Báo cáo khoa học: Modulation of IMPDH2, survivin, topoisomerase I and vimentin increases sensitivity to methotrexate in HT29 human colon cancer cells docx

Modulation of IMPDH2, survivin, topoisomerase I
and vimentin increases sensitivity to methotrexate in HT29
human colon cancer cells
´
´
˜
Silvia Penuelas, Veronique Noe and Carlos J. Ciudad
Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, Spain

Keywords
IMPDH2; methotrexate; survivin; TOP1;
vimentin
Correspondence
´
C. J. Ciudad, Departament de Bioquımica &
`
Biologia Molecular, Facultat de Farmacia,
Universitat de Barcelona, Av. Diagonal 643,
E-08028 Barcelona, Spain
Fax: +34 93 402 4520
Tel: +34 93 403 4455
E-mail:
(Received 3 November 2004, accepted 26
November 2004)
doi:10.1111/j.1742-4658.2004.04504.x

We determined differentially expressed genes in HT29 human colon cancer
cells, both after short treatment with methotrexate (MTX) and after the
resistance to MTX had been established. Screening was performed using
Atlas Human Cancer 1.2K cDNA arrays. The analysis was carried out
using Atlas image 2.01 and genespring 6.1 software. Among the differentially expressed genes we chose for further validation were inosine monophosphate dehydrogenase type II (IMPDH2), inosine monophosphate

cyclohydrolase and survivin as up-regulated genes, and topoisomerase I
(TOP1) and vimentin as down-regulated genes. Changes in mRNA levels
were validated by quantitative RT-PCR. Additionally, functional analyses
were performed inhibiting the products of the selected genes or altering
their expression to test if these genes could serve as targets to modify MTX
cytotoxicity. Inhibition of IMPDH or TOP1 activity, antisense treatment
against survivin, or overexpression of vimentin, sensitized resistant HT29
cells to MTX. Therefore, these proteins could constitute targets to develop
modulators in MTX chemotherapy.

Methotrexate (MTX) is a 4-amino 10-methyl analog
of folic acid that inhibits dihydrofolate reductase
(DHFR), a key enzyme of the folate cycle and the one
carbone unit metabolism [1–3]. MTX was one of the
first antimetabolite drugs developed and even now continues to play an important role in the chemotherapy
of human malignancies such as acute lymphoblastic
leukemia, lymphoma, osteosarcoma, breast cancer, and
head and neck cancer [4]. Unfortunately, the efficacy
of this chemotherapeutic agent is often compromised
by the development of resistance in cancer cells. Typically, MTX resistance is due either to alterations in its
target enzyme, DHFR [5–8], to a decreased drug
import by the reduced folate carrier [9–12], to an
altered polyglutamylation [13,14], or to gene amplification of the dhfr locus [15,16].

The identification of suitable genes to target in combination with MTX could be a strategy to minimize
the development of resistance. To this end, we studied
the gene expression profile produced upon treatment
of cells with MTX, using cDNA arrays that allow the
simultaneous evaluation of expression, at the mRNA
level, of hundreds of genes. Specifically, the Atlas

Human Cancer 1.2K from ClontechÒ, a 1176 gene
array enriched in genes expressed in cancer and proliferation, was used. The human colon adenocarcinoma
cell line HT29 was chosen for this study because it can
be adapted to grow in high concentrations of MTX
[17] and concomitantly develop amplification of the
dhfr gene [18]. We used two experimental approaches:
(a) to analyze the genes differentially expressed upon
short treatment with MTX; and (b) to determine those

Abbreviations
AICARFT, 5-amino-4-imidazolecarboxamide ribonucleotide formyltransferase; aODN, antisense oligonucleotide; APRT, adenosyl
phosphoribosyl transferase; Bcl-2, B-cell leukemia ⁄ lymphoma 2; DHF, dihydrofolate; DHFR, dihidrofolate reductase; GARFT, glycinamide
ribonucleotide formyltransferase; IAP, inhibitor of apoptosis protein; IMPCH, inosine monophosphate cyclohydrolase bifunctional enzyme;
IMPDH2, inosine monophosphate dehydrogenase type II; MTX, methotrexate; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylterazolium
bromide; THF, tetrahydrofolate; TOP1, topoisomerase I.

696

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S. Penuelas et al.
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Gene expression in MTX-resistant HT29 cells

genes whose expression is changed in cells with
acquired resistance to 10)5 m MTX. The aim of this
double strategy was to find out the genes that were differentially expressed once the resistance had been
established and whether their expression had already

changed at early stages of the treatment. Then, the
effect of modulating these gene products on MTX sensitivity was studied.

Results
Analysis of differential gene expression caused
by MTX in HT29 cells using cDNA arrays
The expression profile of the 1176 genes included in
the Atlas Human Cancer 1.2K Array (ClontechÒ) was
analyzed in parental HT29 cells, HT29 cells treated
during 24 h with 10)7 m MTX, or HT29-R cells resistant to 10)5 m MTX. Cells adapted to 10)5 m MTX
presented an enterocyte-like phenotype bearing amplification of the dhfr locus (10-fold increase) and high levels of DHFR protein levels (12-fold increase). Figure 1
shows, as a scatter plot, the distribution of all the
genes in the arrays according to their expression, both
as a result of a short treatment with a low concentration of MTX and once the resistance had been established at high concentrations of MTX, with respect to
control HT29 cells. We focused our attention on genes
that changed their expression in the same direction in
both approximations (short treatment with MTX and
resistance). For that reason, a cut-off of 1.5-fold was
chosen and the differentially expressed genes resulting
from the intersection of the two sets were classified
according to their function and listed in Table 1.
Among the differentially expressed genes there were
a number of genes implicated in nucleotide metabolism
and DNA synthesis, most likely because MTX inhibits
DHFR, an enzyme implicated in these pathways. We
paid special attention to the overexpression of inosine
monophosphate dehydrogenase type II (IMPDH2), the
enzyme that catalyzes the last step in the synthesis of
IMP (the GMP and AMP precursor). IMPDH is a
target for chemical inhibitors already used in cancer

therapy, suggesting the possibility to use them in combination with MTX as modulators. Within the same
pathway we also found inosine monophosphate cyclohydrolase bifunctional enzyme (IMPCH) was overexpressed, a bifunctional enzyme that catalyzes the two
reactions before acting on IMPDH activity. Topoisomerase I (TOP1) was selected among the underexpressed genes because of the importance of topology
for cell viability and because there are inhibitors available for this enzyme.

