Journal of Cancer Therapy, 2012, 3, 442-451
http://dx.doi.org/10.4236/jct.2012.324057 Published Online September 2012 (http://www.SciRP.org/journal/jct)
Therapy-Induced Changes of Gene Expression in a
Matched Pair of Small Cell Lung Cancer (SCLC) Cell
Lines
Gerhard Hamilton, Ernst Ulsperger, Klaus Geissl er, Ulrike Olszewski
Ludwig Boltzmann Cluster of Translational Oncology, Vienna, Austria.
Received July 31st, 2012; revised September 5th, 2012; accepted September 23rd, 2012
ABSTRACT
Extended stage small cell lung cancer (SCLC) responds to platinum/vepeside-based first-line chemotherapy but relapses
early as drug-resistant tumor associated with a dismal prognosis. A pair of SCLC cell lines obtained from a single pa-
tient at different time points during treatment allows for the investigation of the changes in gene expression before
(GLC14) and following cycles of chemotherapy and irradiation (GLC19). GLC19 cells were reported to reveal an in-
creased doubling time and exhibit increased chemoresistance to doxorubicin, etoposide, melphalan and vinblastine.
Upregulated transcripts in GLC19, as assessed by microarray analysis, comprised genes involved in regulation of cellu-
lar growth (NGFRAP1/BEX3), adhesion, glutathione metabolism and, in particular, WNT/Notch pathways and the pu-
tative cancer stem cell phenotype (CD44, ALDH1A1, and AKR1C1/13). Metallothioneins, tubulins TUBA3/4 and tu-
mor protein p53 inducible protein 11 (TP53IP11) were downregulated in this cell line compared to GLC14. Except in-
creased expression of glutathione transferases no classical markers of chemoresistance were found, pointing to a role of
altered growth control/differentiation and reduced accessibility of these SCLC cells that grow as multicellular spheroids.
In conclusion, treatment of this single SCLC patient with cyclophosphamide, doxorubicin and etoposide (CDE) fol-
lowed by radiotherapy ultimately enriched tumor cells that display the typical signature of tumor-initiating cells/cancer
stem cells (CICs/CSCs).
Keywords: Small Cell Lung Cancer; GLC14; GLC19; Chemotherapy; Chemoresistance; Gene Expression;
Cancer-Initiating Cells
1. Introduction
Due to over one million cases diagnosed every year and
corresponding low survival rates, lung cancer is still the
leading cause of cancer death despite a lower incidence
compared to other tumor entities [1]. Small cell lung
cancer (SCLC) represents approximately 13% of all lung
cancer diagnoses and its incidence has reduced over the
last 20 years [2]. These highly malignant neuroendo-
crine tumors of the lung originate from neuroendocrine
cells (Amine Precursor Uptake and Decarboxyla-
tion/APUD cells) in the bronchus called Feyrter cells.
Treatment of SCLC has remained challenging because of
rapid growth, early dissemination and development of
drug resistance during the course of the disease [3]. The
predominant risk factor for SCLC is cigarette smoking
with smokers facing a 20 - 30-fold higher incidence of
SCLC than non-smokers [2]. Although the number of
cases of SCLC is declining in man the frequency is rising
in women due to increased use of tobacco. Within the
context of the next 20 years, the incidence of lung cancer
will grow with an increasing number of cases unrelated
to smoking [4].
Without treatment SCLC has the most aggressive
clinical course of any type of pulmonary tumors, with
median survival from diagnosis of only 2 - 4 months [2].
Compared with other types of lung cancer, SCLC has a
greater tendency to be widely disseminated at time of
diagnosis while being much more responsive to chemo-
therapy and radiation therapy first-line. Because pa-
tients with SCLC tend to develop distant metastases, lo-
calized forms of treatment, such as surgical resection or
radiation therapy, rarely produce long-term survival.
With current chemotherapy regimens survival is un-
equivocally prolonged; however, the overall survival at 5
years is only 5% - 10% [2,5]. At time of diagnosis ap-
proximately 30% of patients with SCLC have a tumor
confined to either the hemithorax of origin, the media-
stinum, or the supraclavicular lymph nodes. These pa-
tients are classified as having limited-stage disease and

Therapy-Induced Changes of Gene Expression in a Matched Pair of Small Cell Lung Cancer (SCLC) Cell Lines 443
hence a median survival of 16 - 24 months with current
forms of treatment can reasonably be expected [1,5].
