Group Title: Molecular Cancer 2008, 7:87
Title: ALDH isozymes downregulation affects cell growth, cell motility and gene expression in lung cancer cells
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Title: ALDH isozymes downregulation affects cell growth, cell motility and gene expression in lung cancer cells
Series Title: Molecular Cancer 2008, 7:87
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Creator: Moreb JS
Baker HV
Chang LJ
Amaya M
Lopez MC
Ostmark B
Chou W
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Volume ID: VID00001
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Molecular Cancer Bioed Central



Research

ALDH isozymes downregulation affects cell growth, cell motility
and gene expression in lung cancer cells
Jan S Moreb*1, Henry V Baker2, Lung-Ji Chang2, Maria Amaya', M
Cecilia Lopez2, Blanca OstmarkI and Wayne Chou2


Address: 'Department of Medicine, University of Florida, Gainesville, Florida, USA and 2Department of Molecular Genetics and Microbiology,
University of Florida, Gainesville, Florida, USA
Email: Jan S Moreb* morebjs@medicine.ufl.edu; Henry V Baker hvbaker@ufl.edu; Lung-Ji Chang lchang@mgm.ufl.edu;
Maria Amaya mariphoo@ufl.edu; M Cecilia Lopez MCLopez@ufl.edu; Blanca Ostmark bostmark@ufl.edu; Wayne Chou wchou@ufl.edu
* Corresponding author



Published: 24 November 2008 Received: 19 June 2008
Molecular Cancer 2008, 7:87 doi:10.1 186/1476-4598-7-87 Accepted: 24 November 2008
This article is available from: http://www.molecular-cancer.com/content/7/l/87
2008 Moreb et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: Aldehyde dehydrogenase isozymes ALDH IAl and ALDH3AI are highly expressed
in non small cell lung cancer. Neither the mechanisms nor the biologic significance for such over
expression have been studied.
Methods: We have employed oligonucleotide microarrays to analyze changes in gene profiles in
A549 lung cancer cell line in which ALDH activity was reduced by up to 95% using lentiviral
mediated expression of siRNA against both isozymes (Lenti 1+3). Stringent analysis methods were
used to identify gene expression patterns that are specific to the knock down of ALDH activity and
significantly different in comparison to wild type A549 cells (WT) or cells similarly transduced with
green fluorescent protein (GFP) siRNA.
Results: We confirmed significant and specific down regulation of ALDH IAI and ALDH3AI in
Lenti 1 +3 cells and in comparison to 12 other ALDH genes detected. The results of the microarray
analysis were validated by real time RT-PCR on RNA obtained from Lenti I +3 or WT cells treated
with ALDH activity inhibitors. Detailed functional analysis was performed on 101 genes that were
significantly different (P < 0.001) and their expression changed by 2 2 folds in the Lenti 1+3 group
versus the control groups. There were 75 down regulated and 26 up regulated genes. Protein
binding, organ development, signal transduction, transcription, lipid metabolism, and cell migration
and adhesion were among the most affected pathways.
Conclusion: These molecular effects of the ALDH knock-down are associated with in vitro
functional changes in the proliferation and motility of these cells and demonstrate the significance
of ALDH enzymes in cell homeostasis with a potentially significant impact on the treatment of lung
cancer.




Background lism of a wide variety of aliphatic and aromatic aldehydes
Aldehyde dehydrogenases (ALDHs) are a group of [1,2]. Many disparate aldehydes are ubiquitous in nature
NAD(P)+-dependent enzymes involved in the metabo- and are toxic at low levels because of their chemical reac-


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tivity. Thus levels of metabolic-intermediate aldehydes
must be carefully regulated which explains the existence
of several distinct ALDH families in most studied organ-
isms with wide constitutive tissue distribution [1,2]. A sys-
tematic nomenclature scheme for the ALDH gene
superfamily based on divergent evolution has been devel-
oped [3] and continues to be updated on paper [4] and on
the internet by Dr. Vasilis Vasiliou and his group htt://
www.aldh.org. According to the latest database, the
human genome contains 19 ALDH functional genes and
three pseudogenes [4].

The role of some of these ALDHs in endobiotic and xeno-
biotic metabolism has been reviewed extensively before
and the specific metabolic pathways affected have been
detailed [2]. Many allelic variants within the ALDH gene
family have been identified, resulting in pharmacogenetic
heterogeneity between individuals which, in most cases,
results in distinct phenotypes [2,5] including intolerance
to alcohol and increased risk of ethanol-induced cancers
(ALDH2 and ALDH1A1), Sjogren-Larson Syndrome
(ALDH3A1), type II hyperprolinemia (ALDH4A1), 4-
hydroxybutyric aciduria (ALDH5A1), developmental
delay (ALDH6A1), hyperammonemia (ALDH18A1), and
late onset of Alzheimer's disease (ALDH2). Furthermore,
knockouts of ALDH1A2 and ALDH1A3 in mouse are
embryonic lethal and newborn lethal, respectively [6-8].
Changes in ALDH activity have been observed during
experimental liver and urinary bladder carcinogenesis and
in a number of human tumors [9].

One of the well studied pathways of ALDH activity is drug
resistance to oxazaphosphorines. We have been interested
in the role ofALDH 1A1 in drug resistance, first in hemat-
opoietic progenitors and more recently in lung cancer.
ALDH1A1, ALDH3A1, and ALDH5A1 have been shown
to catalyze the oxidation of aldophosphamide [10-12].
We and others have shown that overexpression of
ALDH1A1 and ALDH3A1 results in resistance to 4-
hydroperoxycyclophosphamide (4-HC), an active deriva-
tive of cyclophosphamide (CP) [9-11,13,14]. More
recently, ALDH3A1 was recognized as an oxidative stress
response protein and thus can protect against the oxida-
tive damage caused by other chemotherapy drugs such as
etoposide [15]. We have also shown that down regulation
of each enzyme by RNA antisense (AS) [16], all-trans
retinoic acid (ATRA) [17] or siRNA [18] results in
increased sensitivity to 4-HC.

Tetraethylthiuram disulfide (TT) (disulfiram, also known
as Antabuse), an ALDH inhibitor, has been reported to
affect the growth of multiple tumor cells, inhibit cancer
cell invasiveness, and induce apoptosis using in vitro
assays [19]. These effects were thought to be due to differ-
ent mechanisms including inhibition of proteasome


activity [20], increase Cu uptake with pro-oxidant effects
[21,22], inhibition of NF KB [23-25], inhibition of the
relaxation activity of DNA topoisomerases I and II [26],
and inhibition of caspases [27].

All of the above studies indicate the biologic and clinical
significance of these enzymes and, therefore, the need to
better define the regulatory mechanisms involved in
determining their level of expression in normal and
malignant tissues. Multiple studies, mainly in animal
models, have been published on the regulation of the var-
ious ALDH isozymes [28-31]. Functional genomics aim at
analyzing the regulation of genes in response to physio-
logical changes. Microarray technology revolutionized the
analysis of gene expression in biological processes to ena-
ble the assessment of gene activity on a genome-wide
scale. In order to be able to perform such experiment in
relation to ALDH1A1 and ALDH3A1, we have aimed at
achieving "knock-down" of these enzymes using siRNA
approach in vitro. Indeed, we achieved > 95% "knock-
down" of ALDH activity in A549 lung cancer cell line
using lentiviral vectors to permanently express siRNA
sequences specific to each one of the enzymes. The data
presented here reveal multiple genes affected by the
"knock-down" of ALDH activity which will ultimately aid
in identifying the molecular events related to the activity
and regulation of ALDHs expression be it in oxidative
stress, response to carcinogenic aldehydes or malignant
transformation.