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Fig. 1. Scatter plot, in logarithmic scales, of signal intensities representing the gene expression profiles of parental HT29 cells (A),
upon incubation with 10)7 M MTX for 24 h (HT29 treated; B) or
resistant to 10)5 M MTX (C). The values are corrected intensities
upon normalization and averaging of the replicates of genes present
in the Atlas Human Cancer 1.2K cDNA array.

We also found genes implicated in the control of the
cell cycle and the process of apoptosis; among them
survivin, which was up-regulated. This protein is also
involved in tumoral angiogenesis and constitutes a
novel anticancer target.
Oncogenes and tumor suppressors were also present
in the list of differentially expressed genes. Finally, we
selected vimentin as a representative cytoskeleton protein because new functions of these proteins, such as
their role in apoptosis, are emerging.
Changes in mRNA levels for IMPDH2, IMPCH, survivin, topoisomerase I and vimentin in treated HT29
cells or HT29-R cells were validated by quantitative RTPCR (Figs 2–6, panel A). The up-regulation of IMPDH2, IMPCH and survivin and the down-regulation of
topoisomerase I and vimentin were confirmed.
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S. Penuelas et al.
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Table 1. Differentially expressed genes in HT29 cells treated and resistant to MTX. The table shows the GenBank and SwissProt accession
numbers of genes that had their expression up-regulated or down-regulated in both HT29 cells treated with 10)7 M MTX or resistant to
10)5 M MTX. The ratio column corresponds to the expression of each gene relative to the control. Lists of genes were grouped by function.
Results are the mean of three independent experiments performed for each condition.
Overexpressed genes in treated and resistant cells
Gene name

Common

Genbank #

SwissProt #

ratio
treated

ratio
resistant

Apoptosis associated proteins
Sentrin
Survivin

UBL1
survivin

U83117

U75285

Q93068
O15124

2.4
1.9

1.8
2.2

Cell cycle
Stress-activated protein kinase 4

MAPK13

AF004709

O15124

1.7

2.9

Cytoskeleton-motility proteins
B-cell CLL ⁄ lymphoma 7B isoform 1; B-cell CLL ⁄ lymphoma 7B

BCL7B

X89985

Q13845

1.6

1.6

DNA synthesis, recombination, and repair
Proliferating cell nuclear antigen

PCNA

M15796

P12004

3.9

2.9

NP
AICARFT ⁄ IMPCH
TYMS
GARFT
nm23-H4
LDHB
IMPDH2
SLC25A5

X00737

U37436
X02308
X54199
Y07604
Y00711
L33842
J02683

P00491
P31939
P04818
P22102
O00746
P07195
P12268
P05141

2.2
1.5
4.8
1.6
1.9
3.8
1.6
2.1

2.3
2.2
1.6
2.8

2.0
6.4
1.6
2.0

Oncogenes and tumor supressors
FOS-like antigen 1
c-myc transcription factor

FOSL1
NME2

X16707
L16785

P15407
P22392

1.6
2.8

1.9
2.6

Another
Zinc finger transcription factor
Interferon-induced protein with tetratricopeptide repeats 1

EGR alpha
IFIT1

S81439
X03557

none
P09914

3.4
3.0

2.2
2.1

Underexpressed genes in treated and resistant cells
Gene name

Common

Genbank #

SwissProt #

ratio
treated

ratio
resistant

Apoptosis associated proteins
Retinoid X receptor beta; retinoic acid X receptor b

WSL-S2 protein; WSL-S1 protein; WSL-1R protein

RXRB
TNFRSF12

M84820
Y09392

P28702
Q93038

0.11
0.14

0.30
0.11

Cell cycle
pLK
E2F-1
Membrane-associated kinase
Cyclin C
Serine ⁄ threonine protein kinase
P53-like transcription factor
p19INK4d; p19 protein

PLK
E2F1
Myt1
CCNC

CDK6
P73
CDKN2D

U01038
M96577
AF014118
M74091
X66365
Y11416
U40343

P53350
Q01094
O14731
P24863
Q00534
O15350
P55273

0.04
0.14
0.23
0.08
0.01
0.01
0.19

0.50
0.16

0.39
0.08
0.12
0.07
0.12

Cytoskeleton-motility proteins
Growth-arrest-specific protein 2
Vimentin
Keratin
Keratin 1

GAS2
VIM
KRT
KRT1

U95032
X56134
J00124
M98776

O43903
P08670
P02533
P04264

0.5
0.43
0.09

0.14

0.18
0.39
0.17
0.10

DNA synthesis, recombination, and repair
DNA topoisomerase I
Telomerase reverse transcriptase
X-ray repair cross complementing protein 1

TOP1
hTRT
XRCC1

J03250
AF015950
M36089

P11387
O14746
P18887

0.04
0.20
0.25

0.07
0.03

0.04

Metabolism
Purine nucleoside phosphorylase
AICAR formyltransferase ⁄ IMP cyclohydrolase bifunctional enzyme
Tymidylate synthetase
Glycinamide ribonucleotide formyltransferase
Nucleoside-diphosphate kinase
Lactate dehydrogenase B
Inosine monophosphate dehydrogenase type II
Solute carrier family 25 (mitochondrial carrier;
adenine nucleotide translocator)

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Gene expression in MTX-resistant HT29 cells

Table 1. (Continued).
Underexpressed genes in treated and resistant cells
Gene name

Common

Genbank #

SwissProt #

ratio
treated

ratio
resistant

Oncogenes and tumor supressors
Tissue inhibitor of metalloproteinase 3
TIAM1 protein
V-FES feline sarcoma viral ⁄ V-FPS fujinami avian sarcoma viral
oncogene homolog
Pig3
Fibroblast growth factor 5
v-erb-b2 erythroblastic leukemia viral oncogene homolog 3
Small G protein

mig-5
TIAM1
c-fes

Z30183
U16296
X52192

P35625
Q13009
P07332

0.01
0.01
0.06

0.12
0.27
0.25

PIG3
FGF5
ERBB3
Gx

AF010309
M37825
M29366
M64595

O14679
P12034
P21860
P15153

0.15
0.22
0.19
0.05

0.14

0.02
0.51
0.01

Another
Human tenascin-C; hexabrachion
Cadherin 5, type 2 preproprotein; cadherin-5
Cadherin-associated protein-related

