9. Neural Cell Lines (Lineage)

Full text

Neural Cell Lines (Lineage) 169
9
Neural Cell Lines (Lineage)
Qiang Gu
Introduction
The pioneer work by Ross Granville Harrison, who successfully grew cells outside
the body over a century ago, formed the basis of modern cell culture techniques that
allow the study of isolated living cells in an artifi cially controlled environment. Over
the past century, advancements in knowledge, theory, practice and methodologies have
made cell cultures indispensable tools for basic life science, biomedical and clinical
research. To date, many aspects of cell culture techniques such as plate coatings,
growth media and incubation conditions have been standardized, and a wide variety
of cells can be isolated, purifi ed, grown and maintained in vitro. In addition to some
obvious advantages of cell culture systems compared to in vivo situations such as
being simple, homogeneous cell populations, easy-controllable cell exposures and
easy-manipulatable gene expressions, an important aspect of using cell cultures
that deserves a notion here is to set alternative approaches to reduce animal use in
experimentation. Due to the unique character of differentiated neurons that they are
no longer dividing and non-multipliable, a number of cell sources have been explored
with the goal to establish immortalized neural cell lines. These include cells derived
from spontaneous or artifi cially-induced brain tumors, fusion of neurons to tumor cells,
retroviral infection of neural precursor cells, or neurons transfected with oncogenes.
To date a vast variety of cells lines have been developed that continuously express
specifi c neurotransmitters and that are utilized as neuronal cell lines. In addition, several
glial cell lines derived from glioma or glioblastoma have also been established and
Division of Neurotoxicology, HFT-132, National Center for Toxicological Research (NCTR), U.S. Food
and Drug Administration (FDA), 3900 NCTR Rd, Jefferson, AR 72079.
E-mail: qiang.gu@fda.hhs.gov

170 Neural Cell Biology
characterized. Collectively these cell culture systems have been widely used for studies
of neural growth, differentiation, synaptogenesis, gene expression, plasticity, regulation
of transmitter release, signal transduction, pharmacology and toxicology. Given the
wide variety of available cell lines and limited space allowed in this chapter, it would
be impossible to review all neural cell lines. Therefore, this chapter will cover the most
widely studied and well-characterized cell lines in neuroscience research (Table 9.1),
and their applications and important results of those studies. An important objective
and envision of developing neuronal cell lines in those early days was to generate
mass quantities of a particular type of cells or cells carrying certain specifi c genes
for clinical transplantation with the purpose to repair or cure neurological disorders.
However, with the promising features of induced pluripotent stem cells, much effort
and hope have been shifted towards utilizing neural stem cells. Because neural stem
cells have been described in a preceding chapter (Chapter 6) in this book, they will
not be covered here to avoid redundancy.
Table 9.1. Cell lines covered in this chapter.
Cell Origin Species Cell Line Name Year Established*
Pheochromocytoma Rat PC12 1975
Neuroblastoma Human SH-SY5Y 1978
Mouse N1E-115 1972
Neuro-2a 1969
Rat B104 1974
Medulloblastoma Human TE671 1977
Chronic Focal Encephalitis Human HCN-2 1994
Glioma Human U87MG 1968
Rat (Wistar) C6 1968
Rat (Fisher) F98 1971
RG2 1973
Neuroblastoma-Glioma Hybrid Mouse/Rat NG108-115 1971
SV-40 Viral Transfected Brain Tumor Mouse CATH.a 1993
* Year of creation or when the fi rst study utilizing the cell line was published.
Cell Lines of Neuronal Origin
PC12 cell line (rat pheochromocytoma)
The PC12 cell line is by far the most widely used cell line in neuroscience research,
and the most widely used model of neuronal function and differentiation (Fujita et al.,
1989). PC12 cells were derived from rat adrenal gland with pheochromocytoma and
introduced by Arthur Tischler and Lloyd Greene in the 1970s (Greene and Tischler,
1976; Tischler and Greene, 1975). PC12 cells synthesize neurotransmitters such as
dopamine, norepinephrine, and acetylcholine (ACh). PC12 cells express both nicotinic
and muscarinic ACh receptors and are responsive to ACh (Dichter et al., 1977;

Neural Cell Lines (Lineage) 171
Greene and Rein, 1977; Jumblatt and Tischler, 1982). A variety of ion channels have
been detected in PC12 cells including voltage-dependent Na+-, Ca2+- and K+-channels,
and Ca2+-dependent K+ channels (Arner and Stallcup, 1981; O’Lague and Huttner, 1980),
which are localized in plasma membrane and involved in the propagation of electrical
activity. PC12 cells have been shown to build functional synapses with cultured cells
of skeletal muscle origin (Schubert et al., 1977). The processes of PC12 cells contain
varicosities that contain vesicular neurotransmitters which can be released in a quantal
fashion upon electrical or chemical stimulations. Based on these characteristics, the
PC12 cell line is considered a neuroendocrine or neuronal-type cell line and has been
utilized as a model for studying neurosecretion (D’Alessandro and Meldolesi, 2010;
Westerink and Ewing, 2008), and more specifi cally the regulation of neurotransmitter
synthesis, storage and release. PC12 cells also express nerve growth factor (NGF)
receptors and respond reversibly to NGF, i.e., in the presence of NGF, PC12 cells
stop dividing and differentiate to neuronal phenotype, such as induction of electrical
excitability, increase in expression of Na+ channels and muscarinic ACh receptors,
and outgrowth of long processes containing varicosities, whereas withdrawal of NGF
will reverse the phenotype. These phenomena make the PC12 cell line the premiere
model for the study of neurite outgrowth and neuronal differentiation (D’Alessandro
and Meldolesi, 2010; Levi et al., 1988). Since NGF application in a culture dish can
be artifi cially well controlled, PC12 cells have been utilized for delineating molecular
mechanisms underlying NGF action (Levi et al., 1988). For example, NGF has been
shown to increase the level of tubulin, microtubule associated proteins MAP-1, MAP-2
and tau, vimentin, and other neurofi lament proteins (Black et al., 1986; Drubin et al.,
1985; Greene et al., 1983; Lee and Page, 1984). It is conceivable that neurite outgrowth
may directly correlate with the biosynthesis of cytoskeleton proteins. PC12 cells also
have been employed in research of stress, hypoxia, and neurotoxicity (Roth et al., 2002;
Spicer and Millhorn, 2003). Not only has the PC12 cell line helped the discovery of
various phenomena associated with cell growth, differentiation and death, molecular
analysis of these phenomena at different stages further revealed critical elements of
several distinct and independent signal transduction pathways responsible for the
underlying cellular and molecular mechanisms (Kontou et al., 2009; Sombers et al.,
2002; Vaudry et al., 2002). Another signifi cant utilization of the PC12 cell line is in
the area of in vitro pharmacological studies, since PC12 cells are extremely versatile
following pharmacological manipulations and easy to culture. Such studies provide
a wealth of information on cell proliferation and differentiation, on drug effects on
vesicles, and on the regulation of exocytosis (Momboisse et al., 2010). Successful
foreign gene insertion and stable expression in PC12 cells have been achieved
(Li et al., 1999), with the hope that they could be useful for in vivo testing of
transplantation for potential disease treatment (Yoshida et al., 1999). In a recent
study, PC12 cells were employed for developing and validating a new in vitro
cytotoxicity assay (Gu et al., 2014), which utilizes Fluoro-Jade C (FJ-C), a fl uorescent
label previously used for the assessment of neurodegeneration in fi xed brain tissue
sections for the labeling of degenerating cells in cell cultures following toxic insults
(Figure 9.1). This new FJ-C based in vitro approach is simple and fast, has a high
sensitivity and large dynamic range, and has the potential to be utilized in high-
throughput toxicity screening of chemical compounds in vitro.