Methods
Cell Lines
A549 and H522 cell lines were obtained from ATCC and
maintained in -80 C freezer or cultured in RPMI 1640
medium containing 10% FBS before experiments. Cells
were maintained in 5% CO2 incubator at 370C and used
for experiments in the exponential phase of their growth.
These cell lines were specifically chosen for the described
experiments because they are known to have high levels of
ALDH1A1 and ALDH3A1 [32]. These cells were trans-
duced with lentiviral vectors (described below) contain-
ing specific siRNA sequences against ALDH1A1 (Lenti 1
cells), ALDH3A1 (Lenti 3 cells), both vectors (Lenti 1+3
cells), and against the green fluorescent protein (GFP)
gene (GFP cells, used as a control). Overall total of 5 cell
lines from A549 and H522 were used throughout the
experiments described here including the parent wild type
cell line (WT). Microarray gene profiling was performed
only on A549 cells. In order to further validate that the
effects seen in the ALDH knock down cells are directly
related to ALDH activity, we used known ALDH inhibitors
as described below in A549 and H522 cells as well as one
other lung cancer cell line, H1299, that lacks any signifi-
cant expression of ALDH1A1 or ALDH3A1 [32].



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Effects of ALDH Knock-Down on Cell Growth and Motility
The assays described below were used to study the effects
ofALDH knock down by siRNA on cell growth and migra-
tion as well as colony formation. Triplicates of 2 x 105
cells/ml/well were plated in 6-well tissue culture plates
and grown for 72 hours. After this time, the cells were
trypsinized, collected and manually counted using hema-
tocytometer and microscope. Viability was determined by
trypan blue exclusion. For these experiments, we used the
five cell lines from H522 or A549. Means SD of growth
rates were calculated and differences in growth rates were
compared using the Student t-test.

A colorimetric assay kit (MTT based) for the non-radioac-
tive quantification of cell proliferation and viability was
purchased from Roche Diagnostics Corporation (Indiana-
polis, IN) and used for the cell proliferation experiments.
In a 96-well plate, 1 x 103 cells/100 iL/well of each cell
line were cultured for 96 hours. After the 96 hour incuba-
tion period, the labeling reagent was added to each well,
followed by the solubilization solution four hours later.
The cells were left overnight in the incubator, and absorb-
ance was measured the following day using a Molecular
Devices Kinetic Microplate reader at a wavelength of 570
nm as per the manufacturer's protocol. In this experiment,
absorbance directly correlates with number of viable cells
per well. Mean + SD of the absorbance values were calcu-
lated for each experimental group and compared using
the Student t-test.

For each of the cell lines, triplicates of 200 cells/ml/well in
a six-well plate were cultured in RPMI-1640 + 10% FBS.
The cells were allowed to grow for 5 days. The number of
colonies adhered to the bottom of the plate was then
counted using inverted microscope. The mean number of
colonies was calculated and compared among all five cell
lines originating from the A549 or H522 cell lines.

We measured the effect of down regulation of ALDH on
cell migration and motility by using the in vitro scratch
wound migration assay [33]. A549 monolayer confluent
cells from the five different cell lines were scratched using
sterile 200 il pipette tip in 6-well plastic dishes, and after
20 hr of culture in RPMI-1640 +10% FBS, the migration
ability of the cells was evaluated by the width of the
wound under inverted microscope fitted with digital cam-
era. Images of the scratch wound were taken immediately
after the scratch and 20 hr later. The migration distances
(in cm) of the cells were measured on printed images
taken at the same magnification and the difference of the
width of wounds at 0 and 20 hr was determined and the
% wound healing was calculated.


SiRNA Plasmid Constructs
SiRNA constructs were made using pSilencer 2.0-U6 as
vector backbone from Ambion. To generate the lentiviral
siRNA constructs, the pSilencer 2.0-U6 siRNA plasmid
was digested with Pvu II and the U6-siRNA cassette was
cloned into the self-inactivating (SIN) pTYF-EFnlacZ len-
tiviral vector in the Not I site which was blunted with T4
DNA polymerase. For the constructions of ALDH LV
siRNA vectors, the target site for ALDH1A1 (Genbank
NM 000689) is: 5'-gtagccttcacaggatcaa-3' (nt 777-795)
was chosen, and two primers were used to generate the
siRNA construct; primer 1: 5ATA CGC GGA TCC CGT
AGC CTT CAC AGG ATC AAT TCA AGA G AT TGA TC-3',
primer 2: 5'CGC TAG ACT AGT TAA AAA AGT AGC CTT
CAC AGG ATC AAT CTC TTG AA TTG ATC -3'; the target
site for ALDH3A1 (Genbank NM 000691) is: 5'-gaagat-
gattgcagagaca-3' (nt1167-1185), and two primers were
used to generate the siRNA construct: primer 1: 5' ATA
CGC GGA TCC CGA AGA TGA TTG CAG AGA CAT TCA
AGA GAT GTC T-3', primer 2: 5' CGC TAG ACT AGT TAA
AAA AGA AGA TGA TTG CAG AGA CAT CTC TTG AA TGT
CT -3'. As a control, we similarly generated U6 promoter-
driven LV-siRNA targeting GFP, as described before [34].
The lentiviral constructs with the U6-siRNA inserted in the
reverse orientation were chosen for vector preparation
(see Figure 1).

Lentiviral Vector Production, Titration and Transduction
To produce lentiviral vectors, 293T cells were co-trans-
fected with the three vector plasmids: pNHP, pHEFVSVG,
and pTYF siRNA vector, and the virus supernatants were
concentrated and titered as previously described [35]. For
lentiviral transduction, the cells were plated at a concen-
tration of 105 cells per well in 12-well plate to give 60-
80% confluence after overnight culture and then infected
at MOI of 50-100 in the presence of polybrene (8 ig/ml).
Transduction efficiency was determined by the LacZ
reporter gene assay as previously described [35].

After successful transduction with lentiviral constructs, we
plated cells for liquid colony culture assay at 200 cells in
1 ml per 35 mm plate. On approximately day 5 of culture,
we picked single colonies using inverted microscope and
sterile pipette for replating and establishing clones for
each of Lenti 1, Lenti 3, Lenti 1+3, and GFP cell lines. The
100% purity of the clones was validated by LacZ staining
done on similarly cultured colonies from these estab-
lished clones.