Patients with tumors that have spread beyond the supra-
clavicular areas are categorized as having extensive-stage
disease and reveal a worse prognosis than patients with
limited-stage disease. In these cases a median survival of
6 - 12 months is reported with currently available therapy,
whereas long-term disease-free survival is rare.
The majority of patients with SCLC die of their tumor
despite the best available treatment [5,6]. Chemotherapy
improves the survival of patients with limited-stage or
extensive-stage SCLC, but is curative in only a mino-
rity of patients. In patients with limited-stage SCLC
combination chemotherapy produces results that are
clearly superior to single-agent treatment. Current regi-
mens yield overall objective response rates of 65% - 90%
and complete response rates of 45% - 75%. The combi-
nation of etoposide and cisplatin chemotherapy with con-
current chest radiation therapy has now been used in
various single institutional studies and achieved median
survival rates of 18 - 24 months and a 40% - 50% 2-year
survival with less than 3% treatment-related mortality
[1,5,7]. The prognosis for patients with SCLC that has
progressed despite chemotherapy is exceptionally poor
regardless of the stage, with expected median survival of
2 - 3 months. These patients should be considered for
palliative therapy or clinical trials. Though no single che-
motherapy regimen should be regarded standard, those
that have shown activity as second-line treatment include
oral etoposide, etoposide/cisplatin, cyclophosphamide/
doxorubicin/vincristine (CAV), lomustine/methotrexate,
paclitaxel and topotecan [3,5]. Pharmacological inhibit-
tors of receptor tyrosine kinases, including c-Kit, epi-
dermal growth factor receptor (EGFR), insulin-like
growth factor-I receptor (IGFR1) and vascular endothe-
lial growth factor receptor (VEGFR), have been investi-
gated as potential antitumor agents in SCLC with dis-
appointing results [5,8]. Thus, the task of identifying
targeted agents with clinically meaningful activity has so
far proven to be less successful than in other tumors.
In summary, the outcome of treatment of SCLC is
characterized by a lack of progress and extremely short
survival rates in advanced disease exhibiting little im-
provement in the past decades. Despite high initial re-
sponse rates the majority of patients relapse and die of
their disease. Many suggestions discussed the reasons for
this failure, including the presence of resistant stem cells,
development of acquired resistance, redundancy in sig-
naling pathway etc., while the definite underlying cause
has remained unclear. Translational research to correlate
cellular target pathway activation/inhibition with clinical
endpoints is essential to maximize the knowledge gained
from trials and to identify potential predictive markers.
Longitudinal biopsies, viable cells or cell lines that re-
flect the course of chemosensitivity towards resistant
tumors are rarely available for SCLC patients. However,
a series of three tumor biopsies with corresponding cell
lines derived thereof, namely GLC14, GLC16 and
GLC19, was described for a single SCLC patient by
Berendsen et al. in 1988 [9]. There was a good accor-
dance in the morphological, biochemical, and immuno-
histological findings between the cell lines compared to
those obtained in the biopsies This tumor progressed
from an initially responsive state (GLC14) to increased
drug resistance for doxorubicin, etoposide, melphalan
and vinblastine (GLC19), allowing for a gene expression
analysis of the molecular changes acquired during the
relapse and complete treatment failure. In the present
work we describe a comparative analysis of the overex-
pressed gene transcripts and molecular pathways ex-
pected to be linked to tumor progression and chemore-
sistance in this case of SCLC.
2. Materials and Methods
2.1. Cells and Reagents
GLC14 and GLC19 cell lines were obtained from Dr.
Nina Pedersen from the Department of Radiation Bio-
logy, The Finsen Centre, National University Hospital,
Copenhagen, Denmark. Cells were grown in RPMI-1640
bicarbonate medium (Seromed, Berlin, Germany) supple-
mented with 10% fetal bovine serum (Seromed), 4 mM
glutamine and antibiotics (10× stock formulated to con-
tain ~5000 units penicillin, 5 mg streptomycin and 10 mg
neomycin/ml) under tissue culture conditions (37˚C, 5%
CO2, 95% humidity) and checked for mycoplasma con-
tamination (Mycoplasma PCR ELISA, Roche Diagnos-
tics, Vienna, Austria). Cells grow as spheroids in sus-
pension and were subcultures two times a week. Except
where otherwise stated all reagents were from Sig-
ma-Aldrich, St. Louis, MO, USA.
