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: cciudad@ub.edu (Received 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 FEBS Journal 272 (2005) 696–710 ª 2005 FEBS S Penuelas et al ˜ 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 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 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 FEBS Journal 272 (2005) 696–710 ª 2005 FEBS Fig 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 697 Gene expression in MTX-resistant HT29 cells S Penuelas et al ˜ Table 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 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 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 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 Vimentin Keratin Keratin 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 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) 698 FEBS Journal 272 (2005) 696–710 ª 2005 FEBS S Penuelas et al ˜ Gene expression in MTX-resistant HT29 cells Table (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 TIAM1 protein V-FES feline sarcoma viral ⁄ V-FPS fujinami avian sarcoma viral oncogene homolog Pig3 Fibroblast growth factor v-erb-b2 erythroblastic leukemia viral oncogene homolog 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 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 FEBS Journal 272 (2005) 696–710 ª 2005 FEBS 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 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 699 Gene expression in MTX-resistant HT29 cells S Penuelas et al ˜ Fig 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 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 (s) and 10)5 M (d) Results are the mean ± SE obtained from at least three independent experiments A C 700 B D Fig 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 *P < 0.05 compared with the corresponding control situation (B) Effect on IMPCH mRNA levels by using 0.5 and 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 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 FEBS Journal 272 (2005) 696–710 ª 2005 FEBS S Penuelas et al ˜ A Gene expression in MTX-resistant HT29 cells B C Fig 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 *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 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 *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 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 FEBS Journal 272 (2005) 696–710 ª 2005 FEBS 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 (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) 701 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 ˜ Fig 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 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 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 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 FEBS Journal 272 (2005) 696–710 ª 2005 FEBS S Penuelas et al ˜ Gene expression in MTX-resistant HT29 cells Fig 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 lg pSFFV-Bcl2 and 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 FEBS Journal 272 (2005) 696–710 ª 2005 FEBS 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 703 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 [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 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 · 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 The integrity of the RNA was assessed after agarose gel electrophoresis in the presence of formaldehyde Radiolabeled cDNA probes were prepared from lg of total RNA Briefly, RNA was hybridized for at 70 °C followed by at 50 °C with 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 at 50 °C After filtering the unincorporated nucleotide through Sephadex G-50 columns, membrane hybridizations were carried out Nylon filters were prehybridized in mL ExpressHybTM (Clontech) with 100 lgỈmL)1 DNA salmon sperm for 30 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 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 and the cell pellet was washed once with ice-cold NaCl ⁄ Pi The cells were centrifuged again at 10 000 g for min, and after discarding the supernatant, resuspended in 20 lL lysis buffer (20 mm NaCl, mm EDTA, 0.1% SDS, and 50 mm Tris ⁄ HCl, pH 8) plus lL of 10 mgỈmL)1 proteinase K They were then incubated at 55 °C for 15 min, vortexed vigorously and incubated for 15 at the same temperature Finally, the mixture was incubated at 100 °C for and, after cooling, lL was used for PCR amplification [57] The primers used for dhfr amplification were: 5¢-CCTGTTAACGCAGTGTTTCTC-3¢ within intron and 5¢-TCCCACGGGAGACTTCGCACT3¢ within intron For adenosyl phosphoribosyl transferase (aprt) the oligonucleotides were: 5¢-TCACGAGCCAG CAAGGCGTT-3¢ within intron and 5¢-ACGCAGTACT CATCCAGGGT-3¢ within intron 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 lg of total RNA, 125 ng of random hexamers (Roche, Mannheim, Germany), in the presence of 75 mm KCl, 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 and the cDNA product was used for subsequent PCR amplification with specific primers A standard 50 lL mixture contained 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 [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 and 30 s at 94 °C, PCR was performed for the indicated number of cycles Each cycle consisted of denaturation at 92 °C for min, primer annealing at 59 °C, and primer extension at 72 °C for 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 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: 5¢-CTGAATCTCATCCTGCAGGC-3¢ for: 5¢-GCAGCTGGTTGAGCAGCGGAT-3¢ rev: 5¢-AGAGTGGGGCCTGGCAGCTTC-3¢ 480 26 24–28 359 23 21–26 165 27 23–29 333 24 21–25 373 26 22–28 253 20 19–25 IMPCH SURV TOP1 VIM APRT 706 FEBS Journal 272 (2005) 696–710 ª 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 The cell pellet was gently resuspended in mL of hypotonic buffer (15 mm NaCl, 60 mm KCl, 0.5 mm EDTA, mm phenylmethanesulfonyl fluoride, 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 The resulting supernatant corresponded to the whole extract The entire procedure was carried out at °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 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 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, lL of FuGENE6 (Roche Molecular Biochemicals) in 100 lL of serum free –GHT medium was incubated at room temperature for Then, this mixture was added to the DNA and, after 15 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 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 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 at a : 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- 707 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 ˜ 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 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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... 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... 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