ALDH Activity and Protein Measurements
In order to measure the effects of the siRNA expression, we
used the spectrophotometric methods for measurement
of ALDH enzymatic activity as well Western blot analysis
to detect changes in the protein levels of ALDH1A1 and
ALDH3A1. Western blot analysis was performed as


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Transducing vector
pTYF-EZ-siRNA



Helper construct
pNCHP


Envelope
pHEF-VSVG


env deletion


VSV-G nm1rA


hWLp T-siP.NA


dL p g T m ucription


Vlvlgaenione RNA


Host genwue DNA F EI


siRNA


Pol 11.IKZnscrjption

ftlcZ


Figure I
Lentiviral constructs. Schematic presentation of the Lentiviral constructs used in our experiments to express the various
siRNA sequences against ALDH IAI, ALDH3AI, and GFP.


described before [17,18]. In brief, lysates from each of the
5 cell lines mentioned above were size separated in paral-
lel on two 12% denaturing SDS-polyacrylamide gels (Bio-
Rad, Hercules, CA), electrotransferred onto nitrocellulose
membranes, blocked with 5% milk in TBS, and probed
using chicken anti human ALDH1A1 and ALDH3A1 pol-
yclonal antibodies provided generously by Dr. L. Sreer-
ama (St Cloud University, Minneapolis, MN) and Dr. NE
Sladek (University of Minnesota, Minneapolis, MN). The
specificity of these antibodies has been documented by
Dr. Sladek's group [36,37]. Blots were incubated with
chicken anti-human ALDH1A1 and ALDH3A1 primary
antibodies at 1:200 or 1:300 dilution, respectively, for 1
hour at room temperature. After washing, the secondary
antibody (horseradish peroxidase-labeled rabbit anti-
chicken antibody; Sigma Chemical Co., St Louis, MO) was


used at 1:4000 dilution for 1 hour. Chemiluminscence
method (SuperSignal, Pierce, Rockford, IL) was used for
the final visualization of the protein bands on X-ray film
(Super Rx, Fuji Photo Film, Tokyo, Japan). After washing
and blocking, the same blots were labeled again for visu-
alization of actin as a loading control using anti-actin
antibody (Oncogene Research Products, Cambridge, MA).

The cell lysates used for Western blot analysis, were also
freshly used to measure ALDH enzyme activity using the
spectrophotometric assay as described before [17,18].
Briefly, the aliquots of 600 il lysing buffer were incubated
at 37C in Beckman DLC 64 spectrophotometer cuvettes
with the addition of cell lysate, 5 mM NAD+ and 5 mM
propionaldehyde as a substrate. The rate of change in
absorbance at 340 nm was measured in 3 replicates over


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VSV-G nlvA
I


i A


Molecular Cancer 2008, 7:87


hLOB-siRNA~

. ..........
dL gag


LEZLan







http://www. molecular-cancer.com/content/7/1/87


5 min. A control reaction in which the substrate was not
added monitored the endogenous rate ofNAD+ reduction.
The ALDH activity was expressed in nmoles/107 cells.min.

RNA Isolation and cDNA Synthesis
A549 cell lines (WT, GFP, and Lenti 1+3) were plated in
25 cm2 Coming cell culture flasks at a density of 7.5 x 105
cells/ml with RPMI 1640 medium containing 10% FBS at
37 C. Four culture sets were prepared for each experimen-
tal group. Four different clones were used for GFP and
Lenti 1+3. After 24 hr incubation, media was removed
from the dish and RLT buffer was added directly to the
cells. Cells were scraped into this buffer and subsequently
homogenized using the QIAshredder kit (Qiagen, Valen-
cia, CA). Total RNA was then harvested using the RNeasy
RNA isolation kit (Qiagen). Integrity of total RNA was val-
idated using a Bioanalyzer (Agilent Technologies) as well
as OD 260/280 ratios.

Two micrograms of total RNA were utilized for the cDNA
synthesis using the cDNA synthesis kit (Affymetrix
900431) and according to the Affymetrix technical proto-
cols. First-strand synthesis was initiated using Superscript
II Reverse Transcriptase and the T7-(dt) primer (Affyme-
trix 900431), which contains the T7 promoter sequence
followed by an oligo (dt) tract. Second strand synthesis
was carried out using Escherichia coli DNA polymerase I, E.
coli DNA ligase, and RNase H (Affymetrix 900431). Five
microliters of purified ds cDNA was used to generate
biotin-labeled cRNA using the IVT labeling kit (Affymetrix
900449). Biotin labeled cRNA was then purified from
unincorporated nucleotides using the RNA cleanup kit
(Affymetrix 900371). Quantity and quality of cRNA was
determined by OD260/280 ratios and by using a Bioana-
lyzer (Agilent Technologies).

Probe and Preparation/Staining of Array
Before using the array, biotin-labeled cRNA was frag-
mented for 35 minutes at 94 C. The following reagents
were mixed to give the following final concentrations:
fragmented cRNA (0.05 gg/ul), control oligonucleotide
B2 (50 pM, Affymetrix), 20X eukaryotic hybridization
controls (BioB at 1.5 pM, BioC at 5 pM, BioDn at 25 pM,
and CreX at 100 pM, Affymetrix 900454), herring sperm
DNA (0.1 mg/ml, Promega), acetylated BSA (50 mg/ml,
GIBCO-BRL), and 2X hybridization buffer. Probe was
heated to 99 C for 5 min, then transferred to 45 C for 5
min, and finally spun at maximum in a microcentrifuge
for 5 min. Arrays used for this study were the Human
Genome U133 Plus 2.0 Array (Affymetrix) representing
over 47,000 transcripts and variants including 38,500
well-characterized human genes. Chips were interrogated
with material harvested from A549 WT, Lenti 1+3 and
GFP cells and each cell treatment was replicated four times
using 4 chips for each experimental group, making the


data from each array truly independent biological repli-
cas. Each array was injected with 200 ul of target and
rotated in a 45 C oven for 16 hours. Probe was removed
from the array and the array was washed and stained using
the Euk-WS2 fluidics protocol and the streptavidin-phy-
coerythrin (SAPE) reagent (Affymetrix).

Microarray Scanning,, Normalization, Expression Filters,
Cluster Analysis and Statistical Analysis
Scanned images (*.dat files) were analyzed with GCOS
software to generate a detection call for each probe set.
Hybridization single intensities between GeneChips were
normalized and an expression matrix was derived with
dChip [38] using the PM only model model-based expres-
sion index. Probe sets whose hybridization single intensi-
ties were never measured above background levels were
removed from the data set to reduce the noise level in the
data set. Probe sets whose hybridization single intensities
were measured at or below background levels were identi-
fied using the Affymetrix detection call algorithm [39].

Supervised learning, cross validation, and visualization
The modeled-based expression matrix of the probe sets
passing the initial expression filter were analyzed in log
space with BRB Array Tools 3.2.2 (developed by Richard
Simon and Amy Peng Lam and is available online at the
Biometric Research Branch of the National Cancer Insti-
tute website http://linus.nci.nih.gov/BRB-Array
Tools.html to identify genes that differentiated among the
treatment classes at the P < 0.001 significance threshold.
The ability of gene identified at the P < 0.001 significance
threshold to be differentiated among treatment classes
was assessed by using leave-one-out-cross-validation
(LOOCV) using a nearest-neighbor prediction model. For
visualization purposes the modeled-based expression
matrix of probes sets identified as significant among the
treatment groups were mean centered and variance nor-
malized using algorithms implemented in dChip.

Functional Analysis of Transcripts
Probe sets that were significantly different at P < 0.001
and demonstrated > 2 fold change in the level of gene
expression were subject to comprehensive, detailed, six-
level functional analysis, whereby identity was verified
using GenBank accession number, aliases were compiled,
and biochemical and biological functions were deter-
mined. After the identity of the probe set was confirmed,
multiple internet bioinformatics and National Center for
Biotechnology Information (NCBI) search engines,
including PubMed searches, were used to assign the bio-
logical function. Probe sets without confirmed identities
were placed in the "Unknown" category. Probe sets that
could be identified but that did not have ascertainable
function were classified as "Other"



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Validation of Selected Gene Expression Levels by
Quantitative Real-Time RT-PCR
In order to confirm microarray data, we performed real-
time RT-PCR [40] for 7 selected genes measuring the
change in their level of expression. Primer/probe sets for
HMOX1, MAP3K8, MGST1, CXXC5, LHX8, RAB20,
STRA6 and 18S rRNA (reference control) were designed
by Assays-on-Demand (Applied Biosystems, Foster City,
CA, USA) and are listed in Table 1. Total RNA was
obtained as described above from all five A549 cells lines
described under "Cell Lines".