TNC; HXB
VE-cadherin
CTNNA2

X78565
X79981
M94151

P24821
P33151
P26232

0.04
0.01
0.08

0.05
0.01
0.03

Functional validation of IMPDH2

HT29-R cells were incubated with increasing concentrations of chemical inhibitors of IMPDH activity, benzamide riboside, tiazofurin or mycophenolic
acid, in the presence and in the absence of 10)5 m
MTX. The three inhibitors produced an increase in
the cytotoxicity in combination with the concentration of MTX to which the cells were resistant
(Fig. 2B–D).

survivin whereas 21-mer random and four-nucleotide
mismatch oligonucleotides did not show any effect
(Fig. 4B) in accordance with previous results [19]. The
down-regulation of survivin caused by 0.5 lm aODNSURV provoked a sensitization to MTX in HT29-R
cells because the combination of aODN-SURV with
10)5 m MTX produced an increased cytotoxicity in
these resistant cells, more than that produced by
aODN-SURV alone (Fig. 4C).
Functional validation of TOP1

Functional validation of IMPCH
IMPCH was inhibited at the level of mRNA expression using a specific antisense oligonucleotide (aODN)
against its translational start (Fig. 3B). The specificity
of the aODN effect was tested by determining the
mRNA levels of IMPCH after treatment with control
antisenses. Either a random oligonucleotide or an
unrelated aODN did not cause any effect (Fig. 3B). A
dose–response of MTX in combination with 0.5 lm
aODN-IMPCH performed in parental HT29 cells
revealed that down-regulation of IMPCH reverted the
cytotoxicity caused by MTX alone (Fig. 3C). This
result was opposite to the expected and for that reason
the mRNA levels for DHFR were determined when
HT29 cells were treated with aODN-IMPCH. Downregulation of IMPCH increased DHFR expression

(Fig. 3D), thus explaining the desensitization observed
toward MTX.
Functional validation of survivin
Targeting of survivin was carried out by lipofecting
aODN-SURV in HT29 cells. This antisense oligonucleotide caused a decrease of 80% in the expression of
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Down-regulation of TOP1 was confirmed at the
mRNA and protein levels in treated HT29 and
HT29-R cells (Fig. 5A,B). Then, HT29-R cells were
incubated with increasing concentrations of the TOP1
inhibitor, camptothecin, in the presence and in the
absence of 10)5 m MTX. The cytotoxic effect of
camptothecin alone was enhanced by the addition of
the concentration of MTX to which the cells were
resistant (Fig. 5C).
Functional validation of vimentin
By Western blot analysis, the decrease in vimentin
protein produced by MTX treatment (Fig. 6B) that
had already been validated at the level of mRNA by
RT-PCR (Fig. 6A), was confirmed. Then, we performed transient transfections with an expression vector for vimentin (pcDNA-VIM) in parental HT29 and
HT29-R cells in the presence or in the absence of
MTX. It can be observed in Fig. 6C that transfection
with 1 lg of pcDNA-VIM caused an increase in
vimentin protein levels in HT29 cells. The overexpression of vimentin sensitized the cells towards MTX
both in parental and resistant cells, respectively
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Gene expression in MTX-resistant HT29 cells

S. Penuelas et al.
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Fig. 2. Validation of IMPDH2. (A) The levels of mRNA for IMPDH2 in HT29 cells treated for 24 h with MTX 10)7 M (HT29-T) or resistant to
10)5 M MTX (HT29-R) cells were confirmed by quantitative RT-PCR. One microgram of total RNA was used as the starting material for quantitative RT-PCR. The quantification of the intensity of the radioactive bands was carried out by phosphorimaging analysis. *P < 0.05 compared with the corresponding control situation. (B–D) Dose–response curves for IMPDH inhibitors, benzamide riboside (B), tiazofurin (C), or
mycophenolic acid (D), alone and in combination with MTX in HT29-R cells. Cells were exposed to drugs simultaneously, and after 7 days
cell viability was determined by the MTT assay and plotted as a percentage of the control (cells not exposed to drugs). IMPDH inhibitor concentration is shown on the abscissa. The concentrations of MTX were 0 (s) and 10)5 M (d). Results are the mean ± SE obtained from at
least three independent experiments.

A

C

700

B

D

Fig. 3. Validation of IMPCH. (A) The levels of
mRNA for IMPCH in HT29-T and HT29-R cells
were confirmed by quantitative RT-PCR.
Other conditions as in Fig. 2. *P < 0.05
compared with the corresponding control
situation. (B) Effect on IMPCH mRNA levels
by using 0.5 and 1 lM antisense oligonucleotides against its translational start (aODNIMPCH) or using 0.5 lM of aODN-21N or
aODN-NR. IMPCH mRNA levels were determined 48 h after incubation with the aODNs.
(C) Dose–response curves for methotrexate
alone (s) or in combination with 0.5 lM of

aODN-IMPCH (d), in parental HT29 cells.
After 7 days incubation, cell viability was
determined and represented as a percentage of the control. MTX concentration is
shown on the abscissa. (D) Effect of targeting IMPCH on DHFR mRNA levels. Parental
HT29 cells were treated with the indicated
concentrations of aODN-IMPCH. After 24 h
DHFR mRNA levels were determined.
Results are the mean ± SE obtained from
two independent experiments.

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S. Penuelas et al.
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A

Gene expression in MTX-resistant HT29 cells

B

C

Fig. 4. Validation of survivin. (A) The levels of mRNA for survivin in HT29-T and HT29-R cells were confirmed by quantitative RT-PCR. Other
conditions as in Fig. 2. *P < 0.05 and **P < 0.01 compared with the corresponding control situation. (B) Effect of aODN-SURV (21-mer
oligonucleotide against survivin), aODN-21N (a 21-mer random oligonucleotide) and aODN-4MIS (21-mer oligonucleotide against survivin
including four nucleotide mismaches) on survivin mRNA levels. Parental HT29 cells were treated with the indicated concentrations of aODNSURV or 0.5 lM of aODN-21 N or aODN-4MIS. After 48 h survivin mRNA levels were determined. (C) Effect of 0.5 lM aODN-SURV treatment alone or in combination with 10)5 M MTX in HT29-R cells.