172 Neural Cell Biology
Figure 9.1. FJ-C labeling in PC12 cell cultures following 24 hr incubation with various concentrations of
cadmium, a well-known toxicant, showing dose-dependent cytotoxicity as indicated by the increase number
of FJ-C labeled cells. Reprint from Gu et al., 2014.
SH-SY5Y cell line (human neuroblastoma)
The SH-SY5Y cell line was introduced in 1978 (Biedler et al., 1978). It was a
subclone of the SK-N-SH cell line that was derived from a bone marrow biopsy of
a 4-year-old girl with neuroblastoma. SH-SY5Y cells are electrically excitable and
possess a variety of voltage- and ligand-gated ion channels, for example tetrodotoxin-
sensitive Na+ channels, voltage-sensitive K+ channels, and L-type and N-type voltage-
sensitive Ca2+ channels (Forsythe et al., 1992; Kennedy and Henderson, 1992; Morton
et al., 1992; Reeve et al., 1992; Reeve et al., 1994). SH-SY5Y cells also express a
variety of transmitter receptors such as muscarinic ACh M1, M2 and M3 receptors
(Adem et al., 1987; Lambert et al., 1989a), nicotinic ACh receptors (Gould et al., 1992),
mu-opioid receptors (Seward et al., 1991), and neuropeptide-Y receptors (McDonald
et al., 1994). Whole cell patch-clamp and intracellular Ca2+ imaging techniques have
been applied to SH-SY5Y cell cultures and revealed that SH-SY5Y cells possess
purinergic receptors that respond to extracellular ATP, ADT, UTP and UDP. Activation
of purinergic P2X7 receptors in SH-SY5Y cells result in a rapid elevation of intracellular
Ca2+ through the opening of P2X7 receptor-coupled voltage-dependent Ca2+ channels
(Larsson et al., 2002). SH-SY5Y cell line expresses dopamine--hydroxylase
(Biedler et al., 1978) and hence produces the neurotransmitter dopamine. The cell line
also expresses dopamine transporters and receptors. Therefore, the SH-SY5Y cell line
is considered primarily a dopaminergic-type neuron and is often used as a cellular
model for Parkinson’s disease (Xie et al., 2010). SH-SY5Y cells can differentiate
into several different phenotypes depending on differentiation agents such as retinoic
acid (RA), brain derived neurotrophic factor (BDNF), and NGF (Edsjo et al., 2003;
Encinas et al., 2000). When treated by RA and BNDF, SH-SY5Y increased expression
of general cholinergic markers choline acetyltransferase and vesicular ACh transporter
(Edsjo et al., 2003). The SH-SY5Y cell line has been widely used for cellular studies
of neuronal function, differentiation, regulation of transmitter release, degeneration,
cytotoxicity and anti-tumor drug effi cacy (Fasano et al., 2008; Vaughan et al., 1995).
Similar to PC12 cells, SH-SY5Y cells were recently employed for developing and
validating an FJ-C based in vitro assay for cytotoxicity (Figure 9.2).

Neural Cell Lines (Lineage) 173
N1E-115 cell line (mouse neuroblastoma)
The N1E-115 cell line was established in the early 1970s by cloning spontaneous
mouse neuroblastoma tumor (Amano et al., 1972). N1E-115 cells exhibit high levels
of activity of the enzyme tyrosine hydroxylase and express a variety of transmitter
receptors such as muscarinic ACh M1, M2 and M4, histaminergic H1, serotonergic 5-HT3,
and nicotinic ACh receptors, among others (Lenz et al., 1994; Oortgiesen et al., 1997;
Richelson, 1990). N1E-115 cells also express other types of membrane receptors such
as that for adenosine (Murphy and Byczko, 1990), angiotensin (Gilbert et al., 1984),
bradykinin (Snider and Richelson, 1984), melatonin (Bordt et al., 2001), neurotensin
(Gilbert and Richelson, 1984), purinergic P2X7 (Schrier et al., 2002), somatostatin
(McKinney and Barrett, 1989), and tumor necrosis factor (TNF) (Sipe et al., 1996).
At the functional level, it has been shown that the N1E-115 cell line rapidly responds
to melatonin stimulation and forms neurites within 24 hr (Bordt et al., 2001), while
exposure to extracellular TNF, ATP or adenosine can induce apoptosis (Schrier et al.,
2002; Schrier et al., 2001; Sipe et al., 1996). N1E-115 cells can be differentiated under
conditions of low serum in the presence of dimethyl sulfoxide (DMSO) to produce
morphologically mature neuronal-like cholinergic cells (Amano et al., 1972). N1E-
115 cells have been used to study transmitter receptor regulation, second messenger
synthesis, electrophysiological changes, and cell death mechanisms. For example,
most electrophysiological and pharmacological properties of 5-HT3 receptors were
initially characterized in N1E-115 cells (Lambert et al., 1989b). In another study,
muscarinic ACh M1 receptors were detected intracellularly and in the plasma membrane
of N1E-115 cells, and M1 receptors in both locations appeared functionally active,
suggesting additional signaling function of M1 receptor intracellularly (Uwada et al.,
2011). Growth hormone and growth hormone receptors are abundantly expressed in
N1E-115 cells (Grimbly et al., 2009). Exposure of N1E-115 cells to mouse growth
hormone induced neurite sprouting and increased axon growth, thereby providing
a good model for the study of the effects of growth hormone on neurite outgrowth
(Grimbly et al., 2009). N1E-115 cells also have been employed for examining toxicant
effects on viability, structure, and axonal transport of neurons (Brat and Brimijoin,
1992). In this model, N1E-115 cells were induced to extend neurites by elimination
of serum from the medium, and extended neurites 2–5 μm in diameter and up to 400
Figure 9.2. FJ-C labeling in SH-SY5Y cell cultures following 20 M cadmium treatment for different
durations showing time-dependent cytotoxicity as indicated by the increase number of FJ-C labeled cells.
Reprint from Gu et al., 2014.

174 Neural Cell Biology
μm in length on uncoated glass in serum-free medium. By measuring the fraction
of cells bearing neurites and the mean length of the neurites in the presence and
absence of a toxicant, the effect of a test compound on cell growth can be evaluated
(Brat and Brimijoin, 1992). Organellar motility in neurites can be another indicator
of healthiness concerning axonal transport. The N1E-115 cell line appears to be well
suited for time-lapsed optical recording of organellar mobility in neurites, and yields
valuable information on the toxicity of tested compounds (Brat and Brimijoin, 1992).
Neuro-2a cell line (mouse neuroblastoma)
Another mouse neuroblastoma cell line Neuro-2a (also called N2a) was established
from a spontaneous tumor of a strain A albino mouse (Klebe and Ruddle, 1969). Neuro-
2a cells produce large quantities of microtubular proteins including tubulin, actin and
MAP-2, which are believed to play a role in a contractile system which is responsible
for axoplasmic fl ow in nerve cells. Therefore, the Neuro-2a cell line has been
useful for studies of the synthesis, assembly, and turnover of neurofi lament proteins
(Olmsted et al., 1970; Wang et al., 1996; Zimmermann et al., 1987). The Neuro-2a cell
line has made a signifi cant contribution in helping to delineate signaling in regulation
of neurite outgrowth and neuronal differentiation, especially mediated by the Gai/o
pathway (He et al., 2006; Ma’ayan et al., 2009). Neuro-2a cells have also been used
to study neurotoxicity (LePage et al., 2005), Alzheimer’s disease (Provost, 2010),
prion-related diseases (Kaneko et al., 1997), and asymmetric division of mammalian
cell lines (Ogrodnik et al., 2014).
B104 cell line (rat neuroblastoma)
The B104 cell line was cloned from a rat neuroblastoma that was induced
transplacentally with nitrosomethylurea injected 15 d after conception and harvested
when the animal reached adulthood (Schubert et al., 1974). Morphologically B104
cells are very long, thin, and usually bipolar (Schubert et al., 1974). The B104 cell
line exhibits many of the properties characteristic of differentiated neurons, such as
generation of action potentials, synthesis of neurotransmitters such as ACh and GABA,
and the presence of neurotransmitter receptors, neuron-specifi c 14-3-2 protein, and
microtubule-associated proteins MAP-2 and MAP-3 (Huber et al., 1985; Izant and
McIntosh, 1980; Schubert et al., 1975; Schubert et al., 1974). The B104 cell line also
expresses insulin-like growth factor (IGF) receptor I and II (Sturm et al., 1989), and
IGF-I is more potent than insulin at stimulating B104 cell replication in serum-free
medium (Orlowski et al., 1989). Furthermore, B104 cells, like the N1E-115 cell line,
respond to removal of serum by rapidly extending neurites, a phenomenon that has
been correlated with the preceding neuronal properties (Schubert et al., 1974). When
B104 cells were grown in the presence of 1 mM dibutyryl cAMP, they underwent
morphological alterations that mimic neuronal differentiation (Schubert et al., 1975).
In cell culture, basic fi broblast growth factor and platelet-derived growth factor AA
and BB were potent mitogens for B104 cells, because they promoted an increase in
cell number even when the cells were grown in serum-free medium (Luo and Miller,