Two micrograms of total RNA were reverse transcribed to
cDNA in a 50 ptl reaction containing 4 ptl of 100 mM
MgC1, 1.0 il of RNase inhibitor, 4 tl of 10x PCR buffer, 1
1l of 10 mM dNTP mix, 1.0 Al of random hexamers, 1.0
il of reverse transcriptase (Invitrogen). Mixture was incu-
bated for 50 min at 42 C and the reaction was stopped by
heating to 70 C for 15 min. For each of the Genes studied
and 18S, 3 l of 400 ng/mL diluted cDNA was added to
12.5 il of Taqman Universal Master Mix (Applied Biosys-
tems), 1.25 Al of 20x primer/Probe combination, and
8.25 Al of DEPC-treated water per each reaction. RT-PCR
quantification and determination of expression levels
were performed on ABI Prism 7900 and Sequence Detec-
tion Software 1.6 (Applied Biosystems, Foster City, CA,
USA). All PCR reactions were performed in triplicates in
our Gene Expression Core Facility and the results were
analyzed using comparative method following normali-
zation of expression values to 18S rRNA expression.

The Effects of ALDH Inhibitors on ALDH Activity, Cell
Growth and Gene Expression
For comparison to the siRNA induced knock-down of
ALDH activity and as a way of further validation of the

Table I: Primers used for quantitative real time RT-PCR.


microarray results, we also used known inhibitors of
ALDH, Tetraethylthiuram disulfide (TT, disulfiram) or
diethylaminobenzaldehyde (DEAB), to study the effects
of ALDH inhibition on cell growth and expression of
selected genes. These inhibitors were purchased from
Sigma Biochemicals (Milwaukee, WI) dissolved in DMSO
and used in a final DMSO concentration of< 0.1%.

In these experiments, we used the A549 and H522 parent
cell lines as well as H1299 cells, a cell line known not to
have any measurable ALDH activity [32]. Dose response
curves (0, 0.1, 1, 10, and 100 riM) were performed for
each inhibitor measuring ALDH activity, cell growth and
viability. We used either manual cell counts or the calori-
metric (MTT) assay, as described above. In subsequent
experiments only 1 and 100 jiM concentrations were
used. Cells were plated at 2 x 105/well in 6-well plates for
24 hr before adding the inhibitor. After 48 hr incubation
with and without the inhibitors (untreated controls), the
cells were harvested, counted and then used for ALDH
activity assay as described above. The results were
expressed as % decrease in cell proliferation or ALDH
activity in comparison to untreated control.

In order to confirm that changes in the expression of
selected genes are indeed specific to the siRNA induced
knock-down of ALDH activity, we performed real-time
RT-PCR on 3 of the 7 genes mentioned above (CXXC5,
LHX8, RAB20) using RNA obtained from WT A549 cells
after incubation with 5 jiM of either TT or DEAB for 48 hr.
Obtaining similar changes in the expression of these 3
genes after treatment with ALDH inhibitors would
strongly suggest direct relationship between ALDH1A1
and ALDH3A1 activity and the changes in the gene profile
demonstrated by the microarray analysis.


Gene


Gene Product


STRA6 Stimulated by retinoic acid Gene 6 Homolog (mouse)


18S Eukaryotic 18S rRNA


RAB20 Member RAS oncogene family


CXXC5 CXXC finger 5


MAP3K8 Mitogen-activated protein Kinase 8


HMOXI Heme oxygenase I


LHX8 Zinc finger transcription factor

MGSTI Microsomal glutathione S-transferase I


Catalogue #

Hs00223621_ml

Hs99999901_sl

Hs00215134_ml

Hs00212840_ml

Hs00178297_ml

Hs00157965_ml

Hs00418293_ml

Hs00220393 ml


Sequence


GCACCCAGCCAAGATGGGAAAACTG

CATTGGAGGGCAAGTCTGGTGCCAG

ACACCGCAGGGCGGGAGCAGTTCCA

TCCGCTGCTCTGGAGAAGGTGATGC

AACATGGTCATCACTCCCCAAAATG

CGGCTTCAAGCTGGTGATGGCCTCC

AGATCAGCTTCAGGTTATGCAAGCA

CAAGAAAGGTTTTTGCCAATCCAGA


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Results
ALDH Knock-Down Effect on ALDH Activity and Proteins
The expression of the specific siRNAs in A549 cells
resulted in a significant decrease in ALDH activity as dem-
onstrated by spectrophotometric enzyme activity assay
(Figure 2A). The effect of siRNA against each ALDH iso-
zyme was also demonstrated by Western blot analysis
(Figure 2A). With such significant decrease in ALDH activ-
ity and proteins in the Lenti 1+3 cell line, we proceeded
with the microarray gene profiling experiment comparing
gene expression in this cell line to that in GFP and WT
control cell lines. Similar decrease in ALDH activity was
obtained in H522 lung cancer cell line using the same len-
tiviral constructs (Figure 2B). However, we have observed
the loss of siRNA effect after continuous culture of either
cell line for more than 4 weeks. Because of that, fresh cells
were thawed every two weeks, and ALDH activity verified
before any experiments were performed.

ALDH Knock-Down Effects on Cell Growth and Motility
A549 and H522 cells (2 x 105/group) were plated for 72
hours and then counted and viability determined. Figure
3A shows the results which reflect mean + SD of cell
number from at least 4 experiments. The growth rate was
significantly lower (P < 0.024) in Lenti-1, Lenti-3, and
Lenti-1+3 in comparison to GFP, except for A549 Lenti-1
with P value of 0.052. No significant difference was found
between the GFP and the WT. Using the MTT cell prolifer-
ation assay, we were able to show similar effects (data not
shown).

A549 cells (200/petri dish in triplicates per each group)
were plated for 5 days and colonies adhered to the bottom
of the plates were counted after this time period. Results
show that the mean number of colonies decreased signif-
icantly (range, 27-71%) for the Lenti- 1, Lenti-3 and Lenti-
1+3 cells in comparison to GFP and WT cells of both A549
and H522 (P < 0.016 and 0.003, respectively).

An example of the scratch wound migration test results for
A549 cells are shown in Figure 3C which demonstrates
delayed wound healing of the Lenti 1+3 cells in compari-
son to control cells. Summary of at least 4 readings are
given for all the five A549 cell lines (Figure 3B). These
results show significant inhibition (P < 0.005) of cell
migration by knock-down of each enzyme separately as
well as both enzymes (Lenti 1+3).

Significant Changes in Gene Expression Related to
"Knock-down" of ALDH Isozymes
To construct the transcriptional profiles associated with
the "knock-down" of ALDH1A1 and ALDH3A1 in A549
cells (Lenti 1+3), two other cell lines, WT and GFP, were
used to interrogate the Affymetrix U133 Plus human
genome GeneChips containing 38500 well-known genes.