A

B

C

Fig. 5. Validation of topoisomerase I. The levels of mRNA or protein for topoisomerase I in HT29-T and HT29-R cells were confirmed by
quantitative RT-PCR (A) or Western blot (B). Other conditions as in Fig. 2. *P < 0.05 compared with the corresponding control situation. (C)
Dose–response curves for camptothecin alone (s), or in combination with 10)5 M MTX (d) in HT29-R cells. After 7 days, cell viability was
determined by MTT assay and was plotted as a percentage of the control. Each point represents the mean value for two independent
experiments ± SE.

(Fig. 6E,F). We also tested the opposite approximation, that is, down-regulating vimentin in the absence
and in the presence of MTX. Targeting of vimentin
was carried out by lipofecting aODN-VIM into parental HT29 cells causing a decrease of 60% in the
expression of vimentin (Fig. 6D) whereas random or
unrelated control oligonucleotides did not show any
effect (Fig. 6D). The dose–response of aODN-VIM in
the presence or absence of 10)8 m MTX and the
dose–response of MTX in the presence or absence of
0.5 lm aODN-VIM demonstrated that down-regulation of vimentin by aODN-VIM decreased the cytotoxicity of MTX in HT29 cells (Fig. 6G,H). The

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sensitization to MTX caused by overexpression of
vimentin and the decreased cytotoxicity of MTX
caused by down-regulation of vimentin were produced
together with an increase or a decrease in the apoptotic levels, respectively, in both parental or resistant
HT29 cells (Fig. 7A,B). We also determined the levels
of apoptosis after transfection of expression plasmids
for B-cell leukemia ⁄ lymphoma 2 (Bcl-2) and vimentin

in the absence or in the presence of 10)5 m MTX in
HT29-R cells. It can be observed that Bcl-2 overexpression decreased the basal apoptotic level and that overexpression of vimentin reversed the effect of Bcl-2
(Fig. 7C).

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Gene expression in MTX-resistant HT29 cells

A

B

C

D

E

F

G

H

Discussion
The aim of this study was to identify differentially
expressed genes as a result of MTX treatment to
design modulators for this type of therapy. We
focused our attention onto genes that changed their

expression after an initial short incubation of parental
cells with MTX and also once the cells had acquired
resistance to high concentrations of the drug. Presumably, those genes may participate in the mechanism to
develop resistance and, thus, constitute suitable tar702

S. Penuelas et al.
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Fig. 6. Validation of vimentin. The levels of
mRNA and protein for vimentin in HT29-T
and HT29-R cells were confirmed by quantitative RT-PCR (A) or Western blot (B).
*P < 0.05 compared with the corresponding
control situation. (C) Effect of 1 lg pcDNAVIM transfection on vimentin protein levels
in HT29 parental cells. (D) Effect of aODNVIM (21-mer oligonucleotide against
vimentin), aODN-21N (a 21-mer random
oligonucleotide) and aODN-NR (aODN
against a nonrelated gene) on vimentin
mRNA levels. Parental HT29 cells were treated with 0.5 lM of aODN-VIM for 24 or 48 h
or with 0.5 lM of aODN-21 N or aODN-NR
during 48 h. (E,F) Sensitization to MTX by
overexpressing vimentin. The indicated
amounts of pcDNA-VIM were transiently
transfected and incubated in the absence or
presence of MTX. Parental HT29 cells (E)
were incubated with 10)8 M MTX and HT29R cells (F) with 10)5 M MTX. (G) Dose–response curves for aODN-VIM alone (s) or in
combination with 10)8 M of MTX (d) in parental HT29 cells. After 7 days incubation,
cell viability was determined and represented as a percentage of the control. (H)
Dose–response curves for methotrexate
alone (s) or in combination with 0.5 lM of
aODN-VIM (d) in parental HT29 cells. After

7 days incubation, cell viability was determined. MTX concentration is shown on the
abscissa. Results are the mean ± SE obtained from at least two independent
experiments.

gets for combinational therapy with MTX. The
expression analyses were performed using specific cancer cDNA arrays containing 1176 gene cDNAs. Even
though this number might seem limited considering
the total number of coding genes in the human genome, it has to be stated that this array is specifically
designed to contain cDNAs related to proliferation
and cancer. Selected genes were validated by quantitative RT-PCR and their involvement in MTX-sensitivity was tested in functional analyses. Some genes
might have changed their expression associated with
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Gene expression in MTX-resistant HT29 cells

Fig. 7. Changes in apoptosis caused by overexpressing or targeting vimentin. (A,B) One microgram of pcDNA-VIM or 0.5 lM aODN-VIM
were lipofected either in the absence or in the presence of 10)8 M of MTX in parental HT29 cells (A) or 10)5 M of MTX in HT29-R cells (B).
Twenty-four hours after MTX treatment propidium iodide-negative, annexin V-FITC-positive cells were taken as the apoptotic population.
*P < 0.05 with respect to control cells. (C) Propidium iodide-negative, annexin V-FITC-positive cells were determined in HT29-R after transfection of 1 lg pSFFV-Bcl2 and 1 lg pcDNA-VIM, each one alone or together. The same conditions were determined in the presence of
10)5 M of MTX. Results are the mean ± SE obtained from eight values. *P < 0.05 with respect to control cells. #P < 0.05 with respect to
cells with Bcl-2 overexpressed.

the chemotherapeutic treatment, whereas a different
set of genes could modify their expression in order to
compensate for some of the primary changes produced by the chemotherapeutical agent to allow the
cells to survive. However, it is possible to discern the

type of response of each gene by modifying their
expression and testing how this modification affects
MTX cytotoxicity. As tools we used chemical inhibitors of determined gene products, aODNs to decrease
the expression of specific genes and expression vectors
of selected genes.
The first gene subjected to functional validation
was IMPDH2, which actively catalyzes the step from
IMP to XMP and is the rate-limiting step in the
de novo synthesis of guanylates, including GTP and
dGTP [20], and is thus required for DNA synthesis.
In this regard the increased expression of IMPDH2
could be interpreted as a way to compensate for the
inhibition of DHFR by MTX as both enzymes are
in the same pathway. Two isoforms of IMPDH
have been demonstrated. IMPDH type I enzyme is
constitutively expressed in normal cells, whereas the
IMPDH type II is significantly up-regulated in malignant cells [21,22]. Benzamide riboside, tiazofurine and
mycophenolic acid are potent inhibitors of this enzymic activity and phase II ⁄ III clinical trials that have
been conducted with them as anticancer drugs in
patients with leukemias have shown very promising
results. These inhibitors presented a degree of cytotoxicity by themselves, according to the role of
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IMPDH activity in DNA synthesis, which was
increased when combining them with MTX. Therefore, by decreasing the activity of the overexpressed
gene IMPDH2 in HT29-R cells, we were able to sensitize the resistant cells to MTX, thus showing the
modulator effect of IMPDH inhibitors.
As IMPCH expression was also increased, and given
that this RNA encodes the bifunctional enzyme ATIC
[5-amino-4-imidazolecarboxamide ribonucleotide formyltransferase (AICARFT) ⁄ inosine monophosphate

