Inhibition of IMPDH or TOP1 activity, antisense treatment against survivin, or overexpression of vimentin, sensitized resistant HT29 cells to MTX.. Results Analysis of differential gene
Trang 1and vimentin increases sensitivity to methotrexate in HT29 human colon cancer cells
Silvia Pen˜uelas, Ve´ronique Noe´ and Carlos J Ciudad
Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, Spain
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
con-tinues 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
Typic-ally, 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
amplifica-tion of the dhfr locus [15,16]
The identification of suitable genes to target in com-bination 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 Clontech, a 1176 gene array enriched in genes expressed in cancer and prolif-eration, 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
Keywords
IMPDH2; methotrexate; survivin; TOP1;
vimentin
Correspondence
C J Ciudad, Departament de Bioquı´mica &
Biologia Molecular, Facultat de Farma`cia,
Universitat de Barcelona, Av Diagonal 643,
E-08028 Barcelona, Spain
Fax: +34 93 402 4520
Tel: +34 93 403 4455
E-mail: cciudad@ub.edu
(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 differen-tially expressed genes we chose for further validation were inosine mono-phosphate dehydrogenase type II (IMPDH2), inosine monomono-phosphate 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
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.
Trang 2genes whose expression is changed in cells with
acquired resistance to 10)5m MTX The aim of this
double strategy was to find out the genes that were
dif-ferentially 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
sen-sitivity 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 (Clontech) was
analyzed in parental HT29 cells, HT29 cells treated
during 24 h with 10)7mMTX, or HT29-R cells
resist-ant to 10)5m MTX Cells adapted to 10)5m MTX
presented an enterocyte-like phenotype bearing
amplifi-cation of the dhfr locus (10-fold increase) and high
lev-els of DHFR protein levlev-els (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
concentra-tion of MTX and once the resistance had been
estab-lished 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
com-bination with MTX as modulators Within the same
pathway we also found inosine monophosphate
cyclo-hydrolase bifunctional enzyme (IMPCH) was
overex-pressed, a bifunctional enzyme that catalyzes the two
reactions before acting on IMPDH activity
Topoiso-merase I (TOP1) was selected among the
underex-pressed genes because of the importance of topology
for cell viability and because there are inhibitors
avail-able for this enzyme
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 pro-tein because new functions of these propro-teins, such as their role in apoptosis, are emerging
Changes in mRNA levels for IMPDH2, IMPCH, sur-vivin, topoisomerase I and vimentin in treated HT29 cells or HT29-R cells were validated by quantitative RT-PCR (Figs 2–6, panel A) The up-regulation of IMP-DH2, IMPCH and survivin and the down-regulation of topoisomerase I and vimentin were confirmed
Fig 1 Scatter plot, in logarithmic scales, of signal intensities repre-senting the gene expression profiles of parental HT29 cells (A), upon incubation with 10)7M MTX for 24 h (HT29 treated; B) or resistant to 10)5M 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.
Trang 3Table 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)7M MTX or resistant to
10)5M 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
ratio treated
ratio resistant Apoptosis associated proteins
Cell cycle
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
Metabolism
AICAR formyltransferase ⁄ IMP cyclohydrolase bifunctional enzyme AICARFT ⁄ IMPCH U37436 P31939 1.5 2.2
Solute carrier family 25 (mitochondrial carrier;
adenine nucleotide translocator)
Oncogenes and tumor supressors
Another
Interferon-induced protein with tetratricopeptide repeats 1 IFIT1 X03557 P09914 3.0 2.1 Underexpressed genes in treated and resistant cells
ratio treated
ratio resistant Apoptosis associated proteins
Cell cycle
Cytoskeleton-motility proteins
DNA synthesis, recombination, and repair
Trang 4Functional validation of IMPDH2
HT29-R cells were incubated with increasing
con-centrations of chemical inhibitors of IMPDH
acti-vity, benzamide riboside, tiazofurin or mycophenolic
acid, in the presence and in the absence of 10)5m
MTX The three inhibitors produced an increase in
the cytotoxicity in combination with the
concentra-tion of MTX to which the cells were resistant
(Fig 2B–D)
Functional validation of IMPCH
IMPCH was inhibited at the level of mRNA
expres-sion 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
Down-regulation 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
oligonucle-otide caused a decrease of 80% in the expression of
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 aODN-SURV provoked a sensitization to MTX in HT29-R cells because the combination of aODN-SURV with
10)5m MTX produced an increased cytotoxicity in these resistant cells, more than that produced by aODN-SURV alone (Fig 4C)
Functional validation of TOP1 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)5m 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 per-formed transient transfections with an expression vec-tor 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 overexpres-sion of vimentin sensitized the cells towards MTX both in parental and resistant cells, respectively
Table 1 (Continued).