The statistical analyses are detailed above. We performed
a modified F test to identify probe sets whose hybridiza-
tion signal intensities varied significantly as a function of
the ALDH "Knock-down". Overall about 3735 probe sets
out of total of 54675 sets were detected above background
and statistically different (P < 0.001) in the Lenti 1+3
experimental group when compared to the WT group
(Figure 4) (supplemental material is available on http://
www.ncbi.nlm.nih.gov/geo using accession number
GSE8045). Many more changes in gene expression
(17269 probe sets) were seen when 3 way comparisons
were done among the 3 experimental groups mainly due
to non-specific changes seen in the GFP group. In order to
arrange the significant genes into functional framework,
we chose only those genes that were significant (P <
0.001) and differed by > 2 fold in the Lenti 1+3 group
when compared to the WT and GFP groups. There were 26
up-regulated and 77 down-regulated genes including
ALDH1A1 and ALDH3A1. We summarized the chromo-
somal location for these genes in Figure 5. We also
researched their biological and biochemical properties
using multiple internet bioinformatics search engines,
web sites, and NCBI resources including BLAST, PubMed,
etc. The collective results of those searches were synthe-
sized into color-coded, mechanism-based pies of gene
expression (Figure 6). These illustrations provide the rela-
tive dominance of each of the biological-biochemical
processes most affected by the "Knock-down" of the
ALDH isozymes. The names of these genes are tabulated
in Tables 2 and 3. In summary, the affected genes are
found on multiple chromosomes with the largest cluster
(8 genes) observed on chromosome 12. The biological
pathway most affected was signal transduction, organ
development and Transcription among both the induced
and repressed genes, although more pathways were found
to be involved with the repressed genes. On the other
hand, the biochemical pathways most affected in induced
genes were protein binding, cell adhesion and lipid
metabolism, while with the suppressed genes protein
binding, intracellular signaling and DNA related proc-
esses were most affected (see Figure 6). As shown in Table
2, eight of the repressed genes (ID4, DDX3Y, RPS4Y1,
EIF1AY, CCL20, GPC6, GPR37, and HMGA2) had the
highest decrease (> 6 folds) in expression. At least of these
genes, RPS4Y1 and CCL20 are highly expressed in WT
A549 cells.

The Overall Effect on Other ALDH Isozymes
Since the human genome contains 19 ALDH functional
genes and three pseudogenes [5], we sought to analyze the
effect of the siRNA sequences used here on the other
members of the ALDH family. We interrogated the data
set of probes that showed changes in gene expression and
pulled out the ALDH genes and compared changes among
the 3 different experimental groups. The data is summa-


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160
~s-

S140 -

S120 -

I 100-
-
-
S80 -

60-



S20-
0-


GFP


Lerti 1
11522 Cells


Lerti 3 Lent 1+3


Figure 2
A. The effect of siRNA against ALDH IA I and ALDH3AI on ALDH activity and protein. The mean percent SD
of remaining ALDH activity (in comparison to control GFP group) as measured by spectrophotometry enzyme activity assay is
shown in A549 cells transduced by lentiviral vector containing siRNA against ALDH IAl (Lenti I), ALDH3AI (Lenti 3), both
vectors (Lenti 1+3), green fluorescent protein (GFP), and untreated parent cell line (WT). The results represent at least 3 dif-
ferent experiments. Furthermore, Western blot analysis shows the decrease in ALDH IAl and ALDH3AI proteins in Lenti
1+3 group in comparison to WT and GFP control groups. Actin is shown as a loading control. The results are typical of an
experiment performed at least twice. B. Lentivirally mediated expression of siRNA constructs (shown in A.) results in similar
effects on ALDH activity in H522 cell line.




Page 8 of 19
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Lenti 1+3 GFP WT
ALDH1A1 w

ALD(IA311 NO


GFP Lenti 1 Lenti 3 Lenti 1+3
A549 Cells


140-

120



S80-

, 60-

E 40-

* 20-


0-o-


Molecular Cancer 2008, 7:87







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A.
25
20
x


5.

WT GFP Lenti 1 Lenti 3 Lenti 1+3
Experimental Groups

B.
0)80-
r.
S70

40-k

i O- --


=IA549
MH522


WT GFP Lenti 1 Lenti 3 Lenti 1+3
Experimental groups
*(P < 0.005)


Figure 3
The effect of siRNA expression on cell proliferation and motility. A. Knock-down of ALDHIAI, ALDH3AI or both
results in significant decrease in cell proliferation in comparison to WT and GFP controls from A549 and H522 cells (P < 0.024,
except for A549 Lenti- I with P value of 0.052). The horizontal line indicates the starting cell number (2 x I OS/group) incubated,
while the bars represent mean SD of viable cell counts after 72 hr incubation obtained from at least 2 experiments. Scratch
wound migration assay results are shown. B. The bars represent mean SD of 4 measurements of wound healing in the 5 dif-
ferent cell lines of A549 cell. Significant decrease in cell migration into scratch wound is shown after 20 hr in cells with
ALDH IAl (Lenti- ), ALDH3AI (Lenti-3) and both isozymes (Lenti 1+3) knock-down in comparison to WT cells or cells trans-
duced with GFP siRNA (P < 0.005). C. An example of the delayed wound healing seen with the knock-down of both isozymes
(Lenti 1+3 cells) on wound healing in comparison to WT and GFP cells.




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mm








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Table 2: Down regulated Genes (P < 0.001 and > 2 folds) in Lenti 1+3 cells in comparison to WT and GFP control cells.


Gene


SOCS3
SQSTM I
WWOX
JMY
BIRC3
BCL2AI
ITGB5
TENS I
TGFBR3
IGFBP6
UPKIB
ANXAIO
CCL20
NEDDI
GPR37
PMP22
MXI I
INHBB
ALDH3AI
MAP3 K I
SCEL
STEAP2
TGFB2
ALDHIAI
TRAPPC2
TNNTI
ADAM 10


Fold Change in Expression

-2.26
-2.63
-2.67
-2.26
-3.18
-3.07
-2.53
-2
-2.13
-3.25
-2.02
-4.03
-9.52
-2.07
-13.52
-2.11
-2.94
-3.04
-3.73
-2
-3.87
-3.6
-2.12
-8.13
-2
-3.3
-2.69


Gene


Fold Change in Expression


Gene

KCTD12
B4GALT5
CCNGI
BCATI
ASNS
CXCL3
CXCL2
SMADI
MICB
PDGFC
NTS
TFAP2C
SMCY
DDX3Y
THRAP6
NCOA3
USP9Y
PEX13
VCL
ID2
ID4
MAFF
HMGA2
TFAM
TNRC9
MAP3K8
FLJ23749


Fold Change in Expression

-2.26
-2.12
-2
-2.43
-2
-2.82
-2.29
-2.43
-4.35
-2.88
-4.77
-2.35
-2.79
-7.45
-2.48
-2.37
-3.26
-2.26
-2.01
-2.03
-16.12
-5.47
-6.69
-2.49
-3.48
-3.62
-2.22


There were total of 77 genes including ALDH IAl and ALDH3AI (shown in bold).


rized in Table 4, and it shows that the siRNA sequences
used are quite specific which validates our data further.
ALDH1A1 and ALDH3A1 transcripts were decreased by
about 7 and 4 folds, respectively. Total of another 12
ALDH genes showed some expression variability, but it
was either non-specific (similar in GFP and Lenti 1+3) or
minimal.