cyclohydrolase (IMPCH)] we used an aODN to reduce
the expression of both activities. This bifunctional
enzyme catalyzes the penultimate and final steps in
the de novo purine nucleotide biosynthetic pathway
using N10-formyltetrahydrofolate as a reduced folate
cofactor. However, in spite of that aODN-IMPCH
decreased IMPCH mRNA levels (Fig. 3B); when combined with MTX, the cytotoxicity caused by MTX
partially reverted. This effect could be explained by the
observation that when IMPCH mRNA levels were
decreased by aODN-IMPCH, DHFR RNA expression
was increased. It is known that when DHFR activity
is abolished, tetrahydrofolate (THF)-cofactors rapidly
interconvert to 5,10,methylene-tetrahydrofolate which,
in turn, is rapidly oxidized to dihydrofolate (DHF)
[23]. This is followed by a rapid decrease in THFcofactors that, while often incomplete, is associated
with the cessation of THF-cofactor-dependent reactions within a few minutes [24,25]. Thus, overexpression of IMPCH upon MTX treatment could represent
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Gene expression in MTX-resistant HT29 cells

a reaction of the cell to compensate the depletion in
reduced folate cofactors. Similarly, DHFR overexpression caused by IMPCH targeting could be explained
by an attempt of the cell to compensate the low IMPCH activity with a surplus of reduced folates. The high
regulation shown by the enzymes that use THF-cofactors indicates that an improved therapy would involve
a multitargeted antifolate. Indeed, a novel inhibitor,
pemetrexed, now in phase II trials, inhibits at least
four enzymes involved in folate metabolism and purine
and pyrimidine synthesis: thymidylate synthase,
DHFR, glycinamide ribonucleotide formyltransferase

(GARFT) and AICARFT [26]. It is worth mentioning
that the RNAs for GARFT and thymidylate synthase
are also increased in the arrays from cells treated with
MTX or HT29-R. Overexpression of GARFT in treated and resistant cells was also validated by RT-PCR
(data not shown). Thus, the increases in GARFT
and AICARFT expression could be as a result of the
inhibition of these two enzymatic activities by MTX
polyglutamates [27].
Survivin is a member of the inhibitor of apoptosis
protein (IAP) family [28] which directly inhibits
caspase-3, -7 [29–31], and caspase-9 activities [32,33].
Moreover, survivin indirectly inhibits caspase activity
by promoting procaspase-3–p21 complex formation
as a result of an interaction with cyclin-dependent
kinase 4 [34] and by sequestering direct-IAP binding
protein (Smac ⁄ DIABLO), thus preventing Smac ⁄
DIABLO binding to other IAPs [35]. Survivin is largely undetectable in normally differentiated adult tissues but, in contrast, it is dramatically overexpressed
in most human tumors, thus conferring growth and
survival advantages for tumor onset and progression
[36]. The observation that survivin expression is
increased in HT29 cells treated with MTX is in keeping with the antiapoptotic role of this protein, and
would contribute to counteract the apoptosis caused
by MTX. Interestingly, survivin is also overexpressed
in endothelial cells of newly formed blood vessels
found in tumors [37,38]. Therefore, down-regulation or
inhibition of survivin could be considered as an
attractive strategy in cancer therapy. We used the
antitranslational aODN-SURV [19] to decrease the
overexpression of survivin caused upon MTX treatment, observing that the combination of this aODN
plus MTX sensitized the cells towards this drug in

HT29-R cells.
Human TOP1 is a nuclear protein that relaxes
superhelical tension associated with DNA replication,
transcription and recombination by reversibly nicking
one strand of duplex DNA and forming a covalent
3¢-phosphotyrosine linkage. Because many neoplastic
704

S. Penuelas et al.
˜

cells are characterized by high levels of TOP1 activity
[39,40], this enzyme has become one of the cellular targets for anticancer therapy [41,42]. TOP1 is inhibited
by the camptothecin family of anticancer compounds,
which act by stabilizing the covalent protein–DNA
complex and enhancing apoptosis through blocking
the advancement of replication forks. We took advantage of the down-regulation of TOP1 in MTX-resistance as a way to increase the sensitivity to MTX.
Indeed, by combining MTX and camptothecin, a
higher degree of cytotoxicity than with camptothecin
alone was achieved in HT29-R. This illustrates a strategy for cancer therapy based on using as a modulator
an inhibitor of a gene product that it is already underexpressed as a result of the resistance stage toward the
primary chemotherapy agent. A possible explanation
for the increased cytotoxicity of the combination of
MTX plus camptothecin could be based on the observations that treatment with camptothecin, after an initial apoptotic signal, activates nuclear factor j-B
resulting in the expression of genes that have an overall antiapoptotic effect, leading to camptothecin resistance [43]; and that MTX counteracts the binding of
nuclear factor j-B activated by apoptotic stimuli, thus
increasing apoptosis [44].
Finally, we also observed that treatment with MTX
leads to an underexpression of the cytoskeleton protein vimentin, an abundant type III intermediate filament protein, which is cleaved by caspases-3, -7 and
-6 during apoptosis [45]. The proteolysis of vimentin

promotes apoptosis by dismantling intermediate filaments and by generating a proapoptotic aminoterminal cleavage product that interferes with
intermediate filament assembly [45]. On the other
hand, it has been reported that Bcl-2, which has an
antiapoptotic effect, inhibits caspase-3 and the proteolysis of vimentin [46]. Previous reports positivity linked
vimentin to more agressive tumor characteristics [47]
contributing to the invasive phenotype, but cannot
confer it alone [48]. However, a decreased expression
of vimentin has been reported in ERBB2 oncogene
(HER2) overexpressing breast cancer tumors that are
known to be refractory to various types of chemotherapy [49]. Also in breast cancer, either low [50] or high
[51] expression of vimentin has been described in tumors that underexpressed the estrogen receptor (poor
prognostic indicator). Vimentin expression has also
been considered as a marker of resistance in doxorubicin-resistant LoVo cells [52] and in the multidrug
resistant MCF7 subline [53], but parental cells transfected with human vimentin cDNA, did not become
resistant. It is interesting to note that, in addition to
the decrease of vimentin expression, MTX-resistant
FEBS Journal 272 (2005) 696–710 ª 2005 FEBS