2.2. Chemosensitivity Assay
1 × 104 cells in 100 µl medium per well were distrib-
uted in 96-well microtiter plates (Greiner, Kremsmuen-
ster, Austria) and the test compound added in another
100 µl. Cisplatin (initial concentration 20 µg/ml) and
solute con- trols were serially diluted in twofold steps in
triplicate. The microtiter plates were incubated under
tissue culture conditions for four days and cell viability
was measured using a modified MTT (3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay
(EZ4U, Biomedica, Vienna, Austria). Optical density
was measured at 450 nm using a microplate reader with
an empty well as reference. Values obtained from con-
trol wells containing cells and media alone were set to
100% proliferation.

Therapy-Induced Changes of Gene Expression in a Matched Pair of Small Cell Lung Cancer (SCLC) Cell Lines 444
2.3. Gene Expression and Bioinformatic Analysis
Cells were cultivated in 150 cm2 flasks in triplicate under
tissue culture conditions, harvested and pellets of appro-
ximately 35 × 106 cells stored frozen at –80˚C. Briefly,
lysis with extraction buffer (4 M guanidine isothiocy-
anate, 0.5% sodium N-lauroylsarcosinate, 10 mM EDTA,
5 mM sodium citrate, 100 µM β-mercaptoethanol) was
performed at 4˚C, and DNA and RNA of the lysates were
separated by cesium trifluoroacetate ultracentrifugation.
RNA was washed with ice-cold 96% ethanol and dis-
solved in water. Measurements of the optical density at
260/280 nm proved content and purity of the RNA. Gene
expression analysis was performed in duplicate using the
Applied Biosystems Human Genome Survey Microarray
V2.0 (Applied Biosystems, Foster City, CA, USA).
Therefore, 2 - 5 µg mRNA (20 - 50 µg total RNA) were
reverse transcribed to first-strand cDNA and labeled with
digoxigenin-UTP according to the Applied Biosystems
Chemiluminescent Reverse Transcription protocol. Hy-
bridization of cDNA and microarray analysis was per-
formed pursuant to the Applied Biosystems Chemilumi-
nescence Detection Kit protocol and by use of the Ap-
plied Biosystems 1700 Chemiluminescent Microarray
Analyzer. Data were processed by filtering and quantile
normalization. Microarray probe identities were allocated
to the respective gene designations using the microarray
data information provided by Applied Biosystems. Sig-
nificance Analysis of Microarrays (SAM) was used to
identify differentially expressed genes in GLC19 and
GLC14 cells with a false discovery rate of 12% [10].
Pathway analysis was carried out by help of the Reac-
tome database available at http://www.reactome.org [11].
Statistical analysis was performed using two-tailed Stu-
dent’s t-test for normally distributed samples (*p < 0.05
was regarded as statistically significant).
3. Results
During the course of treatment the primary SCLC, rep-
resented by the chemosensitive GLC14 cell line, ac-
quired a resistant phenotype that is retained in form of
the GLC19 SCLC cell line. Since for this patient therapy
had not included platinum-based drugs, we tested both
cell lines in an MTT assay for their sensitivity to cis-
platin in the present study. Dose-response curves re-
vealed IC50 values of 1.3 ± 0.2 and 0.5 ± 0.09 µg/ml for
GLC14 and GLC19, respectively.
Gene transcripts overexpressed in GLC19 in compari-
son to GLC14 cells are listed in Table 1. 440 transcripts
were found to be upregulated more than 2.5-fold in
GLC19 vs. GLC14 SCLC cells. They include a number
of proteins involved in regulation of cell growth, differ-
entiation and cell death/apoptosis. The most prominent
adaptor proteins affected were nerve growth factor re-
ceptor-associated protein (NGFRAP1/BEX3) and CDK5
regulatory subunit associated protein 1 (CDK5RAP1).
Increased expression was detected for the protooncogens
myc and fos, as well as for the growth factors neuro-
tensin (NTS), midkine (MDK; neurite growth-promoting
factor 2), platelet-derived growth factor D (PDGFD), ser-
ine/threonine protein kinase MST4 and Mps one binder
kinase activators (MOBs). Transcripts for vasoactive in-
testinal peptide receptor 1 (VIPR1), fibroblast growth
factor receptor 1 (FGFR1) and ERBB4 exhibited relative
overexpression in GLC19 cells.