Comparison of Apparent Gene Expression among DNA
Array and Real-Time RT PCR
In order to assess the validity of the microarray results, we
used real-time RT-PCR to compare the expression of 8


transcripts with significant expression changes (P < 0.001
and > 2 folds in Lenti 1+3) per the microarray in all 5 cell
lines mentioned in Figure 2A include WT, GFP, Lenti 1,
Lenti 3 and Lenti 1+3. Figure 7 shows results for 3 genes
CXXC5, RAB20, and LHX8 that confirm the results seen in
the 3 experimental groups of the microarray. The increase
in CXXC5 and RAB20 expression was synergistic with the
knock-down of both ALDH1A1 and ALDH3A1, while the
effect seen on LHX8 seems to be related to the lentiviral
vector integration/effect because it is seen equally in all
cell lines except WT. HMOX1 and MGST1 were increased
in Lenti 1+3 and to a lesser degree with either Lenti 1 or


Table 3: Up regulated genes (P < 0.001 and > 2 folds) in Lenti 1+3 cells when compared to WT and GFP control cells.


Fold Change in Expression

2.16
2.51
2.39
2.52
2.59
2.27
2.77
2.05
3.1 1
2.21


Gene Fold Change in Expression


ILI I
CXXC5
RAB20
TFEC
OR51 E2
EHF
THSD2
TM4SF4
FST
ASRGLI


Gene Fold Change in Expression


HMOXI
CFH
BAAT
GATM
FADS2
ANXA8


There were total of 26 genes.


Page 10 of 19
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GPC6
FAM33A
C6orf55
SUSD2
LOC286144
TKT
FILIPI
SMILE
Clorf24
CITED
CST I
FLJ20399
ZFP90
UTY
OSRF
RHOBTB3
KIAA1571
DREI
EMPI
RPS4YI
VCL
EIFIAY
SDPR


-7.47
-2.52
-3.05
-3.61
-4.05
-2.1 1
-5.26
-2.2
-4.02
-2.01
-3.76
-3.63
-2.09
-2.4
-2.21
-2.02
-2.22
-2.42
-2.18
-9.06
-2.01
-18.29
-3.8


Gene


TGFBRI
PAK3
PPARG
MGC11242
FNI
CNTNAP2
VTN
SULTICI
CLDN2
PBEFI


Molecular Cancer 2008, 7:87







http://www. molecular-cancer.com/content/7/1/87


CD CD) CD) C)
C5 0C5


-2.0 -1.3 -0.7 0 0.7 1.3 2.0


Figure 4
Hybridization signal intensities varied significantly as
a function of the ALDH "Knock-down". Four different
Affymetrix U 133 Plus human genome GeneChips were inter-
rogated using labeled cRNA from WT or Lenti 1+3 cells
from 4 different established clones (Vertical columns). Over-
all about 3735 probe sets (horizontal rows) out of total of
54675 sets were detected above background and statistically
different (P < 0.001) in the Lenti 1+3 experimental group
when compared to the WT group.


.%.- C*7-


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1. jr


Molecular Cancer 2008, 7:87


Lenti 3. On the other hand, MAP3K8 expression was
decreased in Lenti 1+3 only, while STRA6 was decreased
in Lenti 1+3 and Lenti 1. Thus, the real-time RT PCR meas-
urements of all 7 transcripts showed agreement of 100%
with the microarray results. LHX8 was the only gene out
of this group exhibiting non specific change in expression,
both by microarray as well real-time RT PCR. On the other
hand, MGST1 and STRA6 change in expression did not
meet the cutoff of > 2 folds.

ALDH Inhibitors Effects on Cell Proliferation and Gene
Expression
To further validate our results, cells (1000/well) were
incubated with ALDH inhibitors, DEAB or TT, for 72 hrs
and cell proliferation measured by the calorimetric MTT
based assay (Figure 8A). Absorbance was significantly
lower (P < 0.0001) after adding any concentration of TT
to A549 cells as well as to H522 cells. Absorbance was sig-
nificantly lower (P < 0.023) after adding 0.1 jiM DEAB to
A549 cells, while for H522 cells, the absorbance was sig-
nificantly lower (P < 0.007) only after adding > 1 jM of
DEAB. The slight recovery of cell proliferation at 10 jiM
concentration of TT has been described before [19]. These
proliferation inhibitory effects seem to be specific to the
inhibition of ALDH activity since the use of these inhibi-
tors in H1299 cells lacking ALDH1A1 and ALDH3A1
expression or any significant ALDH activity [32] resulted
in significantly less effects (P < 0.029) of either TT or
DEAB on H1299 cell growth in comparison to H522 and
A549 cell lines (Figure 8B). The dose-response effect of
both inhibitors (1 and 100 jiM) on ALDH activity in A549
and H522 cells is shown in Figure 8C.

Furthermore, 3 of the genes (CXXC5, RAB20 and LHX8,
shown in Figure 7) were examined using real-time RT-PCR
after 48 hr incubation of A549 cells with 5 tiM DEAB or
TT. The results show similar changes in the expression of
CXXC5 and RAB20 to what was seen in the microarray
results and real-time RT-PCR (Figure 8D). Once again
change in LHX8 expression was affected differently by the
two different inhibitors, which is suggestive of non-spe-
cific effect.

Discussion
In this study, we have used microarray analysis to assess
global changes in gene expression that result from the
knock-down of ALDH1A1 and ALDH3A1 in lung cancer
cell line in order to gain insight into the underlying mech-
anisms that lead to up regulation of these two isozymes in
some cancers. The goal of this study was also to identify
genes or gene networks that interact with these isozymes
and may be responsible for their biological effects. We
have been successful in achieving significant knock-down
of these two isozymes that resulted in abolishing up to
95% of ALDH activity in A549 cells used in these experi-







http://www. molecular-cancer.com/content/7/1/87


4-



2-



S0-

U
46 -2-


4-




-6-



-8-



-10-


II


1 2 3 4 5 6 7 8 9 1011121314 1516171819202122 X Y

Chromosomes


Figure 5
Chromosomal clustering of affected genes. Chromosomal clustering of 101 genes that were found to be significantly dif-
ferent (P < 0.001) and by 2 2 folds in Lenti 1+3 cells in comparison to WT and GFP control groups. The black bars represent
the number of up-regulated genes, while the grey bars represent the number of down-regulated genes on each chromosome.


ments. We have seen significant changes in the rate of
growth of H522 and A549 cell lines as well as significant
decrease in cell motility and migration ofA549 cells with
the knock-down of ALDH1A1 and/or ALDH3A1. Similar
effects have been described before with the in vitro use of
low doses of disulfiram (TT) [41,19], and we were able to
show similar effects on cell growth by the addition of
DEAB or TT, both are known ALDH inhibitors, to cell cul-
tures in experiments presented above. As we mentioned in
the introduction, multiple other mechanisms have been
elucidated for the TT effects on cell growth and apoptosis,
however our results suggest that it may have to do mostly
with ALDH activity either directly or indirectly through
the multiple metabolic effects of ALDH isozymes. This is
mainly based on two facts that are reported here for the


first time: another ALDH inhibitor such as DEAB causes
similar effects on cell growth like TT and the microarray
gene analysis presented here reveals that multiple genes
are affected with the knock-down of ALDH1A1 and
ALDH3A1.

Our results also show that the siRNA sequences used here
are specific to these two isozymes and do not significantly
affect 12 other ALDH genes according to the microarray
analysis. Thus, we feel confident that the findings pre-
sented here are indeed true representation of the changes
in ALDH 1A and ALDH3A1 activity.