Neural Cell Lines (Lineage) 175
1997), while transforming growth factor-1 inhibited the proliferation of B104 cells
(Luo and Miller, 1999). All these properties make the B104 cell line suitable for
studying regulation mechanisms of neuronal proliferation, growth and differentiation.
The B104 cell line has also been studied for neurotoxicity and apoptosis (Heese
et al., 2000; Park et al., 2001).
TE671 cell line (human medulloblastoma)
TE671 is a human cell line derived from a cerebellar tumor of a 6-year-old Caucasian
girl (McAllister et al., 1977), and appears to be composed of six different morphological
types of cells (types I–VI) in varying percentages in culture (Zeltzer et al., 1984).
TE671 cells possess high glutamic acid decarboxylase activity, nicotinic ACh receptors,
dopaminergic D1 and D3 receptors, and glutamatergic receptors, voltage-dependent
Na+- and K+-channels, small conductance Ca2+-activated K+-channel SK3, and are
capable of generating Na+-dependent action potentials (Carignani et al., 2002; Hamel
et al., 1981; Levavi-Sivan et al., 1998; Stepulak et al., 2009; Syapin et al., 1982; Toral
et al., 1995). TE671 cells express neuron-specifi c enolase, gonadotropin-releasing
hormone, and epidermal growth factor receptors, and are negative for the glial marker
glial fi brillary acid protein (GFAP) (Hall et al., 1990; Zeltzer et al., 1984). Since
medulloblastoma is one of the most common brain tumors of childhood, the TE671
cell line has become one of the in vitro experimental models to study neoplastic
cells of brain tumors and therapeutic applications such as chemo-, radiation- and
photodynamic therapy (Houchens et al., 1983; Lipshutz et al., 1994; Merlin et al.,
1991). At the cellular and molecular level, the TE671 cell line has been mostly studied
for the expression, function, pharmacology and regulation of nicotinic ACh receptors,
and because of the possession of functional nicotinic ACh receptors, has been used as a
model for in vitro testing of potential therapeutic compounds against myasthenia gravis
(Voltz et al., 1991). The TE671 cell line has also been employed in pharmacology
studies on ATP-sensitive K+ channels (Miller et al., 1999).
HCN-2 cell line (human encephalitis)
The HCN (human cortical neuron)-2 cell line was derived from cortical tissue removed
from a 7-year-old girl undergoing a hemispherectomy for intractable seizures associated
with chronic focal encephalitis (Ronnett et al., 1994). HCN-2 cells can be induced to
differentiate when cultured with NGF, dibutyryl cAMP, 1-isobutyl-3-methylxanthine,
or phorbol ester (Ronnett et al., 1994), and differentiated HCN-2 cells have a neuron-
like morphology. The cells stain positively for a number of neuronal markers including
neurofi lament proteins and neuron-specifi c enolase. HCN-2 cells are also positive for
tubulin, vimentin, somatostatin, glutamate, GABA, cholecystokinin-8, methionine
enkephalin and vasoactive intestinal peptide, while they are negative for glial markers
such as GFAP, S-100, and myelin basis protein (Ronnett et al., 1994). The HCN-2
cell line has been proposed as a model for amyloid β neurotoxicity as exposure of
differentiated HCN-2 cells to amyloid β can cause cell death (Zhang et al., 1994).
Using the HCN-2 cell line as a model, effects of oxidative stress on major cytoskeleton

176 Neural Cell Biology
fi laments, microfi laments, microtubule, and vimentin, which are thought to play an
important role in neuronal growth and neurite outgrowth, have also been examined
(Allani et al., 2004). Because the HCN-2 cell line was created much later than other
neuronal cell lines of neuroblastoma or medulloblastoma origins, it has yet to gain
similar popularity and utilization.
Cell Lines of Glial Origin
U87MG cell line (human glioma)
The U87MG cell line was derived from a stage-IV glioma of a 44-year-old Caucasian
man (Ponten and Macintyre, 1968). U87MG cells have epithelial morphology
and express membrane receptors for several neurotransmitters including various
glutamatergic subtype receptors NMDA- (NR2A, NR2B, NR2C, NR3A and NR3B),
AMPA- (GluR2, GluR3, GluR4, GluR6 and GluR7), kainite- (KA1 and KA2),
and metabotropic (mGluR1, mGluR2, mGluR3, mGluR5, mGluR6, mGluR7 and
mGluR8) subtype receptors (Arcella et al., 2005; Stepulak et al., 2009; Yoshida et al.,
2006), as well as adrenergic β1- and β2-receptors (Sardi et al., 2013) and muscarinic
M2 receptor (Ferretti et al., 2013). U87MG cells also express plasma membrane
receptors for basic fi broblast growth factor (Murphy and Knee, 1995), interleukin-1
and -6 (Goswami et al., 1998; Gottschall et al., 1991), growth hormone
(Castro et al., 2000), semaphorin (Rieger et al., 2003), platelet-derived growth
factor (Gross et al., 2006), and cholecystokinin-B (Oikonomou et al., 2008), as
well as voltage-gated K+-channels (Ru et al., 2015). U87MG cells possess the glial
marker GFAP (Ito et al., 1989), synthesize and release some important macro-
molecules such as granulocyte colony-stimulating factor (Tweardy et al., 1987), basic
fi broblast growth factor (Sato et al., 1989), glial cell line-derived neurotrophic factor
(Verity et al., 1999), and TNF receptor-associated factor 6 (Peng et al., 2013). U87MG
cells also produce tenascin (Ventimiglia et al., 1992), type VI collagen (Han et al.,
1994), interleukin-6 (Goswami et al., 1998), somatostatin (Hirota et al., 1998), class 3
semaphorins (Rieger et al., 2003), platelet-derived growth factor (Gross et al., 2006),
and cholecystokinin (Oikonomou et al., 2008). The entire genome of the U87MG
cell line has been sequenced (Clark et al., 2010). Activation of NMDA receptors
increased proliferation of the U87MG cell line (Ramaswamy et al., 2014), whereas
TNF- inhibits U87MG cell growth (Chen et al., 1993). Differentiation of U87MG
cells could be induced by treatment with dibutyryl cAMP (Hoffman et al., 1994) or
retinoid (Das et al., 2009). Transplantation studies suggest that transforming growth
factor-2 promotes the invasion of U87MG cells (Wick et al., 2001). The U87MG cell
line also has platelet-activating activity (Bastida et al., 1987). Different types of matrix
metalloproteinases were detected in U87MG cells (Maruiwa et al., 1993; Matsuzawa
et al., 1996), and modulation of matrix metalloproteinase activity has been shown
to play an important role in U87MG cell proliferation, differentiation, and invasion
(Ramaswamy et al., 2014; Wick et al., 2001). For this reason, matrix metalloproteinase
has become a major target for anti-glioma therapy. The U87MG cell line has been
used extensively as a model for studying glioma, including assessing drug toxicity and

Neural Cell Lines (Lineage) 177
anti-tumor effi ciency, cell cycle and apoptosis, glioma invasion, and vascularization.
To date there are well over 1000 published papers utilizing this cell line for glioma
research, both in vitro and xenografts. Experimental therapies have been conducted
in animals using radiation-, chemo-, photodynamic, gene-, and immune-therapy, as
well as caloric restriction, hypoxia, and hyperthermia.
C6 cell line (rat glioma)
The glial cell strain C6 was cloned from an outbred Wistar-Furth rat glial tumor
induced by repeated injection of N-methylnitrosourea (Benda et al., 1968). The C6
cell line expresses S-100 protein, GFAP, vimentin, and somatotrophin, and produces
glyceryl phosphate dehydrogenase in response to glucocorticoids. The cell line
has been extensively used as a brain tumor model in experimental neuro-oncology
(Barth and Kaur, 2009; Grobben et al., 2002). However, a serious side effect of the C6
cell line is the potential of evoking immune-response in host animals, thereby limiting
its applications, especially in brain tumor survival studies. Despite this limitation the
C6 cell line has provided a wealth of information on biological properties of brain
tumors, such as effects of growth factors, extracellular matrix components, proteases,
adhesion molecules, and secretion of tumor-derived factors (Grobben et al., 2002).
Molecular characterizations which compared changes in gene expression patterns
between the C6 glioma and rat stem cell-derived astrocytes revealed that the changes
in gene expressions observed in the C6 cell line were the most similar to those reported
in human brain tumors (Sibenaller et al., 2005). C6 cells have cancer stem cell-like
characteristics, including self-renewal, the potential for multi-lineage differentiation
in vitro and tumor formation in vivo (Shen et al., 2008). The C6 cell line has also been
used extensively for studies of glioblastoma growth, invasion, migration, blood-brain
barrier disruption, capillary permeability, and neovascularization, and for evaluating
new therapeutic modalities such as chemo-, radiation-, photodynamic, anti-angiogenic,
oncolytic viral and gene-therapies, and treatments with proteasome inhibitors and
various toxins (Barth and Kaur, 2009).
F98 and RG2 cell lines (rat glioma)
The F98 and RG2 cell lines were produced in the same laboratory in 1971 (Ko et al.,
1980). A pregnant Fischer rat on day 20 of gestation was inoculated with a single 50
mg/kg dose of N-ethyl-N-nitrosourea. The tumors were harvested and cloned in vitro.
One of them was designated F98, and another RG2 (rat glioma 2) or RG-2 (the same
clone was also called D74-RG2 or D74). The biological characteristics of both tumors
closely resemble those of human glioblastoma, and have been used extensively for both
in vitro and in vivo studies of a rat brain tumor. While both cell lines express vimentin,
one difference between the two cell lines is that the F98 cell line demonstrated only
a fraction of GFAP positive cells, whereas the RG2 cell line exhibited virtually no
GFAP immunoreactivity in culture (Mathieu et al., 2007; Reifenberger et al., 1989).
Another difference between the two cell lines is that F98 cells are weakly immunogenic
while RG2 cells are non-immunogenic in syngeneic rats. Thus, both cell lines are

