 - These studies also present the first analysis
of a somatostatin congener with affinity for both ssti and ssti. WOC3b is the first
tyrosinated somatostatin analogue to demonstrate affinity Kd’s in the nanomolar range for
both ssti and ssti. The range of affinities that are effective and efficient for imaging and
detection of neuroblastoma has not been demonstrated.
Expression of sstj in several other human tumors has been recently reported.
Kubota et al. studied glucagonoma, insulinoma, pheochromocytoma and carcinoid tumor
types (84) using RT-PCR analysis and found sst] expression in all 12 tumors. Vikiv-
Topic et al. reported sst] expression in 73% of 55 human breast tumor specimens and in
85% of 28 human carcinoid and renal cell tumor specimens analyzed by RT-PCR (93).
While expression of ssti in Vikiv-Topic’s study was higher than the expression of sst],
115 these combined results suggest an analogue with affinity to ssti, a combination of both
sstj and sst 2 specific analogues, or an analogue such as WOC3b, which recognizes both
ssti and sst 2 , would offer more effective receptor based imaging.
Further studies of the physiologic regulation of ssti in neuroblastoma are needed.
While ssti mRNA has been identified in several tumors, the mRNA expression does not
necessarily correlate with identification of ssti receptor protein. Positive sst 2 mRNA
expression has been demonstrated in neuroendocrine tumors which were negative by
octreoscan scintigraphy (94). My studies demonstrate ssti mRNA in SKNSH cells, but
negligible binding of l25I-SS| 4 . However, high affinity binding of 125I-SSi 4 is observed
with ssti upregulation, in both transiently transfected COS-7 cells and in stably
transfected SKNSH cells. The half life of the ssti protein may be short in vivo due to the
presence of PEST sequences unique to ssti among somatostatin receptors (136). The
effect of high receptor turnover on in vivo scintigraphy is not presently known.
The third series of experiments studying the biology of somatostatin receptors in
neuroblastoma exploited a xenograft model developed by Dr. O’Dorisio (137). Athymic
mice received injections of 30 million neuroblastoma cells from the three cell lines
SKNSH, SKNSH/ssti, and SKNSH/sst 2 . Cell line tumorigenesis was observed closely and time to tumor development recorded. Survival analysis for tumorigenesis detected no significant difference in time to tumor development for the three cell lines. However, at the close of the study, only the SKNSH/sst 2 cell line had mice that remained tumor free beyond 50 days. Somatostatin levels in the athymic mice at the time of cell injection and during tumor development are not known. Endogenous somatostatin may generate either
116 a positive or negative feedback loop for regulatory peptides (i.e. somatostatin) which is
dependent on sst expression. Such a feedback loop might regulate production of a
number of growth factors including GH, insulin-like growth factor-1 (IGF-1), epidermal
growth factor (EGF), leukemia inhibitory factor (LIF), NGF, and platelet derived growth
factor (PDGF) as well as neuropeptides such as NPY, VIP, and somatostatin.
The in vivo pharmacologic characteristics of the somatostatin analogues octreotide
and the iodinated CH288 (identical to CH275 except for the tyrosine at position 1 that
facilitates iodination) were studied in the xenograft model. Twenty jo,Ci of l25I-labeled
analogue (0.01 nM) was injected intraperitoneally and Neoprobe counts per 2 seconds
monitored. Radio-receptor guided surgery (RRGS) was monitored at 5, 15, 30, and 60 minutes post radiolabel injection. The counts were analyzed for statistical difference between the ratio of tumor counts to right leg, or background counts, for the four timepoints. The p value for difference between the ratios of counts was greater than 0.05, however tumor counts were always higher than right leg counts. Clinically significant ratios of 2 or higher indicate tumor uptake of radiolabel, and values of >2 were seen for the xenograft tumors studied with either ,25I-CH288 or l25I-tyr3-octreotide. However, no specificity for upregulated receptor subtype - specific somatostatin analogue was observed. Longer timepoints for RRGS may be necessary to detect significant tumor localization in the xenograft model because in vivo receptor binding characteristics remain to be determined. Zamora et al. analyzed bio-distribution of 188Re-RC-160
(Rhenium labeled, cyclic D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH 2 , a SS analogue with high affinity for sst 2 > at 2 hours and 24 hours post injection (138). The model used
117 was a xenograft of PC-3 prostate cancer cell tumors in athymic mice (138). High uptake
was measured in liver and gut, indicating clearance through the hepatobiliary route.
Uptake was higher in tumor lesion than in muscle of the opposite normal leg. Uptake
could be significantly reduced by addition of excess octreotide or RC-160. These studies
determined that tumor specific retention of the radiolabel could be detected at 2 and 24
hours post intralesion injection (138). Intraperitoneal injection of radiolabeled peptide
might have higher tumor specificity at such longer timepoints.