S. Penuelas et al.
˜

HT29 cells show enhanced synthesis and secretion of
mucin 1 transmembrane (MUC1) [54], which has been
linked to tumor aggressiveness. Conversely, low levels
of MUC1 expression were associated with increased
expression of vimentin [55], suggesting an inverse relationship between vimentin and MUC1. Mimicking the
down-regulation of vimentin by aODN a decrease in
MTX sensitivity was observed, while overexpression of
vimentin turned parental and resistant HT29 cells sensitive to MTX. These changes in MTX sensitivity were

also reflected in the apoptotic levels. On one hand,
overexpression of vimentin counteracts the antiapoptotic effect of Bcl-2, leading to an increase in the
apoptotic levels caused by MTX in resistant cells, and
on the other hand, down-regulation of vimentin leads
to a decrease in apoptosis, which is a well known
mechanism of resistance.
In summary, we performed a study using cDNA
arrays to screen for genes that are differentially
expressed upon short treatment with MTX and in
cells resistant to this drug. We sought to modulate
methotrexate therapy using either (a) chemical inhibitors against gene products that are overexpressed
(such as IMPDH2); (b) antisense oligonucleotides that
reverted the up-regulation of genes that increased their
expression (like IMPCH and survivin); (c) chemical
inhibitors that are already used as anticancer drugs
against gene products that are underexpressed
(TOP1); or (d) expression vectors for gene products
that are underexpressed (vimentin). The functional
targets defined in this study could contribute to the
development of new therapeutic protocols in combinations with MTX.

Experimental procedures
Cell culture
Human colon adenocarcinoma cell line HT29 were routinely grown in Ham’s F12 selective DHFR medium
(–GHT medium) lacking glycine, hypoxanthine and thymidine, the final products of DHFR activity. This medium
was supplemented with 7% dialyzed fetal bovine serum
(Gibco, Paisley, UK) at 37 °C in a 5% CO2 humidified
atmosphere. Resistant cells 10)5 m MTX (HT29-R) were
obtained upon incubation with stepwise concentrations of
MTX (Lederle, Madrid, Spain).

cDNA arrays
Gene expression was analyzed by hybridization to cDNA
arrays (AtlasTM Human Cancer Array 1.2K from Clontech
Laboratories Inc., Palo Alto, CA, USA). Total RNA for

FEBS Journal 272 (2005) 696–710 ª 2005 FEBS

Gene expression in MTX-resistant HT29 cells

cDNA arrays was prepared using the AtlasTM pure total
RNA Labeling kit (Clontech) from 4 · 106 cells, either
parental HT29, HT29 treated with 10)7 m MTX for 24 h,
or HT29-R. RNA was treated with RNAse-free DNAse
(10 U per 50 lg of RNA) at 37° for 30 min. The integrity
of the RNA was assessed after agarose gel electrophoresis
in the presence of formaldehyde. Radiolabeled cDNA
probes were prepared from 5 lg of total RNA. Briefly,
RNA was hybridized for 2 min at 70 °C followed by 2 min
at 50 °C with 1 lL of the primer mix, containing only the
1176 primers for the genes present in the array, thus conferring a 10-fold increase in sensitivity and a concomitant
reduction in nonspecific background. The reverse transcription reaction was carried out using 100 U MMLV RT,
40 U rRNasinÒ (Promega, Madison, WI, USA) and 30 lCi
of [32P]dATP[lP] (Amersham Bioscience, Freiburg, Germany) for 25 min at 50 °C. After filtering the unincorporated nucleotide through Sephadex G-50 columns, membrane
hybridizations were carried out. Nylon filters were prehybridized in 5 mL ExpressHybTM (Clontech) with
100 lgỈmL)1 DNA salmon sperm for 30 min in roller bottles with continuous agitation in an oven at 68 °C. Then,
the 32P-labelled probe was added and hybridization continued overnight in the same conditions. Afterwards, membranes were washed four times, lowering the astringency
progressively to 0.5· NaCl ⁄ Cit, 0.5% SDS at 65 °C and
placed in contact with europium screens (Kodak, Rochester, NY, USA) for 7 days and scanned with a Storm 840
phosphorimager (Molecular Dynamics, Sunnyvale, CA,

USA).

Array data analysis
Image analysis and quantification were carried out with
Atlas image 2.0.1. After grid assignment the adjusted
intensity for each gene was calculated by subtracting the
background. This value was used as the input for the
genespring 6.1 program (Silicon Genetics, Redwood City,
CA, USA), which allows multifilter comparisons using
data from different experiments to perform the normalization, generation of restriction lists and the functional
classification of the differentially expressed genes. Normalization was applied in two steps: (a) ‘per chip normalization’ in which each measurement was divided by the 50th
percentile of all measurements in its array; and (b) ‘per
gene normalization’ in which all the samples were normalized against the median of the control samples. The
expression of each gene is reported as the ratio of the
value obtained after each condition relative to control
conditions after normalization of the data. Then data were
filtered using the control strength, a control value calculated using the Cross-Gene Error Model based on replicates
[56]. Measurements with higher control strength are relatively more precise than measurements with lower control

705


Gene expression in MTX-resistant HT29 cells

S. Penuelas et al.
˜

strength. Genes that didn’t reach this value were discarded. Lists of differentially expressed genes considering a
1.5-fold expression were generated using data from at least
three independent experiments for each condition. The

genes in these lists were further classified according to
their function.