178 Neural Cell Biology
very attractive models to investigate the mechanisms underlying glioma resistance to
immunotherapy (Barth and Kaur, 2009). F98 and RG2 cell lines have been used for
a variety of in vivo transplantation studies including vascular permeability, regional
blood fl ow, tumor metabolism, tumor growth, and blood brain barrier disruption, anti-
angiogenic, chemo-, radiation-, and gene-therapy (Barth and Kaur, 2009).
Other Neural Cell Lines
NG108-15 cell line (mouse/rat neuroblastoma-glioblastoma hybrid)
The NG108-15 cell line, originally named 108CC15, was developed by fusing mouse
N18TG2 neuroblastoma cells with rat C6-BU-1 glioma cells in the presence of
inactivated Sendai virus (Hamprecht, 1977). These cells display a considerable range
of neuronal features and possess many of the functions of differentiated neurons,
including extension of long processes, clear and dense core vesicles, excitable
membranes, formation of functional synapses, express neurotransmitter synthesizing
enzymes such as choline acetyltransferase and dopamine--hydroxylase, uptake
systems for catecholamines, depolarization-induced Ca2+-dependent release of ACh,
and expression of transmitter receptors (Hamprecht et al., 1985). NG108-15 cells have
been widely used as a model system to examine transmembrane signaling processes
in the nervous system. These cells are particularly suitable for such studies as they
express a considerable range of receptors which can be demonstrated to couple to
a variety of effector systems. The transmembrane signaling systems which have
been examined in greatest detail in NG108-15 cells are stimulation and inhibition
of adenylate cyclase (Klee et al., 1985), activation of phosphoinositidase C, and
regulation of voltage-sensitive Ca2+ channels (Tsunoo et al., 1986). NG108-15 cells
contain opioid receptors and adrenergic 2 receptors (Klee and Nirenberg, 1974).
Opiates initially inhibit adenylate cyclase activity in these cells, but after a period of
adaptation the levels of cAMP return to normal (Sharma et al., 1975). The cells are
desensitized to the acute effects of opiates (tolerance) and if they are withdrawn, or an
opioid antagonist is added, the cells respond with an overshoot of adenylate cyclase
activity (Lee et al., 1988). The adenylate cyclase overshoot response after chronic
exposure to opiates has been proposed as a biochemical model for opiate dependence
(Sharma et al., 1975). Adaptation mechanisms similar to those for opiates have been
described for 2 receptor agonists in NG108-15 cells (Sabol and Nirenberg, 1979).
NG108-15 cells also express bradykinin receptors and an external application of
bradykinin produces a biphasic change in membrane potential (a hyperpolarization
followed by a depolarization) accompanied by an increase in ACh release
(Reiser and Hamprecht, 1982). This cell model allowed examination of physiological
and biochemical consequences in the receptor-mediated regulation of neurotransmitter
release at a single cell level (Higashida and Ogura, 1991). NG108-15 cells express
several guanine nucleotide binding proteins (G-proteins) and a variety of heterotrimeric,
plasma membrane-associated G-proteins coupled receptors, including Gs, Gi2, Gi3,
Gq/11, and at least two variants of G0, which allow the conversion of extracellular signals
into intracellular responses (Milligan et al., 1990). Overall, the NG108-15 cell line

Neural Cell Lines (Lineage) 179
has been mostly used for studying expression and function of cross-membrane and
intracellular signaling pathways.
CATH.a cell line (Transgenic mouse)
The CATH.a cell line was established from cultures of a tumor that arose in the brain of
a transgenic mouse carrying the SV-40 T antigen oncogene (Suri et al., 1993). Although
the CATH.a cell line continues to express T antigen, the cells exhibit many properties
of locus coeruleus neurons. The cells express the catecholaminergic biosynthetic
enzymes tyrosine hydroxylase and dopamine -hydroxylase, neurofi laments, and
synaptophysin (Suri et al., 1993). The cell line possesses kappa- and delta-opioid
receptors and responds to opioids by suppression of voltage-activated K+-current
(Baraban et al., 1995; Bouvier et al., 1998). Interestingly, dopamine produces a dose-
and time-dependent increase in cell death in the CATH.a cell line, and the cell death is
not mediated through dopaminergic receptors since selective receptor agonists have no
effect on CATH.a cell viability (Masserano et al., 1996). Brain-derived neurotrophic
factor and glia cell line-derived neurotrophic factor reduced dopamine-induced cell
death, while NGF, basic fi broblast growth factor, neurotrophin-4/5 and insulin had no
protective effect on dopamine-induced cell death (Gong et al., 1999). Since agents that
activate the cAMP pathway lead to a decrease in expression of the cAMP response
element-binding protein (CREB) mRNA in CATH.a cells, the cell line has made
signifi cant contributions in studying regulation mechanisms of CREB expression
(Coven et al., 1998; Widnell et al., 1996; Widnell et al., 1994).
Concluding Remarks
Available neural cell lines have provided relatively simple and well-controlled systems
for elucidating basic biological processes and mechanisms governing neural growth,
differentiation, signal transduction, and cell death. In addition, it can be concluded
that all neural cell lines have been utilized in pharmacological and toxicological
studies. Both in vivo transplantation and in vitro experiments further helped
therapeutic development and evaluation for disease treatment. Despite the success and
achievements of various cell lines, it remains essential to recognize their limitations,
which include but are not limited to: (1) tumor origin and continued expression of
oncogenes, which may have changed many cellular and molecular properties; (2)
artifi cial media and growth conditions, which may have infl uenced signal transduction
pathways and gene expression patterns; (3) mono-type of cells in the culture dish,
which may lack certain features that require interactions with other types of cells;
and (4) cells in a 2-dimensional mono-layer, which may behave differently than in a
3-dimensional environment. Looking forward it is anticipated that more genetically
engineered cell lines will be developed that more closely represent neurons and glial
cells, respectively, and the expression of particular genes can be artifi cially controlled.
Co-cultures of multi-types of cells and 3-dimensional cell cultures will further mimic
in vivo situations. Their applications could lead to a better understanding of molecular
mechanisms underlying biological processes such as growth, differentiation, signal

180 Neural Cell Biology
transduction and cell death, as well as more effective therapeutic treatments for brain
tumors, neurological disorders and diseases beyond the nervous system.
Acknowledgements
The author’s work cited in this chapter was supported by the U.S. Food and Drug
Administration (FDA), National Center for Toxicological Research protocol numbers
E746001 and E752401. This document has been reviewed in accordance with FDA
policy and approved for publication. Approval does not signify that the contents
necessarily refl ect the position or opinions of the FDA nor does mention of trade
names or commercial products constitute endorsement or recommendation for use. The
fi ndings and conclusions in this report are those of the authors and do not necessarily
represent the views of the FDA.
References
Adem, A., M.E. Mattsson, A. Nordberg, and S. Pahlman. 1987. Muscarinic receptors in human SH-SY5Y
neuroblastoma cell line: regulation by phorbol ester and retinoic acid-induced differentiation. Brain
Res 430: 235–242.
Allani, P.K., T. Sum, S.G. Bhansali, S.K. Mukherjee, and M. Sonee. 2004. A comparative study of the effect
of oxidative stress on the cytoskeleton in human cortical neurons. Toxicol Appl Pharmacol 196: 29–36.
Amano, T., E. Richelson, and M. Nirenberg. 1972. Neurotransmitter synthesis by neuroblastoma clones
(neuroblast differentiation-cell culture-choline acetyltransferase-acetylcholinesterase-tyrosine
hydroxylase-axons-dendrites). Proc Natl Acad Sci U S A 69: 258–263.
Arcella, A., G. Carpinelli, G. Battaglia, M. D’Onofrio, F. Santoro, R.T. Ngomba, V. Bruno, P. Casolini,
F. Giangaspero, and F. Nicoletti. 2005. Pharmacological blockade of group II metabotropic glutamate
receptors reduces the growth of glioma cells in vivo. Neuro Oncol 7: 236–245.
Arner, L.S., and W.B. Stallcup. 1981. Two types of potassium channels in the PC12 cell line. Brain Res
215: 419–425.
Baraban, S.C., E.W. Lothman, A. Lee, and P.G. Guyenet. 1995. Kappa opioid receptor-mediated suppression
of voltage-activated potassium current in a catecholaminergic neuronal cell line. J Pharmacol Exp
Ther 273: 927–933.
Barth, R.F., and B. Kaur. 2009. Rat brain tumor models in experimental neuro-oncology: the C6, 9L, T9,
RG2, F98, BT4C, RT-2 and CNS-1 gliomas. J Neurooncol 94: 299–312.
Bastida, E., L. Almirall, G.A. Jamieson, and A. Ordinas. 1987. Cell surface sialylation of two human
tumor cell lines and its correlation with their platelet-activating activity. Cancer Res 47: 1767–1770.
Benda, P., J. Lightbody, G. Sato, L. Levine, and W. Sweet. 1968. Differentiated rat glial cell strain in tissue
culture. Science 161: 370–371.
Biedler, J.L., S. Roffl er-Tarlov, M. Schachner, and L.S. Freedman. 1978. Multiple neurotransmitter synthesis
by human neuroblastoma cell lines and clones. Cancer Res 38: 3751–3757.
Black, M.M., J.M. Aletta, and L.A. Greene. 1986. Regulation of microtubule composition and stability
during nerve growth factor-promoted neurite outgrowth. J Cell Biol 103: 545–557.
Bordt, S.L., R.M. McKeon, P.K. Li, P.A. Witt-Enderby, and M.A. Melan. 2001. N1E-115 mouse
neuroblastoma cells express MT1 melatonin receptors and produce neurites in response to melatonin.
Biochim Biophys Acta 1499: 257–264.
Bouvier, C., D. Avram, V.J. Peterson, B. Hettinger, K. Soderstrom, T.F. Murray, and M. Leid. 1998.
Catecholaminergic CATH.a cells express predominantly delta-opioid receptors. Eur J Pharmacol
348: 85–93.
Brat, D.J., and S. Brimijoin. 1992. A paradigm for examining toxicant effects on viability, structure, and
axonal transport of neurons in culture. Mol Neurobiol 6: 125–135.
Carignani, C., R. Roncarati, R. Rimini, and G.C. Terstappen. 2002. Pharmacological and molecular
characterisation of SK3 channels in the TE671 human medulloblastoma cell line. Brain Res 939: 11–18.