A second group of upregulated messengers comprised
proteins involved in cell adhesion/cell membrane signal
transduction including cell surface associated mucin 15
(MUC15), epithelial membrane protein 2 (EMP2), Pleck-
strin homology domain-containing family C member
(PLEKHC1), receptor for hyaluronic acid CD44, cal-
cium-dependent cell-adhesion protein protocadherin 18
(PCDH18), claudin 1 (CLDN1), cwcv and kazal-like
domains proteoglycan sparc/osteonectin (SPOCK) and
the neural cell adhesion molecule 2 (NCAM2). In-
creased transcription of glutathione S-transferases glu-
tathione S-transferase theta 1 (GSTT1), glutathione S-
transferase alpha 4 (GSTA4), microsomal glutathione
S-transferase 2 (MGST2), and thioredoxin interacting
protein (TXNIP) function in elevated conjugation of re-
duced glutathione to a wide number of exogenous and
endogenous hydrophobic electrophiles for elimination
and in balancing the redox state, respectively.
Several mediators upregulated in GLC19 cells belong
to the wingless-type MMTV integration site family (Wnt)
and to Notch signaling pathways. They comprise se-
creted frizzled-related protein 1 (SFRP1), WNT5B, coac-
tivator mastermind-like 2 (MAML2), transducin-like
enhancer of split 2 (E(sp1)) homolog (TLE2) and the
Notch receptor ligands jagged 1/2 (JAG1 and JAG2).
Putative markers of CSCs overexpressed in GLC19 cells
moreover include CD44, aldehyde dehydrogenase 1 fam-
ily, member A1 (ALDH1A1) and the aldo-keto reductase
family 1 members C1/13 (AKR1C1 and AKR1C13).
Furthermore, several members of the MAGE family that
may play a role in embryonal development, tumor trans-
formation or aspects of tumor progression were elevated
in GLC19 cells. 60 gene transcripts were downregulated
more than 3-fold in GLC19 vs. GLC14 cells and include
several metallothioneins (MTs; 1A, 1F, 1X, 3 and 4),
tumor protein 53 inducible protein 11 (TP53I11), as well
as members of the tubulin family (TUBA3, TUBB4) and
tubulin folding cofactor A (TBCA; data not shown).
The expression data was further analyzed for overrep-
resented pathways with help of the Reactome software
(www.reactome.org) and the significant results are listed
in Table 2. Increases in sphingolipid metabolism, expre-
ssion of mediators of neuronl, Notch, nitric oxide and a

Therapy-Induced Changes of Gene Expression in a Matched Pair of Small Cell Lung Cancer (SCLC) Cell Lines 445
Table 1. Gene transcripts overexpressed in GLC19 vs GLC14.
Identity (ID) ID Expression ratio Gene symbol
Applied Biosystems Celera Genomics GLC19/GLC14
Growth regulators/factors/factor receptors
210438 hCG1992111 573.5 NGFRAP1
132666 hCG15917.3 504.9 MYC
160562 hCG21128.4 150.2 NTS
209085 hCG18178.2 139.1 POF1B
115497 hCG2039648.2 90.9 CDK5RAP1
112198 hCG1783468.2 67.8 MST4
128869 hCG32359.4 24.3 MOB
159434 hCG28852.3 12.8 VIPR1
221237 hCG23105.3 9.5 FGFR1
131372 hCG28052.3 7.2 GUCY1B3
170165 hCG28051.3 3.2 GUCY1A3
205128 hCG22355.3 6.5 FOS
111257 hCG25765.4 4.4 MDK
192974 hCG2012284 3.5 ERBB4
108301 hCG40536.2 3.9 PDGFD
186955 hCG2039636 2.7 SDF2
Cell adhesion
167910 hCG27270.4 303.2 MUC15
173909 hCG14644.2 41.8 EMP2
183236 hCG20966.3 37.8 PLEKHC1
133604 hCG1811182.2 13.6 CD44
199474 hCG16335.3 10.8 PCDH18
152957 hCG17574.3 6.4 CLDN1
125730 hCG39479.3 4.4 SPOCK
199050 hCG2007901.1 3.4 NCAM2
Glutathione metabolism
118930 hCG37372.3 6.9 TXNIP
163516 hCG39955.5 3.2 GSTT1
180333 hCG37948.3 2.6 MGST2
173212 hCG21940.3 2.5 GSTA4
Wnt-Notch signaling pathways
143998 NM_003012.2 51.4 SFRP1
172164 hCG24219.3 6.7 WNT5B
167731 hCG2043008 4.9 MAML2
141263 hCG2039528 3.12 TLE2
123012 hCG38131.3 3.1 JAG1
184984 hCG1748359. 2.8 JAG2
MAGE
159590 hCG38709.2 32.6 MAGEA4
189042 hCG1644199.2 15.8 MAGEA9
181315 hCG38711.2 6.1 MAGEA10
Putative stem cell markers
162248 hCG17306.2 8.6 ALDH1A1
122158 hCG2017792 5.9 AKR1C1
129282 hCG19343.44.9AKR1C13
Table includes the Applied Biosystems identity number, human Celera Genomics clone designation, relative overexpression ratio and gene symbol. Trans-
cripts are grouped according to their main cellular functions.