Determination of the dhfr copy number
HT29 or HT29-R cells (10 000) were centrifuged at
10 000 g for 5 min and the cell pellet was washed once with
ice-cold NaCl ⁄ Pi. The cells were centrifuged again at
10 000 g for 5 min, and after discarding the supernatant,
resuspended in 20 lL lysis buffer (20 mm NaCl, 1 mm
EDTA, 0.1% SDS, and 50 mm Tris ⁄ HCl, pH 8) plus 2 lL
of 10 mgỈmL)1 proteinase K. They were then incubated at
55 °C for 15 min, vortexed vigorously and incubated for
15 min at the same temperature. Finally, the mixture was
incubated at 100 °C for 5 min and, after cooling, 1 lL was
used for PCR amplification [57]. The primers used for dhfr
amplification were: 5¢-CCTGTTAACGCAGTGTTTCTC-3¢
within intron 1 and 5¢-TCCCACGGGAGACTTCGCACT3¢ within intron 2. For adenosyl phosphoribosyl transferase
(aprt) the oligonucleotides were: 5¢-TCACGAGCCAG
CAAGGCGTT-3¢ within intron 1 and 5¢-ACGCAGTACT
CATCCAGGGT-3¢ within intron 2.
The dhfr copy number was calculated according to the
ratio of the dhfr and aprt signals for each MTX-resistant
clone.

RT-PCR
Levels of specific mRNAs were determined by RT-PCR
under quantitative conditions. Total RNA was extracted
from cells (4 · 106) using UltraspecTM RNA reagent (Biotecx, Houston, TX, USA) following the recommendations of
the manufacturer. Complementary DNA was synthesized in

a total volume of 20 lL from RNA samples by mixing 1 lg
of total RNA, 125 ng of random hexamers (Roche,
Mannheim, Germany), in the presence of 75 mm KCl, 3 mm
MgCl2, 10 mm dithiothreitol, 20 U RNasin (Promega),
0.5 mm dNTPs (AppliChem, Darmstedt, Germany), 200 U
M-MLV reverse transcriptase (BRL-Gibco, Paisley, UK)
and 50 mm Tris ⁄ HCl buffer, pH 8.3. The reaction mixture
was incubated at 37 °C for 60 min and the cDNA product
was used for subsequent PCR amplification with specific
primers. A standard 50 lL mixture contained 5 lL of the
cDNA mixture, 1.2 mm MgCl2, 0.2 mm dNTPs, 2.5 lCi of
´
[32P]dATP[aP] (3000 CiỈmmol)1, Amersham Iberica, Madrid,
Spain), 1.5 U Taq polymerase (Ecogen, Barcelona, Spain)
and 500 ng of each primer, and 20 mm Tris ⁄ HCl, pH 8.5. To
avoid unspecific annealing, cDNA and Taq DNA polymerase
were separated from primers and dNTPs by using a layer of
paraffin (Fluka, Basel, Switzerland) (reaction components
contact only when paraffin fuses, at 60 °C). PCR was performed in an MJ Research (South San Francisco, CA, USA)
thermocycler equipped with peltier system and temperature
probe. Preliminary experiments were carried out using different numbers of cycles to determine the linear conditions of
PCR amplification for all the genes studied. The sequences of
the forward and reverse primers used for PCR amplification,
the length of the PCR product, the number of cycles used in
the PCR, and the linear range for the amplification are given
in Table 2.
[32P]dATP[aP] was used in the PCR to produce a radioactive product that could be detected with great sensitivity
during the exponential phase of the reaction. After an initial denaturation for 1 min and 30 s at 94 °C, PCR was
performed for the indicated number of cycles. Each cycle
consisted of denaturation at 92 °C for 1 min, primer

annealing at 59 °C, and primer extension at 72 °C for
1 min. A final 5-min extension step at 72 °C was performed. Five microliters of each PCR sample was electrophoresed on a 1-mm-thick 5% polyacrylamide gel. The gels

Table 2. Sequences of the forward and reverse primers used for PCR amplification, the length of the PCR product, the number of cycles
used in the PCR, and the linear range for the amplification.
Gene

Primer sequence (5¢ to 3¢)

Product length (bp)

Number of cycles

Linear cycle range

IMPDH2

for: 5¢-AACCTCATTGATGCAGGTGTG-3¢
rev: 5¢-AATGTCCTGGCATGAGTGTTG-3¢
for: 5¢-ACTCCTTGGAGACTAGACGC-3¢
rev: 5¢-TTTGGCCTCATCTTCACTGAG-3¢
for: 5¢-GGACCGCCTAAGAGGGCGTGC-3¢
rev: 5¢-AATGTAGAGATGCGGTGGTCC-3¢
for: 5¢-CAAGCAGCCCGAGGATGATC-3¢
rev: 5¢-GCACTTTTCAGGTCTCTCCG-3¢
for: 5¢-GGCTCAGATTCAGGAACAGC-3¢
rev: 5Â-CTGAATCTCATCCTGCAGGC-3Â
for: 5Â-GCAGCTGGTTGAGCAGCGGAT-3Â
rev: 5Â-AGAGTGGGGCCTGGCAGCTTC-3Â

480

26

2428

359

23

2126

165

27

2329

333

24

2125

373

26

2228

253

20

1925

IMPCH
SURV
TOP1
VIM
APRT

706

FEBS Journal 272 (2005) 696710 ê 2005 FEBS


S. Penuelas et al.
˜

were dried and placed on contact with europium screens
that were scanned using phosphorimaging. The expression
of specific mRNAs is reported upon normalization using
the APRT mRNA as the internal control.

Total extracts and Western blots
Whole extracts were obtained from HT29 or HT29-R cells
following the method of Kraus et al. [58]. Cells were collected in ice-cold F12 medium and centrifuged at 1000 g for
5 min. The cell pellet was gently resuspended in 5 mL of
hypotonic buffer (15 mm NaCl, 60 mm KCl, 0.5 mm