Underexpressed genes in treated and resistant cells
ratio treated
ratio resistant Oncogenes and tumor supressors
V-FES feline sarcoma viral ⁄ V-FPS fujinami avian sarcoma viral
oncogene homolog
v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 ERBB3 M29366 P21860 0.19 0.51
Another
Trang 5Fig 2 Validation of IMPDH2 (A) The levels of mRNA for IMPDH2 in HT29 cells treated for 24 h with MTX 10)7M (HT29-T) or resistant to
10)5M MTX (HT29-R) cells were confirmed by quantitative RT-PCR One microgram of total RNA was used as the starting material for quan-titative RT-PCR The quantification of the intensity of the radioactive bands was carried out by phosphorimaging analysis *P < 0.05 com-pared 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 con-centration is shown on the abscissa The concon-centrations of MTX were 0 (s) and 10)5M (d) Results are the mean ± SE obtained from at least three independent experiments.
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 l M antisense oligonucle-otides against its translational start (aODN-IMPCH) or using 0.5 l M of aODN-21N or aODN-NR IMPCH mRNA levels were deter-mined 48 h after incubation with the aODNs (C) Dose–response curves for methotrexate alone (s) or in combination with 0.5 l M of aODN-IMPCH (d), in parental HT29 cells After 7 days incubation, cell viability was determined and represented as a percent-age of the control MTX concentration is shown on the abscissa (D) Effect of target-ing 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.
Trang 6(Fig 6E,F) We also tested the opposite
approxima-tion, 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
paren-tal 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)8m MTX and the
dose–response of MTX in the presence or absence of
0.5 lm aODN-VIM demonstrated that
down-regula-tion of vimentin by aODN-VIM decreased the
cyto-toxicity of MTX in HT29 cells (Fig 6G,H) The
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 apop-totic 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)5m MTX in HT29-R cells It can be observed that Bcl-2 overexpres-sion decreased the basal apoptotic level and that over-expression of vimentin reversed the effect of Bcl-2 (Fig 7C)
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)5M 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 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 aODN-SURV or 0.5 l M of aODN-21 N or aODN-4MIS After 48 h survivin mRNA levels were determined (C) Effect of 0.5 l M aODN-SURV treat-ment alone or in combination with 10)5M MTX in HT29-R cells.
Trang 7The 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
Presum-ably, those genes may participate in the mechanism to
develop resistance and, thus, constitute suitable
tar-gets for combinational therapy with MTX The expression analyses were performed using specific can-cer cDNA arrays containing 1176 gene cDNAs Even though this number might seem limited considering the total number of coding genes in the human gen-ome, it has to be stated that this array is specifically designed to contain cDNAs related to proliferation and cancer Selected genes were validated by quantita-tive RT-PCR and their involvement in MTX-sensitiv-ity was tested in functional analyses Some genes might have changed their expression associated with
Fig 6 Validation of vimentin The levels of mRNA and protein for vimentin in HT29-T and HT29-R cells were confirmed by quanti-tative RT-PCR (A) or Western blot (B).
*P < 0.05 compared with the corresponding control situation (C) Effect of 1 lg pcDNA-VIM transfection on vimentin protein levels
in HT29 parental cells (D) Effect of aODN-VIM (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 trea-ted with 0.5 l M of aODN-VIM for 24 or 48 h
or with 0.5 l M 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)8M MTX and
HT29-R cells (F) with 10)5M MTX (G) Dose–resp-onse curves for aODN-VIM alone (s) or in combination with 10)8M of MTX (d) in par-ental HT29 cells After 7 days incubation, cell viability was determined and represen-ted as a percentage of the control (H) Dose–response curves for methotrexate alone (s) or in combination with 0.5 l M of aODN-VIM (d) in parental HT29 cells After
7 days incubation, cell viability was deter-mined MTX concentration is shown on the abscissa Results are the mean ± SE
obtain-ed from at least two independent experiments.