Neural Cell Lines (Lineage) 181
Castro, J.R., J.A. Costoya, R. Gallego, A. Prieto, V.M. Arce, and R. Senaris. 2000. Expression of growth
hormone receptor in the human brain. Neurosci Lett 281: 147–150.
Chen, T.C., D.R. Hinton, M.L. Apuzzo, and F.M. Hofman. 1993. Differential effects of tumor necrosis
factor-alpha on proliferation, cell surface antigen expression, and cytokine interactions in malignant
gliomas. Neurosurgery 32: 85–94.
Clark, M.J., N. Homer, B.D. O’Connor, Z. Chen, A. Eskin, H. Lee, B. Merriman, and S.F. Nelson. 2010.
U87MG decoded: the genomic sequence of a cytogenetically aberrant human cancer cell line. PLoS
Genet 6: e1000832.
Coven, E., Y. Ni, K.L. Widnell, J. Chen, W.H. Walker, J.F. Habener, and E.J. Nestler. 1998. Cell type-
specifi c regulation of CREB gene expression: mutational analysis of CREB promoter activity. J
Neurochem 71: 1865–1874.
D’Alessandro, R., and J. Meldolesi. 2010. In PC12 cells, expression of neurosecretion and neurite outgrowth
are governed by the transcription repressor REST/NRSF. Cell Mol Neurobiol 30: 1295–1302.
Das, A., N.L. Banik, and S.K. Ray. 2009. Molecular mechanisms of the combination of retinoid and
interferon-gamma for inducing differentiation and increasing apoptosis in human glioblastoma T98G
and U87MG cells. Neurochem Res 34: 87–101.
Dichter, M.A., A.S. Tischler, and L.A. Greene. 1977. Nerve growth factor-induced increase in electrical
excitability and acetylcholine sensitivity of a rat pheochromocytoma cell line. Nature 268: 501–504.
Drubin, D.G., S.C. Feinstein, E.M. Shooter, and M.W. Kirschner. 1985. Nerve growth factor-induced neurite
outgrowth in PC12 cells involves the coordinate induction of microtubule assembly and assembly-
promoting factors. J Cell Biol 101: 1799–1807.
Edsjo, A., E. Lavenius, H. Nilsson, J.C. Hoehner, P. Simonsson, L.A. Culp, T. Martinsson, C. Larsson, and
S. Pahlman. 2003. Expression of trkB in human neuroblastoma in relation to MYCN expression and
retinoic acid treatment. Lab Invest 83: 813–823.
Encinas, M., M. Iglesias, Y. Liu, H. Wang, A. Muhaisen, V. Cena, C. Gallego, and J.X. Comella. 2000.
Sequential treatment of SH-SY5Y cells with retinoic acid and brain-derived neurotrophic factor gives
rise to fully differentiated, neurotrophic factor-dependent, human neuron-like cells. J Neurochem
75: 991–1003.
Fasano, M., T. Alberio, M. Colapinto, S. Mila, and L. Lopiano. 2008. Proteomics as a tool to investigate
cell models for dopamine toxicity. Parkinsonism Relat Disord 14 Suppl 2: S135–138.
Ferretti, M., C. Fabbiano, M. Di Bari, C. Conte, E. Castigli, M. Sciaccaluga, D. Ponti, P. Ruggieri, A. Raco,
R. Ricordy, A. Calogero, and A.M. Tata. 2013. M2 receptor activation inhibits cell cycle progression
and survival in human glioblastoma cells. J Cell Mol Med 17: 552–566.
Forsythe, I.D., D.G. Lambert, S.R. Nahorski, and P. Lindsdell. 1992. Elevation of cytosolic calcium by
cholinoceptor agonists in SH-SY5Y human neuroblastoma cells: estimation of the contribution of
voltage-dependent currents. Br J Pharmacol 107: 207–214.
Fujita, K., P. Lazarovici, and G. Guroff. 1989. Regulation of the differentiation of PC12 pheochromocytoma
cells. Environ Health Perspect 80: 127–142.
Gilbert, J.A., and E. Richelson. 1984. Neurotensin stimulates formation of cyclic GMP in murine
neuroblastoma clone N1E-115. Eur J Pharmacol 99: 245–246.
Gilbert, J.A., M.A. Pfenning, and E. Richelson. 1984. The effect of angiotensins I, II, and III on formation
of cyclic GMP in murine neuroblastoma clone N1E-115. Biochem Pharmacol 33: 2527–2530.
Gong, L., R.J. Wyatt, I. Baker, and J.M. Masserano. 1999. Brain-derived and glial cell line-derived
neurotrophic factors protect a catecholaminergic cell line from dopamine-induced cell death. Neurosci
Lett 263: 153–156.
Goswami, S., A. Gupta, and S.K. Sharma. 1998. Interleukin-6-mediated autocrine growth promotion in
human glioblastoma multiforme cell line U87MG. J Neurochem 71: 1837–1845.
Gottschall, P.E., K. Koves, K. Mizuno, I. Tatsuno, and A. Arimura. 1991. Glucocorticoid upregulation of
interleukin 1 receptor expression in a glioblastoma cell line. Am J Physiol 261: E362–368.
Gould, J., H.L. Reeve, P.F. Vaughan, and C. Peers. 1992. Nicotinic acetylcholine receptors in human
neuroblastoma (SH-SY5Y) cells. Neurosci Lett 145: 201–204.
Greene, L.A., and A.S. Tischler. 1976. Establishment of a noradrenergic clonal line of rat adrenal
pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci U S A
73: 2424–2428.
Greene, L.A., and G. Rein. 1977. Release of (3H)norepinephrine from a clonal line of pheochromocytoma
cells (PC12) by nicotinic cholinergic stimulation. Brain Res 138: 521–528.