Therapy-Induced Changes of Gene Expression in a Matched Pair of Small Cell Lung Cancer (SCLC) Cell Lines 446
Table 2. Cellular pathways overrepresented in GLC19 vs GLC14 SCLC cells.
p-value Identifier Name of event
0.001 REACT_19323 Sphingolipid metabolism
0.004 REACT_13685 Neuronal system
0.005 REACT_118859 Signaling by Notch
0.008 REACT_23862 Nitric oxide stimulates guanylate cyclase
0.018 REACT_24989 ATPase type IV transporters - phospholipid transfer
0.027 REACT_75770 Voltage gated potassium channels
0.03 REACT_15518 Transmembrane transport of small molecules
0.048 REACT_21310 Phospholipase C-mediated cascade
The list of overexpressed gene transcripts in GLC19 vs GLC14 was used to identify overrepresented cellular pathways.
phospolipase C signaling, as well as of ion/small mole-
cules/phospolipid membrane transporters were observed.
4. Discussion
The GLC14 cell line was established using a biopsy from
a right supraclavicular node of a 55-year-old woman with
extended SCLC. Treatment was started with cyclophos-
phamide, doxorubicin and etoposide (CDE), which re-
sulted in a complete response [9]. After seven months
tumor recurrence was detected in chest X-ray and rein-
duction chemotherapy with CDE led to a partial response.
Radiotherapy was given to the left hilar region and me-
diastinum and led to a normal chest X-ray, before SCLC
reappeared in the left upper lobe of the lung. At this stage
another cell line (GLC19) was established from a biopsy.
The tumor progressed despite two additional cycles of
CDE and the patient died after discontinuation of therapy,
17 months after first presentation. The electron micro-
scopic appearance of all cell lines was compatible with
SCLC of the “classic type”. Additionally, dense core
vesicles were present in moderate amounts in all cell
lines, whereas desmosomes with associated tonofila-
ments were identified in GLC19 cells only. The IC50 for
doxorubicin and etoposide increased 1.7-fold from 0.44
for GLC14 to 0.75 mM for GLC19 and 5.5-fold from 4.5
for GLC14 to 24.8 ng/ml for GLC19, respectively. Simi-
larly, IC50 values for GLC14 and GLC19 for vinblastine
were 445 and 1936 ng/ml, for vincristine, 145.7 and
179.9 ng/ml and for actinomycin, 0.084 and 0.337 µg/ml,
revealing an increased drug resistance for GLC19 cells
for doxorubicin, etoposide, Vinca alkaloids and actino-
mycin. Whereas in RPMI-1640 with 10% heat-inactivated
fetal calf serum GLC14 cells grew almost completely
attached to the culture flask, GLC19 cells formed float-
ing aggregates and exhibited a prolonged doubling time
of 44 hrs compared to 26 hrs for GLC14 cells [12]. Cis-
platin was not used for treatment of this patient and we
found a slight decrease of the chemosensitivity for this
drug in GLC19 vs GLC14 cells in good agreement with a
previous report [12].
In order to detect global changes in gene expression
linked to SCLC tumor progression and chemoresistance,
we compared gene transcripts of GLC14 and GLC19
cells using analysis by the Applied Biosystems Human
Genome Survey Microarrays V2.0. Approximately 440
genes were more than 2.5-fold overexpressed in GLC19
cells and 100 genes were more than 3-fold downregu-
lated in this cell line compared to GLC14 cells. Of the
440 upregulated transcripts, approximately 300 could be
assigned to annotated genes. 40 of these with strongest
elevations and highest relevance for the SCLC phenotype
under investigation are listed in Table 1. 37/60 down-
regulated transcripts could be traced to annotated genes.