The role ofALDH isozymes in many endobiotic and xeno-
biotic metabolic pathways has been described in details


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Repressed Genes, Biological Pathways


Induced Genes, Biological Pathway


Unknown
12%
L._


Signal transduction
- 17%


Transcription Ne--l-




S ...- .veopment

Reproductive Inune
9% Metabolism 5%
1%


Metabolism
7%
Reproductive
4%
Inmune
4%




Organ
Development
26%


Signal transduction
r 29%


SNeural
11%


Transcription
15%


Repressed Genes. Biochemical Pathways
Oxyreductase Activity ATP Binding Cell
4% % Adheson/Migration
Chemokine/Cytokine 8%
Activity .. ...
10% C ,ell Cycle
5%
other
4%
Metabolism other.-- DNA related
4% 12%



Intracellular Signaling
l 12%

Protein Biri'].r. .
33% Lt ^, rated
5%


Induced Genes, Biochemical Pathway
Cytokine Related
8%


Lipid Metabolism /
15% '.







\ .
Protein Bindin. '
26%
2G% [.---^-i
r
Intracellular
Signaling
4%


ATP/GTP Binding
r 12%


Cell Differentiation
8%


Cell Adhesion
15%


(Cell Proliferation
r 4%

DNA related
8%


Figure 6
Color-coded pies showing the distribution of induced and repressed genes into functional categories. There
were 26 significantly induced genes and 75 significantly repressed genes as a result of ALDH activity knock down in Lenti 1+3
cells compared to control groups WT and GFP. Both groups of genes were categorized once into various known biochemical
pathways and again into the different biological pathways.


before. ALDH1A1 and/or ALDH3A1 have been impli-
cated in retinoic acid metabolism, metabolism of alde-
hydes produced during lipid metabolism, alcohol
metabolism, cyclophosphamide metabolism, and oxida-
tive stress response [5]. We have reported the upregualtion
of ALDH1A1 mRNA and protein in hematopoietic pro-
genitors by interleukin-1 and tumor necrosis factor alpha
[42]. Furthermore, some mutations in ALDH isozymes
have been described that lead to specific clinical pheno-
types or diseases, and many of these diseases are charac-
terized by neurologic abnormalities [5]. Disulfiram
(Antabuse, TT), an ALDH inhibitor used to treat alcohol-
ism, has been reported to cause significant neurologic tox-
icity in patients [43,44]. Thus, it is not surprising to detect


such wide spread changes in the levels of multiple genes
as revealed in our microarray analysis. These include
down and up regulated genes (2 2 fold) that are involved
in neural biological pathways, such as GPR37, PMP22,
NTS, KCTD12 (PFET1), SMAD1 (all down regulated 2 2
fold), as well as PAK3, CNTNAP2, and OR51E2 (up regu-
lated 2 2 fold). Furthermore, several biochemical path-
ways are affected in the A549 cells expressing siRNA
against both ALDH1A1 and ALDH3A1 such as protein
binding, cell adhesion, intracellular signaling, and lipid
metabolism. These pathways are inter-related since pro-
tein binding could be in the center of intracellular signal-
ing; while lipid metabolism could lead to changes in


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Table 4: Changes in the expression of the different ALDH genes
profiled in the microarray including ALDH IAI and ALDH3AI.


Gene Symbol Accession No.


ALDH3AI
ALDHIAI
ALDH2
ALDH3BI
ALDH3B2
ALDHIA3
ALDH IA2
ALDH9AI
ALDH I8AI
ALDHILI
ALDH7AI
ALDH5AI
ALDH4AI
ALDH6AI


NM 000691
NM 000689
NM 000690
NM 000694
NM 000695
NM 000693
AB015228
NM 000696
U76542
NM 012190
BC002515
NM 001080
NM 003748
AW612403


Mean of Intensities
Lenti + 3 GFP WT


900.2
1366.7
2172.6
619.7
81.2
94.5
8 1.1
693.2
807.3
120.6
450.4
139.1
231.9
77


2300.5
9197.6
828
349.4
99.5
229.3
125.9
563.8
554.4
74
395
72.5
188.1
40.2


3363.7
9390.9
3083.2
617.1
113.6
144.5
125.1
809.5
549
108.4
542.1
112.2
265.4
74.5


Note: ALDH3BI, ALDH3B2, ALDH4AI, ALDH6AI, and ALDH 18AI
had two sets of probes, which demonstrated similar changes as shown
in the table above.


membrane structure and, as a result, changes in adhesion
properties.

The most important data we hoped to obtain from the
microarray analysis was identifying genes (transcription
factors) that may have a role in the regulation of expres-
sion of these two enzymes and may give clues to the
mechanisms involved in their up regulation in the differ-
ent types of cancer. Previous studies have shown that
ALDH1A1 can be up-regulated by HOX11 [45,46], and
that peroxisome proliferator activated receptor gamma
(PPAR-y) affect the expression of ALDH3A1 via other
unknown transcription factors [28]. Furthermore, the
involvement of the Ah receptor nuclear translocator
(ARNT) in xenobiotic induction of ALDH3A1 has been
established [47]. Our results confirm the involvement of
PPAR-y in ALDH regulation which was found to be signif-
icantly up regulated by ALDH knock down (Table 3). Also
in this study, we have identified significant changes in
some transcription factors such as TFEC which was up reg-
ulated (2 2 folds), and TFAM, LISCH7, HBP1 and HOXA5
which were down regulated (> 2 folds). HBP1 is highly
expressed in bronchial epithelium [48], while HOXA5
was reported to be abnormally expressed in NSCLC [49].
Again, these observations will open the door for further
studies in order to define mechanisms involved in the reg-
ulation of ALDH1A1 and ALDH3A1.

Most importantly, our finding showing significant
decrease in the levels of 8 genes by > 6 folds implies the
importance ofALDH activity in cell homeostasis and can-
cer transformation. These include CCL20, GPR37,
DDX3Y, ID4, GPC6, RPS4Y1, EIF1AY, and HMGA2.


Figure 7
Quantitative real-time RT-PCR to validate the
results of microarray analysis. Results of quantitative
real-time RT-PCR for 3 (CXXC5, RAB20, LHX8) out of 7
seven genes studied are shown here. These genes were
found to be significantly different (P > 0.001) by microarray
analysis in Lenti I +3 experimental group, in which both
ALDH IAl and ALDH3AI were knocked down by siRNA
expression, in comparison to WT control group. The real-
time RT-PCR was performed on RNA obtained from 5 cell
lines including cells that express siRNA against ALDH IAl
only (Lenti I), against ALDH3AI only (Lenti 3), or against
green fluorescent protein (GFP) as another control, in addi-
tion to the Lenti 1+3 and WT groups. The results are
expressed as the log 10 of relative change in each gene
expression with the denominator being the 18S as a refer-
ence control and using the fold increase (+) or decrease (-)
compared to the WT/18S ratio as the baseline expression
value. The direction (up- or down-regulation) for all 7 genes
confirmed the results of microarray analysis even in the Lenti
I and Lenti 3 cells. LHX8 was the only gene out of the 7
tested that was non-specifically down-regulated since it
showed similar changes in the GFP control group as well.