182 Neural Cell Biology
Greene, L.A., R.K. Liem, and M.L. Shelanski. 1983. Regulation of a high molecular weight microtubule-
associated protein in PC12 cells by nerve growth factor. J Cell Biol 96: 76–83.
Grimbly, C., B. Martin, E. Karpinski, and S. Harvey. 2009. Growth hormone production and action in
N1E-115 neuroblastoma cells. J Mol Neurosci 39: 117–124.
Grobben, B., P.P. De Deyn, and H. Slegers. 2002. Rat C6 glioma as experimental model system for the
study of glioblastoma growth and invasion. Cell Tissue Res 310: 257–270.
Gross, D., G. Bernhardt, and A. Buschauer. 2006. Platelet-derived growth factor receptor independent
proliferation of human glioblastoma cells: selective tyrosine kinase inhibitors lack antiproliferative
activity. J Cancer Res Clin Oncol 132: 589–599.
Gu, Q., S. Lantz-McPeak, H. Rosas-Hernandez, E. Cuevas, S.F. Ali, M.G. Paule, and S. Sarkar. 2014. In
vitro detection of cytotoxicity using FluoroJade-C. Toxicol In Vitro 28: 469–472.
Hall, W.A., M.J. Merrill, S. Walbridge, and R.J. Youle. 1990. Epidermal growth factor receptors on
ependymomas and other brain tumors. J Neurosurg 72: 641–646.
Hamel, E., I.E. Goetz, and E. Roberts. 1981. Glutamic acid decarboxylase and gamma-aminobutyric acid in
Huntington’s disease fi broblasts and other cultured cells, determined by a [3H]muscimol radioreceptor
assay. J Neurochem 37: 1032–1038.
Hamprecht, B. 1977. Structural, electrophysiological, biochemical, and pharmacological properties of
neuroblastoma-glioma cell hybrids in cell culture. Int Rev Cytol 49: 99–170.
Hamprecht, B., T. Glaser, G. Reiser, E. Bayer, and F. Propst. 1985. Culture and characteristics of hormone-
responsive neuroblastoma X glioma hybrid cells. Methods Enzymol 109: 316–341.
Han, J., J.C. Daniel, N. Lieska, and G.D. Pappas. 1994. Immunofl uorescence and biochemical studies
of the type VI collagen expression by human glioblastoma cells in vitro. Neurol Res 16: 370–375.
He, J.C., S.R. Neves, J.D. Jordan, and R. Iyengar. 2006. Role of the Go/i signaling network in the regulation
of neurite outgrowth. Can J Physiol Pharmacol 84: 687–694.
Heese, K., Y. Nagai, and T. Sawada. 2000. Induction of rat L-phosphoserine phosphatase by amyloid-beta
(1-42) is inhibited by interleukin-11. Neurosci Lett 288: 37–40.
Higashida, H., and A. Ogura. 1991. Inositol trisphosphate/calcium-dependent acetylcholine release evoked
by bradykinin in NG108-15 rodent hybrid cells. Ann N Y Acad Sci 635: 153–166.
Hirota, N., K. Matsumoto, M. Iida, H. Sakagami, and M. Takeda. 1998. Expression of somatostatin messenger
RNA and receptor in cultured brain tumor cells. Anticancer Res 18: 3295–3297.
Hoffman, L.M., S.E. Brooks, M.R. Stein, and L. Schneck. 1994. Cyclic AMP causes differentiation and
decreases the expression of neutral glycosphingolipids in cell cultures derived from a malignant
glioma. Biochim Biophys Acta 1222: 37–44.
Houchens, D.P., A.A. Ovejera, S.M. Riblet, and D.E. Slagel. 1983. Human brain tumor xenografts in nude
mice as a chemotherapy model. Eur J Cancer Clin Oncol 19: 799–805.
Huber, G., D. Alaimo-Beuret, and A. Matus. 1985. MAP3: characterization of a novel microtubule-associated
protein. J Cell Biol 100: 496–507.
Ito, M., T. Nagashima, and T. Hoshino. 1989. Quantitation and distribution analysis of glial fi brillary acidic
protein in human glioma cells in culture. J Neuropathol Exp Neurol 48: 560–567.
Izant, J.G., and J.R. McIntosh. 1980. Microtubule-associated proteins: a monoclonal antibody to MAP2
binds to differentiated neurons. Proc Natl Acad Sci U S A 77: 4741–4745.
Jumblatt, J.E., and A.S. Tischler. 1982. Regulation of muscarinic ligand binding sites by nerve growth
factor in PC12 phaeochromocytoma cells. Nature 297: 152–154.
Kaneko, K., L. Zulianello, M. Scott, C.M. Cooper, A.C. Wallace, T.L. James, F.E. Cohen, and S.B. Prusiner.
1997. Evidence for protein X binding to a discontinuous epitope on the cellular prion protein during
scrapie prion propagation. Proc Natl Acad Sci U S A 94: 10069–10074.
Kennedy, C., and G. Henderson. 1992. Chronic exposure to morphine does not induce dependence at the
level of the calcium channel current in human SH-SY5Y cells. Neuroscience 49: 937–944.
Klebe, R.J., and F.H. Ruddle. 1969. Neuroblastoma: Cell culture analysis of a differentiating stem cell
system. J Cell Biol 43: 69A.
Klee, W.A., and M. Nirenberg. 1974. A neuroblastoma times glioma hybrid cell line with morphine receptors.
Proc Natl Acad Sci U S A 71: 3474–3477.
Klee, W.A., G. Milligan, W.F. Simonds, and B. Tocque. 1985. Opiate receptors in neuroblastoma x glioma
hybrid cell lines: a system for investigating N,/N, interactions. Mol Asp Cell Regul 4: 117–129.
Ko, L., A. Koestner, and W. Wechsler. 1980. Morphological characterization of nitrosourea-induced glioma
cell lines and clones. Acta Neuropathol 51: 23–31.

Neural Cell Lines (Lineage) 183
Kontou, M., W. Weidemann, K. Bork, and R. Horstkorte. 2009. Beyond glycosylation: sialic acid precursors
act as signaling molecules and are involved in cellular control of differentiation of PC12 cells. Biol
Chem 390: 575–579.
Lambert, D.G., A.S. Ghataorre, and S.R. Nahorski. 1989a. Muscarinic receptor binding characteristics of a
human neuroblastoma SK-N-SH and its clones SH-SY5Y and SH-EP1. Eur J Pharmacol 165: 71–77.
Lambert, J.J., J.A. Peters, T.G. Hales, and J. Dempster. 1989b. The properties of 5-HT3 receptors in clonal
cell lines studied by patch-clamp techniques. Br J Pharmacol 97: 27–40.
Larsson, K.P., A.J. Hansen, and S. Dissing. 2002. The human SH-SY5Y neuroblastoma cell-line expresses a
functional P2X7 purinoceptor that modulates voltage-dependent Ca2+ channel function. J Neurochem
83: 285–298.
Lee, S., C.R. Rosenberg, and J.M. Musacchio. 1988. Cross-dependence to opioid and alpha 2-adrenergic
receptor agonists in NG108-15 cells. FASEB J 2: 52–55.
Lee, V.M., and C. Page. 1984. The dynamics of nerve growth factor-induced neurofi lament and vimentin
fi lament expression and organization in PC12 cells. J Neurosci 4: 1705–1714.
Lenz, W., C. Petrusch, K.H. Jakobs, and C.J. van Koppen. 1994. Agonist-induced down-regulation of the
m4 muscarinic acetylcholine receptor occurs without changes in receptor mRNA steady-state levels.
Naunyn Schmiedebergs Arch Pharmacol 350: 507–513.
LePage, K.T., R.W. Dickey, W.H. Gerwick, E.L. Jester, and T.F. Murray. 2005. On the use of neuro-2a
neuroblastoma cells versus intact neurons in primary culture for neurotoxicity studies. Crit Rev
Neurobiol 17: 27–50.
Levavi-Sivan, B., B.H. Park, S. Fuchs, and C.S. Fishburn. 1998. Human D3 dopamine receptor in the
medulloblastoma TE671 cell line: cross-talk between D1 and D3 receptors. FEBS Lett 439: 138–142.
Levi, A., S. Biocca, A. Cattaneo, and P. Calissano. 1988. The mode of action of nerve growth factor in
PC12 cells. Mol Neurobiol 2: 201–226.
Li, S.H., A.L. Cheng, H. Li, and X.J. Li. 1999. Cellular defects and altered gene expression in PC12 cells
stably expressing mutant huntingtin. J Neurosci 19: 5159–5172.
Lipshutz, G.S., D.J. Castro, R.E. Saxton, R.P. Haugland, and J. Soudant. 1994. Evaluation of four new
carbocyanine dyes for photodynamic therapy with lasers. Laryngoscope 104: 996–1002.
Luo, J., and M.W. Miller. 1997. Basic fi broblast growth factor- and platelet-derived growth factor-mediated
cell proliferation in B104 neuroblastoma cells: effect of ethanol on cell cycle kinetics. Brain Res
770: 139–150.
Luo, J., and M.W. Miller. 1999. Transforming growth factor beta1-regulated cell proliferation and expression
of neural cell adhesion molecule in B104 neuroblastoma cells: differential effects of ethanol. J
Neurochem 72: 2286–2293.
Ma’ayan, A., S.L. Jenkins, A. Barash, and R. Iyengar. 2009. Neuro2A differentiation by Galphai/o pathway.
Sci Signal 2:cm1.
Maruiwa, H., Y. Sasaguri, M. Shigemori, M. Hirohata, and M. Morimatsu. 1993. A role of matrix
metalloproteinases produced by glioma-cells. Int J Oncol 3: 1083–1088.
Masserano, J.M., L. Gong, H. Kulaga, I. Baker, and R.J. Wyatt. 1996. Dopamine induces apoptotic cell
death of a catecholaminergic cell line derived from the central nervous system. Mol Pharmacol
50: 1309–1315.
Mathieu, D., R. Lecomte, A.M. Tsanaclis, A. Larouche, and D. Fortin. 2007. Standardization and detailed
characterization of the syngeneic Fischer/F98 glioma model. Can J Neurol Sci 34: 296–306.
Matsuzawa, K., K. Fukuyama, P.B. Dirks, S. Hubbard, M. Murakami, L.E. Becker, and J.T. Rutka. 1996.
Expression of stromelysin 1 in human astrocytoma cell lines. J Neurooncol 30: 181–188.
McAllister, R.M., H. Isaacs, R. Rongey, M. Peer, W. Au, S.W. Soukup, and M.B. Gardner. 1977.
Establishment of a human medulloblastoma cell line. Int J Cancer 20: 206–212.
McDonald, R.L., P.F. Vaughan, and C. Peers. 1994. Muscarinic (M1) receptor-mediated inhibition of K(+)-
evoked [3H]-noradrenaline release from human neuroblastoma (SH-SY5Y) cells via inhibition of
L- and N-type Ca2+ channels. Br J Pharmacol 113: 621–627.
McKinney, M., and R.W. Barrett. 1989. Biochemical evidence for somatostatin receptors in murine
neuroblastoma clone N1E-115. Eur J Pharmacol 162: 397–405.
Merlin, J.L., P. Chastagner, C. Marchal, B. Weber, and P. Bey. 1991. In vitro combination of high dose
busulfan with radiotherapy on medulloblastoma cells: additive effect without potentiation. Anticancer
Drugs 2: 465–468.