A panel of genes upregulated in GLC19 vs. GLC14
cells are involved in regulation of cell growth, differenti-
ation and cell death/apoptosis. Overexpression of NGFR-
AP1/BEX3, a p75NTR (low-affinity neurotrophin recep-
tor p75)-associated cell death executor initially cloned
from a human ovarian granulosa cell cDNA library as an
unknown protein termed pHGR74, was likewise reported
by Lawson et al. and constituted the most upregulated
gene in our data set [13,14]. BEX3 belongs to a family of
genes, including BEX1, NGFRAP1 (alias BEX3),
BEXL1 (alias BEX4), and NGFRAP1L1 (alias BEX5)
[15]. Both BEX1 and NGFRAP1 interact with p75NTR
and modulate NGF signaling through NF-κB to regulate
cell cycle, apoptosis and differentiation in neural tissues
[16]. Enforced expression of NGFRAP1 in Chinese ham-
ster ovary cells and MDA-MB-231 human breast cancer
cells had little effect on the growth of the cells in vitro,
while cellular growth was dramatically suppressed in
vivo [17]. Furthermore, the cells stably transfected with
NGFRAP1 did not respond neither to NGF nor TNF.
These findings may be linked to the prolonged doubling
time of GLC19 vs. GLC14 cells. In breast cancer cells,
NGF inhibits ceramide-homolog-induced apoptosis through
binding of p75NTR and NF-κB activation via BEX2.
Additionally, overexpression of NGFRAP1 enhances the
antiproliferative effect of tamoxifen at pharmacological
dose. Furthermore, NGFR-AP1 was reported to consti-

Therapy-Induced Changes of Gene Expression in a Matched Pair of Small Cell Lung Cancer (SCLC) Cell Lines 447
tute a marker of androgen-dependent primary prostate
cancer [18]. A second cell cycle regulator, namely
CDK5RAP1, specifically inhibits CDK5 activation by
CDK5R1, which is the catalytic subunit of neuronal
Cdc2-like kinase (Nclk) involved inneuronal cell differ-
entiation, and apoptosis [19].
Increased expression was detected for the protoonco-
gens myc and fos, as well for the growth factors neuro-
tensin (NTS), MDK, PDGFD, serine/threonine protein
kinase MST4 and Mps one binder kinase activators
(MOBs). Upregulation of the expression of myc in
GLC19 was previously described by Kok et al. [20].
Amplification and overexpression of the MYC family of
oncogenes, MYC (c-Myc), MYCN (N-Myc) or MYCL1
(L-Myc), occurs in 18% - 31% of SCLCs, and is more
common in chemorefractory disease [21]. Transcripts for
VIPR1, FGFR1 and ERBB4 exhibited relative overex-
pression in GLC19 cells, which indicates cellular re-
sponses to their cognate growth factors VIP, FGF and
EGF, respectively. VIP in SCLC is involved in upregula-
tion of VEGF via fos, and FGFs as well as EGF are sup-
port the proliferation of CSCs under serum-depleted
conditions [22,23]. In general, neuropeptides can func-
tion as autocrine growth factors in cancer cells or modu-
late the microenvironment [24,25].
GLC19 cells overexpress WNT5B, a ligand for mem-
bers of the frizzled family of seven transmembrane re-
ceptors, SFRP1 which functions as modulator of Wnt
signaling through direct interaction with Wnts, and TLE2,
which inhibits the transcriptional activation mediated by
β-catenin (CTNNB1) and TCF/LEF family members in
Wnt signaling [26]. The Notch pathway powerfully in-
fluences stem cell maintenance, development and cell
fate and is increasingly recognized for the key roles it
plays in cancer [27]. Notch promotes cell survival, an-
giogenesis and resistance in numerous cancers, making it
a promising target for cancer therapy. In respect to the
Notch signaling pathway, MAML2, which acts as a tran-
scriptional coactivator for Notch proteins as well as in-
creased expression of JAG1 and JAG2, that are ligands
for multiple Notch receptors are distinctive characteris-
tics of GLC14 and GLC19 cells. Notch activation in-
duces the transcription of both subunits of the soluble
guanylyl cyclase (sGC) heterodimer, namely GUCY1A3
and GUCY1B3, which form the nitric oxide receptor [28].