In my neuroblastoma xenograft studies, the maximal timepoint of 60 minutes was
chosen for autoradiography for the tumors because this was the optimal time period for
,25I-tyr3-octreotide binding to colonic ganglia and l25I-CH288 binding to neuronal cells in
the submandibular interstitium. In the ganglion cells of the colon, grain deposition was
strongly positive for 125I-tyr3-octreotide labeled animals. This grain deposition could be
effectively reduced with addition of 10,000 fold excess unlabeled octreotide added 5
minutes prior to radiolabeled peptide injection.
Sixty minutes demonstrated specific uptake of ,25I-CH288 in discreet cells of the
submandibular interstitium that co-localize with neuronal ganglia. This grain deposition
was specific to l2SI-CH288 labeled animals, and was not present on the same cells from
125I-tyr3-octreotide injected animals. Interestingly, 125I-CH288 injected animals had no grain deposition on colonic ganglion cells. The specific labeling of 125I-CH288 demonstrates in vivo binding for this newly developed somatostatin congener.
No tumor uptake was noted in tumor autoradiography of tumor tissue harvested at
1 7 ^ 1 7C 'i 60 minutes in either I-CH288 or I-tyr -octreotide injected mice. Because blood flow
118 to the increased tissue mass (the vascularized neuroblastoma) may account for the tumor
uptake of radioactivity, co-injection of SS analogue and 131I-albumin should be studied.
The radiolabeled albumin or any compound that would be contained in the circulation
would indicate whether blood flow is the cause of higher counts for the tumor versus the
background right leg.
Gene expression in the xenograft tumors was analyzed using RT-PCR. The gene
expression of ssti, sst 2 , SS, and c-abl in total RNA isolated from xenograft mouse tumors
was determined as follows, RNA was isolated from three tumors for each cell line
injected using the methods applied to the neuroblastoma samples. The RNA was DNased
as described and RT-PCR run to determine whether expression of the upregulated
receptor genes persisted in vivo, and whether SS was upregulated by in vivo factors. Gene
expression was preserved in vivo as in vitro. SS was not upregulated in the xenograft
tumor. The results of the RT-PCR analysis of the xenografts indicate that the receptor
genes that were upregulated in vitro remained upregulated in vivo.
Questions generated from these studies concern transcriptional regulation of sst and pharmacologic characteristics in vivo. Whether mRNA (or cDNA) reflect functional receptor proteins remains unclear. In vitro versus in vivo affinity analysis for somatostatin congeners and specific receptor subtypes has not been determined. The Kp for a somatostatin congener-receptor in vitro that will yield effective imaging/binding in vivo remains unclear; but could be tested using the model developed in the studies described in this dissertation. Gene expression upregulated in vitro is preserved in vivo in the xenograft model of neuroblastoma, offering an in vivo model for specific receptor-
119 analogue interaction and analysis. With clonal expansion of cell lines expressing ssl 3 , ssL*, and sst5, in vitro and subsequent in vivo somatostatin congener studies are possible.
Longer timepoints must be studied in vivo, for example at 4, 8, 12, and 24 hours post radiolabel injection. These experiments would help describe what steady state binding affinity is reached and at what timepoint for a somatostatin congener and each of the five somatostatin receptor subtypes.
This work presents the first molecular analysis of sst expression in neuroblastoma.
The in vitro binding of somatostatin analogues to viable neuroblastoma cells constitutively expressing a specific receptor subtype offers an effective means for studying the pharmacologic characteristics of each sst in vitro in neuroblastoma cells. All neuroblastoma samples expressed sst). Expression of sst 2 explains the false negative rate seen in somatostatin receptor based scintigraphy of neuroblastoma. Pharmacologic studies of CH275, octreotide, and WOC3b indicate that CH275 has high affinity to human ssti, octreotide has high affinity to human sst 2 , and WOC3b is the first congener to demonstrate high affinity to both ssti and sst 2 .
In vivo analysis of the growth of ssti and sst 2 upregulated SKNSH neuroblastoma cells demonstrated that tumorigenic potential is preserved with sst upregulation. This initial study does not demonstrate inhibition of xenograft tumor formation by upregulation of either ssti or sst 2 - However, in vivo RRGS detected neuroblastoma xenograft tumors with two different somatostatin analogues and suggests that a combination of I251-CH288 and 125I-tyr3-octreotide, would improve in vivo detection of neuroblastoma. Additionally, the use of a multityrosinated analogue such as the tri-
120 tyrosinated WOC3b, which binds to both sst] and sst 2 offers potential for scintigraphic imaging and tumoricidal radiotherapy by increased radiodose to the tumor site or to residual tumor.
Further studies of plasticity in the transfected neuroblastoma cells, whether subject to retinoic acid exposure, various hormones, or leukemia inhibitory factor, will build on our ideas of neural crest development. Signaling between neuroblastoma cells and the peripheral nervous system is implied in studies of maturing neuroblastomas that show different ploidy between Schwann and neuronal cells in one tumor (139).
Somatostatin receptor subtype expression in neuroblastoma merits analysis as a significant part of understanding the interactions of genes and peptides in the development of the neural crest. Additionally, the application of receptor biology to diagnosis and therapy in neuroblastoma offers exciting prospects in understanding the biology and improving the clinical management of this devastating tumor of childhood.
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