184 Neural Cell Biology
Miller, T.R., R.D. Taber, E.J. Molinari, K.L. Whiteaker, L.M. Monteggia, V.E. Scott, J.D. Brioni, J.P. Sullivan,
and M. Gopalakrishnan. 1999. Pharmacological and molecular characterization of ATP-sensitive K+
channels in the TE671 human medulloblastoma cell line. Eur J Pharmacol 370: 179–185.
Milligan, G., F.R. McKenzie, S.J. McClue, F.M. Mitchell, and I. Mullaney. 1990. Guanine nucleotide
binding proteins in neuroblastoma x glioma hybrid, NG108-15, cells. Regulation of expression and
function. Int J Biochem 22: 701–707.
Momboisse, F., S. Ory, M. Ceridono, V. Calco, N. Vitale, M.F. Bader, and S. Gasman. 2010. The Rho
guanine nucleotide exchange factors Intersectin 1L and beta-Pix control calcium-regulated exocytosis
in neuroendocrine PC12 cells. Cell Mol Neurobiol 30: 1327–1333.
Morton, A.J., C. Hammond, W.T. Mason, and G. Henderson. 1992. Characterisation of the L- and N-type
calcium channels in differentiated SH-SY5Y neuroblastoma cells: calcium imaging and single channel
recording. Brain Res Mol Brain Res 13: 53–61.
Murphy, M.G., and Z. Byczko. 1990. Effects of membrane polyunsaturated fatty acids on adenosine receptor
function in intact N1E-115 neuroblastoma cells. Biochem Cell Biol 68: 392–395.
Murphy, P.R., and R.S. Knee. 1995. Basic fi broblast growth factor binding and processing by human glioma
cells. Mol Cell Endocrinol 114: 193–203.
O’Lague, P.H., and S.L. Huttner. 1980. Physiological and morphological studies of rat pheochromocytoma
cells (PC12) chemically fused and grown in culture. Proc Natl Acad Sci U S A 77: 1701–1705.
Ogrodnik, M., H. Salmonowicz, R. Brown, J. Turkowska, W. Sredniawa, S. Pattabiraman, T. Amen,
A.C. Abraham, N. Eichler, R. Lyakhovetsky, and D. Kaganovich. 2014. Dynamic JUNQ inclusion
bodies are asymmetrically inherited in mammalian cell lines through the asymmetric partitioning of
vimentin. Proc Natl Acad Sci U S A 111: 8049–8054.
Oikonomou, E., M. Buchfelder, and E.F. Adams. 2008. Cholecystokinin (CCK) and CCK receptor expression
by human gliomas: Evidence for an autocrine/paracrine stimulatory loop. Neuropeptides 42: 255–265.
Olmsted, J.B., K. Carlson, R. Klebe, F. Ruddle, and J. Rosenbaum. 1970. Isolation of microtubule protein
from cultured mouse neuroblastoma cells. Proc Natl Acad Sci U S A 65: 129–136.
Oortgiesen, M., R.G. van Kleef, and H.P. Vijverberg. 1997. Dual, non-competitive interaction of lead with
neuronal nicotinic acetylcholine receptors in N1E-115 neuroblastoma cells. Brain Res 747: 1–8.
Orlowski, C.C., S.D. Chernausek, and R. Akeson. 1989. Actions of insulin-like growth factor-I on the
B104 neuronal cell line: effects on cell replication, receptor characteristics, and infl uence of secreted
binding protein on ligand binding. J Cell Physiol 139: 469–476.
Park, S.A., H.J. Park, B.I. Lee, Y.H. Ahn, S.U. Kim, and K.S. Choi. 2001. Bcl-2 blocks cisplatin-induced
apoptosis by suppression of ERK-mediated p53 accumulation in B104 cells. Brain Res Mol Brain
Res 93: 18–26.
Peng, Z., Y. Shuangzhu, J. Yongjie, Z. Xinjun, and L. Ying. 2013. TNF receptor-associated factor 6 regulates
proliferation, apoptosis, and invasion of glioma cells. Mol Cell Biochem 377: 87–96.
Ponten, J., and E.H. Macintyre. 1968. Long term culture of normal and neoplastic human glia. Acta Pathol
Microbiol Scand 74: 465–486.
Provost, P. 2010. Interpretation and applicability of microRNA data to the context of Alzheimer’s and
age-related diseases. Aging (Albany NY) 2: 166–169.
Ramaswamy, P., N. Aditi Devi, K. Hurmath Fathima, and N. Dalavaikodihalli Nanjaiah. 2014. Activation
of NMDA receptor of glutamate infl uences MMP-2 activity and proliferation of glioma cells. Neurol
Sci 35: 823–829.
Reeve, H.L., P.F. Vaughan, and C. Peers. 1992. Glibenclamide inhibits a voltage-gated K+ current in the
human neuroblastoma cell line SH-SY5Y. Neurosci Lett 135: 37–40.
Reeve, H.L., P.F. Vaughan, and C. Peers. 1994. Calcium channel currents in undifferentiated human
neuroblastoma (SH-SY5Y) cells: actions and possible interactions of dihydropyridines and omega-
conotoxin. Eur J Neurosci 6: 943–952.
Reifenberger, G., T. Bilzer, R.J. Seitz, and W. Wechsler. 1989. Expression of vimentin and glial fi brillary
acidic protein in ethylnitrosourea-induced rat gliomas and glioma cell lines. Acta Neuropathol
78: 270–282.
Reiser, G., and B. Hamprecht. 1982. Bradykinin induces hyperpolarizations in rat glioma cells and in
neuroblastoma X glioma hybrid cells. Brain Res 239: 191–199.
Richelson, E. 1990. The use of cultured cells in the study of mood-normalizing drugs. Pharmacol Toxicol
66 Suppl 3: 69–75.
Rieger, J., W. Wick, and M. Weller. 2003. Human malignant glioma cells express semaphorins and their
receptors, neuropilins and plexins. Glia 42: 379–389.