EDTA, 1 mm phenylmethanesulfonyl fluoride, 1 mm
2-mercaptoethanol, 15 mm Tris ⁄ HCl, pH 8). After centrifugation in the same conditions as above, the pellet was
resuspended in 100 lL of a buffer containing deoxycholate
[100 mm NaCl, 10 mm NaH2PO4 (pH 7.4), 0.5% (w ⁄ v)
sodium deoxycholate, 0.1% (w ⁄ v) SDS, 1% (v ⁄ v) Triton X100] and centrifuged at 13 000 g for 10 min. The resulting
supernatant corresponded to the whole extract. The entire
procedure was carried out at 4 °C. Five microliters of the
extract was used to determine the protein concentration by
the Bradford assay (Bio-Rad, Madrid, Spain). The extracts
were frozen in liquid N2 and stored at )80 °C. One hundred micrograms of total extracts from cells was resolved
on SDS polyacrylamide gels (12% for DHFR; 10% for
vimentin and 7% for TOP1) [59] and transferred to
poly(vinylidene diflouride) membranes (Immobilon P, Millipore, Billerica, MA, USA) using a semidry electroblotter.
The membranes were probed with antibodies against
DHFR [60] to determine the levels of DHFR protein due
to gene amplification in HT29-R cells, and with antibodies
against vimentin or TOP1 (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA) when validating these targets. Signals were detected by secondary horseradish
peroxidase-conjugated antibody and enhanced chemiluminiscence, as recommended by the manufacturer (Amersham). To normalize the results, blots were reprobed with
antibodies against actin (for DHFR) or Oct-1 (for vimentin
and TOP1).

Functional validations using chemical inhibitors
of the gene target
Twenty-five thousand HT29-R cells were seeded in 35-mm
diameter wells in –GHT medium. Cells were cultured for
18 h without treatment and then incubated with different
IMPDH inhibitors, benzamide riboside, tiazofurine, mycophenolic acid, supplied by H Jayaram (IUPUI, Indianapolis,
IN, USA), or the TOP1 inhibitor camptothecin (Sigma,
Madrid, Spain), in the presence or absence of MTX. IMPDH
activity inhibitors were added simultaneously to MTX.

Camptothecin, was added 24 h after MTX. After 7 days the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide
(MTT) test [61] was performed to determine cell viability.

FEBS Journal 272 (2005) 696–710 ª 2005 FEBS

Gene expression in MTX-resistant HT29 cells

Functional validation by transfecting expression
plasmids of the gene target
HT29 or HT29-R cells were seeded into 6-well plates at a
density of 105 cellsỈwell)1 in 2 mL of Ham’s F12 selective
medium. Eighteen hours later, transfections with an expression plasmid for vimentin (pcDNA3-VIM) (RM Evans,
University of Colorado, Denver, CO, USA) in the presence
or absence of MTX were performed. For each well, 3 lL of
FuGENE6 (Roche Molecular Biochemicals) in 100 lL of
serum free –GHT medium was incubated at room temperature for 5 min. Then, this mixture was added to the DNA
and, after 15 min at room temperature, added to the cells.
When combining pcDNA-VIM transfection and MTX
treatment, MTX was added at the same time of the transfection. The effect of FuGENE in MTX effectiveness was
assessed with the corresponding controls. Seven days later,
the viability was measured by the MTT assay. The level of
apoptosis was determined 24 h after MTX treatment as
described below. Overexpression of Bcl-2 was achieved by
transient transfection of 1 lg pSFFV-Bcl2 (P Perego, Instituto Nazionale Tumori, Milan, Italy). In the experiments of
cotransfection with Bcl-2 and vimentin, the expression plasmids were transfected simultaneously.

Functional validation by transfecting aODNs
against the target RNAs
HT29 or HT29-R cells (2.5 · 104) were plated in 35-mm

wells in 1 mL of –GHT medium. Two hours later, antisense
oligonucleotides were delivered to cells in the form of complexes with the transfection reagent DOTAPÒ (Roche), in
the presence or absence of MTX. Previously, the oligonucleotides and the liposome were mixed in Eppendorf tubes
at room temperature for 15 min at a 1 : 10 molar ratio
(DNA ⁄ DOTAPÒ) as described previously [62]. Then the
mixture and MTX were added simultaneously to the cell
culture and cells were incubated for 24 h before apoptotic
levels were determined, or for seven days before the MTT
assays were performed. Oligonucleotides aODN-IMPCH,
aODN-SURV and aODN-VIM, were directed against
nucleotides 1–21 relative to the translational start site of
the IMPCH, survivin and vimentin RNAs, respectively.
The sequences of the antisense oligonucleotides were:
aODN-IMPCH, 5¢-T*A*A*GGC*TGAGAGAGAAGA*
C*A*T-3¢; aODN-SURV, 5¢-G*G*G*CAACG*T*C*GG
GGCAC*C*C*A*T-3¢; aODN-VIM, 5¢-G*G*A*CAC*GG
AC*C*T*GGT*GGA*C*A*T-3¢.
Specific bonds (marked with an asterisk) contained
phosphorothioate modifications to render the aODNs
(Sigma) more resistant to the nucleases. To check for
specificity, three types of control oligonucleotides were
used: (a) an unrelated aODN (aODN-NR) directed
against the translational start of the livin mRNA, (b) a
random 21-mer (aODN-21 N) and (c) a four-nucleo-

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Gene expression in MTX-resistant HT29 cells

tide mismatch oligonucleotide (aODN-4MIS): aODNNR, 5¢-G*G*C*AC*TGT*CT*T*TAGGT*C*C*C*A*T-3¢;
aODN-4MIS, 5¢-G*G*G*CAACG*T*C*GC*C*C*GAC*
C*C*A*T-3¢.

S. Penuelas et al.
˜

8

Apoptosis assays
Apoptosis was assessed by annexin V-FITC (Bender MedSystems, Vienna, Austria) binding. Twenty-four hours after
MTX treatment, floating and adherent cells were collected,
washed in NaCl ⁄ Pi and resuspended in annexin-binding buffer. Cells were collected, washed in NaCl ⁄ Pi and resuspended
in binding buffer containing a final concentration of
0.5 lgỈmL)1 annexin V-FITC and incubated at room temperature for 1 h in the dark. Afterwards, cells were centrifuged at 800 g 5 min and resuspended in annexin-binding
buffer containing propidium iodide (Sigma) at 1 lgỈmL)1.
After 15 min of incubation, cells were analyzed with an
EPICS-XL flow cytometer (Coulter Corp., Miami, FL,
USA). Propidium iodide-negative, annexin V-FITC-positive
cells were taken as the apoptotic population.

9

10

11

Acknowledgements
This work was supported by grants 2002SAF-0363
´

from Comision Interministerial de Ciencia y Tecnologia and 2001SGR-0141 from Generalitat de Catalunya.
S.P. was a recipient of a fellowship from the Ministerio
de Ciencia y Tecnologı´ a (MCYT).

12

13

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