Trang 8the chemotherapeutic treatment, whereas a different
set of genes could modify their expression in order to
compensate for some of the primary changes
pro-duced 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
inhibi-tors 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
malig-nant cells [21,22] Benzamide riboside, tiazofurine and
mycophenolic acid are potent inhibitors of this
enzy-mic 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
cyto-toxicity by themselves, according to the role of
IMPDH activity in DNA synthesis, which was increased when combining them with MTX There-fore, by decreasing the activity of the overexpressed gene IMPDH2 in HT29-R cells, we were able to sen-sitize 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 for-myltransferase (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 com-bined 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 THF-cofactors that, while often incomplete, is associated with the cessation of THF-cofactor-dependent reac-tions within a few minutes [24,25] Thus, overexpres-sion of IMPCH upon MTX treatment could represent
Fig 7 Changes in apoptosis caused by overexpressing or targeting vimentin (A,B) One microgram of pcDNA-VIM or 0.5 l M aODN-VIM were lipofected either in the absence or in the presence of 10)8M of MTX in parental HT29 cells (A) or 10)5M 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 trans-fection 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)5M 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.
Trang 9a reaction of the cell to compensate the depletion in
reduced folate cofactors Similarly, DHFR
overexpres-sion caused by IMPCH targeting could be explained
by an attempt of the cell to compensate the low
IMP-CH activity with a surplus of reduced folates The high
regulation shown by the enzymes that use
THF-cofac-tors 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
trea-ted and resistant cells was also validatrea-ted 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
lar-gely undetectable in normally differentiated adult
tis-sues 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
keep-ing 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
treat-ment, 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
cells are characterized by high levels of TOP1 activity [39,40], this enzyme has become one of the cellular tar-gets 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 advant-age of the down-regulation of TOP1 in MTX-resist-ance 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 stra-tegy for cancer therapy based on using as a modulator
an inhibitor of a gene product that it is already under-expressed 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 obser-vations that treatment with camptothecin, after an ini-tial apoptotic signal, activates nuclear factor j-B resulting in the expression of genes that have an over-all antiapoptotic effect, leading to camptothecin resist-ance [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 pro-tein vimentin, an abundant type III intermediate fil-ament protein, which is cleaved by caspases-3, -7 and -6 during apoptosis [45] The proteolysis of vimentin promotes apoptosis by dismantling intermediate fila-ments and by generating a proapoptotic amino-terminal 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 proteo-lysis 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 chemother-apy [49] Also in breast cancer, either low [50] or high [51] expression of vimentin has been described in tum-ors that underexpressed the estrogen receptor (poor prognostic indicator) Vimentin expression has also been considered as a marker of resistance in doxorubi-cin-resistant LoVo cells [52] and in the multidrug resistant MCF7 subline [53], but parental cells trans-fected with human vimentin cDNA, did not become resistant It is interesting to note that, in addition to the decrease of vimentin expression, MTX-resistant
Trang 10HT29 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
rela-tionship 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
sen-sitive to MTX These changes in MTX sensitivity were
also reflected in the apoptotic levels On one hand,
overexpression of vimentin counteracts the
antiapop-totic 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
inhibi-tors 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
combina-tions with MTX
Experimental procedures
Cell culture
Human colon adenocarcinoma cell line HT29 were
rou-tinely grown in Ham’s F12 selective DHFR medium
(–GHT medium) lacking glycine, hypoxanthine and
thymi-dine, the final products of DHFR activity This medium
was supplemented with 7% dialyzed fetal bovine serum
(Gibco, Paisley, UK) at 37C in a 5% CO2 humidified
atmosphere Resistant cells 10)5m 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
cDNA arrays was prepared using the AtlasTM pure total RNA Labeling kit (Clontech) from 4· 106
cells, either parental HT29, HT29 treated with 10)7m 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 70C followed by 2 min
at 50C with 1 lL of the primer mix, containing only the
1176 primers for the genes present in the array, thus confer-ring a 10-fold increase in sensitivity and a concomitant reduction in nonspecific background The reverse transcrip-tion reactranscrip-tion was carried out using 100 U MMLV RT,
40 U rRNasin (Promega, Madison, WI, USA) and 30 lCi
of [32P]dATP[lP] (Amersham Bioscience, Freiburg, Ger-many) for 25 min at 50C After filtering the
unincorporat-ed nucleotide through Sephadex G-50 columns, membrane hybridizations were carried out Nylon filters were prehy-bridized in 5 mL ExpressHybTM (Clontech) with
100 lgÆmL)1 DNA salmon sperm for 30 min in roller bot-tles with continuous agitation in an oven at 68C Then, the 32P-labelled probe was added and hybridization contin-ued overnight in the same conditions Afterwards, mem-branes 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, Roche-ster, 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 normali-zation, generation of restriction lists and the functional classification of the differentially expressed genes Normal-ization was applied in two steps: (a) ‘per chip normaliza-tion’ 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 normal-ized 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 calcula-ted using the Cross-Gene Error Model based on replicates [56] Measurements with higher control strength are relat-ively more precise than measurements with lower control