Additionally, the core stem cell signaling networks, such
as the Wnt, Notch and Hedgehog pathways, also criti-
cally regulate the self-renewal and survival of CSCs [29].
A number of upregulated transcripts in GLC19 cells
comprise genes involved in cell adhesion/cell membrane
signaling in accordance with the exclusive finding of
desmosomes in GLC19 exclusively in this series of
SCLC lines [9]. CD44 cell-surface glycoprotein, which
represents the receptor for hyaluronic acid (HA), is in-
volved in cell-cell interactions, cell adhesion and migra-
tion [30]. It mediates cell-cell and cell-matrix interact-
tions through its affinity for HA and possibly also for
other ligands such as osteopontin, collagens, and matrix
metalloproteinases (MMPs). Furthermore, CD44 has
been defined as a CIC marker in many tumor entities,
promoting epithelial-to-mesenchymal transition (EMT),
motility, endothelial cell and niche adhesion, thereby
contributing to niche generation and modulation [31].
MUC15 may play a role in cell adhesion to the extracel-
lular matrix (ECM) and promotes the oncogenic poten-
tial of human colon cancer cells [32]. The upregulated
transcripts PLEKHC1, SPOCK, CLDN1 and EMP2 con-
nect ECM adhesion sites to the actin cytoskeleton, medi-
ate calcium-independent cell-adhesion and regulate the
surface display and signaling from selected integrin pairs,
respectively [33-35]. NCAM2 is a close homolog of
neural cell adhesion molecule NCAM1 (CD56), which is
expressed in several types of tumors, constitutes a diag-
nostic marker of SCLCs, and is associated with tumori-
genesis in salivary gland tumors [36]. Compared with
NCAM1, the function of NCAM2 in tumors, such as
prostate and breast cancer, is unknown, but may repre-
sent a therapeutic target [37].
Many principal antitumor agents, such as platinum
complexes and doxorubicin are electrophiles or re-
dox-active agents and may preferentially react with sulf-
hydryl groups [38]. The elevated amount of glutathione
(GSH) and the increased activity of GSTs can explain the
resistance to alkylating agents and other drugs that act by
releasing free radicals. Our finding of increased tran-
scription of the GSTs GSTT1, GSTA4 and MGST2 in
GLC19 cells is in good agreement with data published in
a previous analysis of its mechanisms of resistance [10].
The GSH level of GLC19 and global GST activity were
significantly increased compared to GLC14, whereas the
amount of total sulfhydryl groups remained the same and,
furthermore, L-buthionine-sulfoximine (BSO), which
depletes the cellular GSH pool, increased both doxorubi-
cin- and cisplatin-induced cytotoxicity. This seems to
constitute an important mechanism of chemoresistance
for GLC19 cells, since other effectors like P-gp are ab-
sent in GLC19 [10].
ALDH1A1 was expressed at low level in GLC14 cells
with the amount rising in the GLC19 cells. This gene is
used as a CSC marker and is also associated with cyclo-
phosphamide resistance [39-41]. The AKR1C1/13 genes
encode members of the aldo/keto reductase super-family,
that consists of more than 40 known enzymes, which
catalyze the conversion of aldehyde and ketone moieties
of metabolites and xenobiotics to corresponding alcohols
by utilizing NADH and/or NADPH as cofactors.
AKR1C1 and C2 were found as genes linked to poor
clinical prognosis in non-small cell lung cancer (NSCLC)

Therapy-Induced Changes of Gene Expression in a Matched Pair of Small Cell Lung Cancer (SCLC) Cell Lines 448
following characterization of elevated transcripts of the
A549 CSCs Hoechst 33342-positive side population [42].