Neural Cell Lines (Lineage) 185
Ronnett, G.V., L.D. Hester, J.S. Nye, and S.H. Snyder. 1994. Human cerebral cortical cell lines from
patients with unilateral megalencephaly and Rasmussen’s encephalitis. Neuroscience 63: 1081–1099.
Roth, J.A., C. Horbinski, D. Higgins, P. Lein, and M.D. Garrick. 2002. Mechanisms of manganese-induced
rat pheochromocytoma (PC12) cell death and cell differentiation. Neurotoxicology 23: 147–157.
Ru, Q., X. Tian, M.S. Pi, L. Chen, K. Yue, Q. Xiong, B.M. Ma, and C.Y. Li. 2015. Voltagegated K+ channel
blocker quinidine inhibits proliferation and induces apoptosis by regulating expression of microRNAs
in human glioma U87MG cells. Int J Oncol 46: 833–840.
Sabol, S.L., and M. Nirenberg. 1979. Regulation of adenylate cyclase of neuroblastoma x glioma hybrid
cells by alpha-adrenergic receptors. I. Inhibition of adenylate cyclase mediated by alpha receptors.
J Biol Chem 254: 1913–1920.
Sardi, I., L. Giunti, C. Bresci, A.M. Buccoliero, D. Degl’innocenti, S. Cardellicchio, G. Baroni, F.
Castiglione, M.D. Ros, P. Fiorini, S. Giglio, L. Genitori, M. Arico, and L. Filippi. 2013. Expression
of beta-adrenergic receptors in pediatric malignant brain tumors. Oncol Lett 5: 221–225.
Sato, Y., P.R. Murphy, R. Sato, and H.G. Friesen. 1989. Fibroblast growth factor release by bovine endothelial
cells and human astrocytoma cells in culture is density dependent. Mol Endocrinol 3: 744–748.
Schrier, S.M., E.W. van Tilburg, H. van der Meulen, A.P. Ijzerman, G.J. Mulder, and J.F. Nagelkerke. 2001.
Extracellular adenosine-induced apoptosis in mouse neuroblastoma cells: studies on involvement of
adenosine receptors and adenosine uptake. Biochem Pharmacol 61: 417–425.
Schrier, S.M., B.I. Florea, G.J. Mulder, J.F. Nagelkerke, and I.J. AP. 2002. Apoptosis induced by extracellular
ATP in the mouse neuroblastoma cell line N1E-115: studies on involvement of P2 receptors and
adenosine. Biochem Pharmacol 63: 1119–1126.
Schubert, D., S. Heinemann, W. Carlisle, H. Tarikas, B. Kimes, J. Patrick, J.H. Steinbach, W. Culp, and
B.L. Brandt. 1974. Clonal cell lines from the rat central nervous system. Nature 249: 224–227.
Schubert, D., W. Carlisle, and C. Look. 1975. Putative neurotransmitters in clonal cell lines. Nature
254: 341–343.
Schubert, D., S. Heinemann, and Y. Kidokoro. 1977. Cholinergic metabolism and synapse formation by a
rat nerve cell line. Proc Natl Acad Sci U S A 74: 2579–2583.
Seward, E., C. Hammond, and G. Henderson. 1991. Mu-opioid-receptor-mediated inhibition of the N-type
calcium-channel current. Proc Biol Sci 244: 129–135.
Sharma, S.K., W.A. Klee, and M. Nirenberg. 1975. Dual regulation of adenylate cyclase accounts for
narcotic dependence and tolerance. Proc Natl Acad Sci U S A 72: 3092–3096.
Shen, G., F. Shen, Z. Shi, W. Liu, W. Hu, X. Zheng, L. Wen, and X. Yang. 2008. Identifi cation of cancer
stem-like cells in the C6 glioma cell line and the limitation of current identifi cation methods. In Vitro
Cell Dev Biol Anim 44: 280–289.
Sibenaller, Z.A., A.B. Etame, M.M. Ali, M. Barua, T.A. Braun, T.L. Casavant, and T.C. Ryken. 2005.
Genetic characterization of commonly used glioma cell lines in the rat animal model system.
Neurosurg Focus 19: E1.
Sipe, K.J., D. Srisawasdi, R. Dantzer, K.W. Kelley, and J.A. Weyhenmeyer. 1996. An endogenous 55 kDa
TNF receptor mediates cell death in a neural cell line. Brain Res Mol Brain Res 38: 222–232.
Snider, R.M., and E. Richelson. 1984. Bradykinin receptor-mediated cyclic GMP formation in a nerve cell
population (murine neuroblastoma clone N1E-115). J Neurochem 43: 1749–1754.
Sombers, L.A., T.L. Colliver, and A.G. Ewing. 2002. Differentiated PC12 cells: a better model system
for the study of the VMAT’s effects on neuronal communication. Ann N Y Acad Sci 971: 86–88.
Spicer, Z., and D.E. Millhorn. 2003. Oxygen sensing in neuroendocrine cells and other cell types:
pheochromocytoma (PC12) cells as an experimental model. Endocr Pathol 14: 277–291.
Stepulak, A., H. Luksch, C. Gebhardt, O. Uckermann, J. Marzahn, M. Sifringer, W. Rzeski, C. Staufner,
K.S. Brocke, L. Turski, and C. Ikonomidou. 2009. Expression of glutamate receptor subunits in human
cancers. Histochem Cell Biol 132: 435–445.
Sturm, M.A., C.A. Conover, H. Pham, and R.G. Rosenfeld. 1989. Insulin-like growth factor receptors and
binding protein in rat neuroblastoma cells. Endocrinology 124: 388–396.
Suri, C., B.P. Fung, A.S. Tischler, and D.M. Chikaraishi. 1993. Catecholaminergic cell lines from the brain
and adrenal glands of tyrosine hydroxylase-SV40 T antigen transgenic mice. J Neurosci 13: 1280–1291.
Syapin, P.J., P.M. Salvaterra, and J.K. Engelhardt. 1982. Neuronal-like features of TE671 cells: presence
of a functional nicotinic cholinergic receptor. Brain Res 231: 365–377.
Tischler, A.S., and L.A. Greene. 1975. Nerve growth factor-induced process formation by cultured rat
pheochromocytoma cells. Nature 258: 341–342.

186 Neural Cell Biology
Toral, J., W. Hu, D. Critchett, A.J. Solomon, J.E. Barrett, P.T. Sokol, and M.R. Ziai. 1995. 5-HT3 receptor-
independent inhibition of the depolarization-induced 86Rb effl ux from human neuroblastoma cells,
TE671, by ondansetron. J Pharm Pharmacol 47: 618–622.
Tsunoo, A., M. Yoshii, and T. Narahashi. 1986. Block of calcium channels by enkephalin and somatostatin
in neuroblastoma-glioma hybrid NG108-15 cells. Proc Natl Acad Sci U S A 83: 9832–9836.
Tweardy, D.J., L.A. Cannizzaro, A.P. Palumbo, S. Shane, K. Huebner, P. Vantuinen, D.H. Ledbetter,
J.B. Finan, P.C. Nowell, and G. Rovera. 1987. Molecular cloning and characterization of a cDNA for
human granulocyte colony-stimulating factor (G-CSF) from a glioblastoma multiforme cell line and
localization of the G-CSF gene to chromosome band 17q21. Oncogene Res 1: 209–220.
Uwada, J., A.S. Anisuzzaman, A. Nishimune, H. Yoshiki, and I. Muramatsu. 2011. Intracellular distribution
of functional M(1) -muscarinic acetylcholine receptors in N1E-115 neuroblastoma cells. J Neurochem
118: 958–967.
Vaudry, D., P.J. Stork, P. Lazarovici, and L.E. Eiden. 2002. Signaling pathways for PC12 cell differentiation:
making the right connections. Science 296: 1648–1649.
Vaughan, P.F., C. Peers, and J.H. Walker. 1995. The use of the human neuroblastoma SH-SY5Y to study
the effect of second messengers on noradrenaline release. Gen Pharmacol 26: 1191–1201.
Ventimiglia, J.B., C.J. Wikstrand, L.E. Ostrowski, M.A. Bourdon, V.A. Lightner, and D.D. Bigner. 1992.
Tenascin expression in human glioma cell lines and normal tissues. J Neuroimmunol 36: 41–55.
Verity, A.N., T.L. Wyatt, W. Lee, B. Hajos, P.A. Baecker, R.M. Eglen, and R.M. Johnson. 1999. Differential
regulation of glial cell line-derived neurotrophic factor (GDNF) expression in human neuroblastoma
and glioblastoma cell lines. J Neurosci Res 55: 187–197.
Voltz, R., R. Hohlfeld, A. Fateh-Moghadam, T.N. Witt, M. Wick, C. Reimers, B. Siegele, and H. Wekerle.
1991. Myasthenia gravis: measurement of anti-AChR autoantibodies using cell line TE671. Neurology
41: 1836–1838.
Wang, L.J., R. Colella, G. Yorke, and F.J. Roisen. 1996. The ganglioside GM1 enhances microtubule
networks and changes the morphology of Neuro-2a cells in vitro by altering the distribution of MAP2.
Exp Neurol 139: 1–11.
Westerink, R.H., and A.G. Ewing. 2008. The PC12 cell as model for neurosecretion. Acta Physiol (Oxf)
192: 273–285.
Wick, W., M. Platten, and M. Weller. 2001. Glioma cell invasion: regulation of metalloproteinase activity
by TGF-beta. J Neurooncol 53: 177–185.
Widnell, K.L., D.S. Russell, and E.J. Nestler. 1994. Regulation of expression of cAMP response element-
binding protein in the locus coeruleus in vivo and in a locus coeruleus-like cell line in vitro. Proc Natl
Acad Sci U S A 91: 10947–10951.
Widnell, K.L., J.S. Chen, P.A. Iredale, W.H. Walker, R.S. Duman, J.F. Habener, and E.J. Nestler. 1996.
Transcriptional regulation of CREB (cyclic AMP response element-binding protein) expression in
CATH.a cells. J Neurochem 66: 1770–1773.
Xie, H.R., L.S. Hu, and G.Y. Li. 2010. SH-SY5Y human neuroblastoma cell line: in vitro cell model of
dopaminergic neurons in Parkinson’s disease. Chin Med J (Engl) 123: 1086–1092.
Yoshida, H., I. Date, T. Shingo, K. Fujiwara, Y. Miyoshi, T. Furuta, and T. Ohmoto. 1999. Evaluation of
reaction of primate brain to grafted PC12 cells. Cell Transplant 8: 427–430.
Yoshida, Y., K. Tsuzuki, S. Ishiuchi, and S. Ozawa. 2006. Serum-dependence of AMPA receptor-mediated
proliferation in glioma cells. Pathol Int 56: 262–271.
Zeltzer, P.M., S.L. Schneider, and D.D. Von Hoff. 1984. Morphologic, cytochemical and neurochemical
characterization of the human medulloblastoma cell line TE671. J Neurooncol 2: 35–45.
Zhang, Z., G.J. Drzewiecki, J.T. Hom, P.C. May, and P.A. Hyslop. 1994. Human cortical neuronal (HCN)
cell lines: a model for amyloid beta neurotoxicity. Neurosci Lett 177: 162–164.
Zimmermann, H.P., U. Plagens, and P. Traub. 1987. Infl uence of triethyl lead on neurofi laments in vivo
and in vitro. Neurotoxicology 8: 569–577.