According to the microarray results, RPS4Y1 and CCL20
are highly expressed in A549 cells, while the other 5 genes
are expressed at lower levels. Information about the bio-
logical functions of these 8 genes was extracted form
reviewing relevant publications and is summarized in
Table 5. Some of these genes are involved in transcription


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P1


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A. C.
> 100-
1.05-
S75-
c 0.80-


0 o.ss-f 25-


0.05- I 0

DEAB TT DEAB TT
Concentration (pM) Concentration (pM)

cB. D.
.= 2
T 100 H1299 0.4
0 75 IH522
75 A549 0.3,
a. I' A549






S DEAB TT O.02.

Concentration (pIoM) oJ
Genes Monitored


Figure 8
The effects of ALDH inhibitors DEAB and TT on ALDH activity, cell proliferation and gene expression. A.
Dose-response effect of DEAB and TT on cell proliferation of A549 and H522 lung cancer cell lines. A colorimetric non-radio-
active (MTT based) assay was used as described in Methods to quantify cell proliferation. The results are expressed as the
mean SD of absorbance and reflect 8 measurements from 2 experiments. Overall, significant decrease (P < 0.023) in cell pro-
liferation was seen with both inhibitors. B &C. H 1299 lung cancer cell line with no measurable ALDH activity and A549 and
H522 cells were cultured with or without ALDH inhibitors and cell proliferation and ALDH activity were measured as
described in Methods. The results are expressed as % decrease (mean SD of at least 2 experiments) in cell proliferation (B)
or ALDH activity (C) in comparison to untreated controls. The results demonstrate that the effect of ALDH inhibitors (I and
100 piM) on H 1299 cell proliferation is significantly (P < 0.029) less than that on A549 and H522. These results suggest that the
effect on cell proliferation is mostly due to the inhibition of ALDH activity. D. After 48 hr incubation of A549 cells with 5 ipM
of DEAB (dotted bars) or TT (clear bars), RNA was extracted and real-time RT-PCR was performed, as described in Methods,
for the 3 genes shown in Figure 7. The results are expressed as the log 10 of relative change in gene expression when com-
pared to untreated WT cells (see Figure 7 legend). These results confirm that the increase in CXXC5 and RAB20 expression
is most likely related to the decrease in ALDH activity. The effect on LHX8 was different by the two inhibitors and therefore
non specific to ALDH activity inhibition.






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Table 5: Genes that were highly affected by the knock-down ofALDH IAI and ALDH3AI in A549 lung cancer cell line.

Symbol of Gene GB Accession Number Chromosome Location Fold Change in Expression Known Function

CCL20 (MIP-3a) NM 004591 2q33-q37 -9.5 Chemokine ligand 20 is a ligand for
CCR6. Upregulated in
hepatocellular carcinoma. Part of
the immune and inflammatory
response in the lungs. Lack of
expression of CCR6 on Lewis lung
cancer decreases metastasis.
Important for invasiveness of
pancreatic and colon cancers.
Expressed in oral SCCA.

GPR37 U87460 7q31 13.5 Highly expressed in brain neurons.
If parking absent, it accumulates and
causes cell death & degeneration
(Parkinson's disease). The
neuropeptide head activator (HA)
is a high-affinity ligand for GPR37.
Hypermethylated frequently in
AML.

DDX3Y NM 004660 Yq Il -7.45 Belongs to the DEAD-box RNA
helicase family. Alter RNA
secondary structure. Role in RNA
splicing and transport. A candidate
for tumor suppressor gene.

ID4 AW157094 6p22-p21 -16.12 Transcription factor. It inhibits
binding to DNA and transcriptional
transactivation by
heterodimerization with bHLH
protein. Expressed in small cell lung
cancer. It is deregulated in ALL
with t(6; 14), gastric adenoca, and
bladder cancer. Part of TGF-p
pathway. Possible tumor
suppressor gene

HMGA2 NM 003483 12q15 -6.6 Belongs to non-histone
chromosomal high mobility group
protein family. It affects chromatin
structure & DNA binding. Involved
in obesity, pituitary adenomas,
lipomas, serious cancer of very,
MDS, malignant transformation of
oral cancer. Overexpressed in
NSCLC, thyroid cancer, CML,
prostate and possibly others.
Detection of its mRNA in blood,
prognostic for breast cancer
patients.

GPC6 A1651255 13q32 -7.5 A cell surface proteoglycan.
Involved in cell growth and
division. Co-receptor for growth
factors

RPS4Y I NM 001008 Yp 11.3 -9.06 Ribosomal protein 4 belongs to the
S4E family. Involved in translation

EIFIAY NM 004681 Yql 1.222 -18 Eukaryotic initiation factor IA,
required for the binding of 43S (a
40S subunit) to the 5' end of
capped RNA.



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regulation, cell growth, differentiation and apoptosis [50-
56]. Furthermore, seven of these genes (not including
RPS4Y1) were implicated and studied in various cancers,
but only HMGA2 was recently reported to be over
expressed in lung cancer and inversely associated with sur-
vival [56]. HMGA2 is found on chromosome 12q13-15,
and indeed clinically relevant chromosome 12 abnormal-
ities have been reported among the frequent chromo-
somal abnormalities described in non-small cell lung
cancer [57-59]. Furthermore, our data show that the larg-
est cluster of genes affected by ALDH knock-down was on
chromosome 12. Furthermore, 3 of the highly affected
genes are located on Y chromosome (Table 5). Deletion of
Y chromosome is one of the frequent abnormalities
reported in NSCLC [60-63] and is associated with malig-
nant transformation and development of lung cancer [64-
66]. We have recently reported our immunohistochemis-
try results on archived pathological specimens of patients
with lung cancer which showed that both isozymes are
highly expressed in non-small cell primary lung cancers
[67]. Overall, these findings indicate that some of the
genes identified in this study are good candidates for
future investigation aiming at defining gene networks in
which ALDH isozymes play an important role in cancer
biology and may, in turn, enhance our knowledge of over-
all disease process.

Conclusion
We have been able to show that reduction in ALDH activ-
ity by siRNA or ALDH inhibitors may have significant
effects on cell growth and proliferation possibly through
effects on wide spectrum of genes with different biological
roles in lung cancer cells. We predict that these results will
have significant implications on defining the biological
significance and mechanisms involved in high expression
of ALDH isozymes in NSCLC, and therefore, impact the
treatment of lung cancer.

List of abbreviations
ALDH: aldehdye dehydrogenase; ALDH 1A and
ALDH3A1: aldehyde dehydrogenase class 1A1 and 3A1;
DEAB: diethylaminobezaldehyde; TT: tetraethylthiuram
disulfide or disulfiram; NSCLC: non small cell lung can-
cer; Lenti 1 or Lenti 3: cells expressing siRNA against
ALDH1A1 or ALDH3A1 using lentivirus; Lenti 1+3: cells
expressing siRNA against ALDH1A1 and ALDH3A1; WT:
refers to wild type cells; GFP: refers to cells expressing
siRNA against the green fluorescence protein.

Competing interests
The authors declare that they have no competing interests.

Authors' contributions
JSM was behind the hypothesis tested here, designed and
analyzed the studies, his laboratory (MA and BO) per-


formed the cell transduction, RNA extraction and cell cul-
ture experiments; WC prepared the lentiviral constructs
with the specific siRNA in Dr Chang's Laboratory; HVB
and his laboratory technician (MCL) performed the
microarray hypridization and analysis. All authors read
and approved the final manuscript.

Acknowledgements
Financial support for this work was provided by a grant (to JSM) from the
Flight Attendant Medical Research Institute (Miami, FL).

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