AKR1C13 transcription was increased in leukemia cells
following transcription factor Meis1-triggered induction
of a CSC phenotype [43]. In our data set MEIS1
(hCG1818196.1) was upregulated 3.5-fold in GLC19
compared to GLC14 cells. A family of cancer-related
molecules are the MAGEs that are in normal tissues ex-
clusively found in testis and often highly expressed in
cancer cell lines and tumors. They are targets for many
studies on immunotherapy for melanoma; however, since
tumor xenografts of SCLC cell lines had a markedly
lower amount of MAGEs, expression of these genes
seems to be activated in tissue culture only and therefore
not to represent a promising target for this tumor entity
[44]. Lawson et al. analyzed the GLC14/16/19 series of
SCLC cell lines using the Illumina Human WGv3.0 Ex-
pression BeadChip microarray and identified polymerase
B (POLB) and NKX2.2 as transcripts linked to resistance
to etoposide [14]. Of the selected unique gene transcripts
that were upregulated in GLC19 compared to GLC14 as
published by Lawson et al. 18/23 probes were as well
found. Additionally, we found overexpression of growth
factors and their cognate receptors that was not reported
before.
Downregulationed gene expression in GLC19 in com-
parison to GLC14 cells was detected for a compara-
tively low number of approximately 60 transcripts. Sur-
prisingly, GLC14 cells expressed significant amounts of
MTs (1A, 1F, 1X, 3 and 4), which showed lower levels
of transcripts after several cycles of therapy in GLC19
cells. Presence of these MTs in the chemonaive GLC14
cells is could be explained by a smoking history of the
affected patient, which is known to be associated with
increased expression of these heavy metal-binding pro-
teins [45]. The tumor suppressor p53 regulates the ex-
pression of various genes that promote apoptosis.
TP53I11 is a direct target gene of p53 and was markedly
upregulated in arsenic trioxide-induced apoptosis and,
thus, downregulation of this protein may impede drug-
induced apoptosis [46]. Downregulation of the transcrip-
tion of TUBA3, TUBB4 and tubulin folding cofactor A
TBCA seems to be a consequence of the crossresistance
of GLC19 cells to Vinca alkaloids [47].
Sphingolipid metabolism constituted the most over-
represented pathway in GLC19 vs. GLC14 cells. Sphin-
golipids are known to play important roles in the regula-
tion of cell proliferation, response to chemo-therapeutic
agents, and/or prevention of cancer. [48,49]. Bioactive
sphingolipids like ceramide, sphingosine 1-phosphate
and globotriaosylceramide initiate and process cellular
signaling to alter cell behaviour to respond immediately
to oncogenic stress or treatment challenges. More than
ten sphingolipids and glycosphingolipids selectively me-
diate the expression of approximately 50 genes encoding
c-myc, p21, c-fos, telomerase reverse transcriptase, cas-
pase-9, Bcl-x, cyclooxygenase-2, MMPs, integrins, Oct-4,
glucosylceramide synthase and P-gp [50]. Through di-
verse functions of these genes, sphingolipids enduringly
affect cellular processes of mitosis, apoptosis, migration,
stemness of CSCs and cellular resistance to therapies.
Mechanistic studies indicate that sphingolipids regulate
gene expression by modulation of phosphorylation and
acetylation of proteins that serve as transcription factors
(β-catenin, Sp1), repressor of transcription (histone H3)
and regulator (SRp30a) of RNA splicing.
In conclusion, GLC19 cells are distinguished from
GLC14 by a reduced growth rate, formation of multicel-
lular spheroids, expression of CD44, increased expres-
sion of components of the Wnt and Notch signaling
pathways, as well as receptors of the growth factors
FGFs and EGF and the elevated transcription of the CSC
markers ALDH1A1 and AKR1C1/13. Although the mi-
croarray results need to be confirmed by PCR, our find-
ings are compatible with the typical signature estab-
lished for CICs or CSCs and different tumor entities
[51,52]. Characterization of CICs in SCLC is being on-
going and increased expression of ABCG2, FGF1, IGF1,
MYC, SOX1/2, WNT1, as well as genes involved in an-
giogenesis, Notch and Hedgehog pathways was reported
for this type of tumor cells that comprise a fraction of
less than one percent in SCLC cell lines [53]. The gene
expression profile of GLC19 cells indicates a ther-
apy-induced increase of typical stem cell markers in vivo
and enhanced cell adhesion and formation of spheroids
may constitute an important part in the acquisition of
chemoresistance through a reduced access of chemo-
therapeutic drugs [54].
5. Acknowledgements
This work was funded by the “Medical Scientific Fund of
the Mayor of the City of Vienna“, project number 11016.
We wish to thank the team of the “Oncology Lab” of the
University Clinic of Gynecology, Medical University of
Vienna, Austria, for help with the microarry experiments.
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