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1 Department of Medical Oncology, Beatson Institute for Cancer Research, Glasgow, United Kingdom; and 2 Department of Medicine and Therapeutics, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, United Kingdom
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B (NFB) p65 and related antiapoptotic c-Flip gene was detected in resistant cells. The 5-FU-resistant cell lines also showed high NFB DNA-binding activity. Cotransfection of NFB p50 and p65 cDNA induced 5-FU resistance in MCF-7 cells. Both NFB- and 5-FU-induced resistant cell lines manifested reduced expression of genes governing G1-S and S-phase transition. Expression of genes involved in DNA replication was also down-regulated in resistant cell lines. These findings were highly consistent with the slower growth rate, higher proportion of G1, and lower proportion of S-phase cells in the resistant cell lines. This phenotype may protect resistant cells from cell death induced by incorporation of 5-FU into DNA chains, by allowing time to repair 5-FU-induced damage. Our findings may provide novel targets for tackling 5-FU resistance. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The sensitivity of cancer cells to 5-FU is influenced by multiple molecular events. Global analysis of the molecular alterations in 5-FU-resistant cancer cells is required to unravel the complex mechanisms of 5-FU chemoresistance. Microarray technology, developed in recent years, has enabled analysis of pan-genomic expression profiles in cells or tissues of interest (6) . When DNA microarray technology is used, complex pathologic events can be intricately probed. A combination of microarray and traditional molecular technologies will enable us to functionally characterize genes related to anticancer drug resistance and identify novel molecular targets for anticancer drug development.
Using Affymetrix HG-U133A oligonucleotide microarrays consisting of 22,283 transcripts representing approximately 16,000-classified human genes, we have analyzed the expression profiles of five pairs of 5-FU-resistant and relevant drug sensitive parental cancer cell lines. Specific molecular factors and cellular pathways mediating and/or predictive of 5-FU resistance were elucidated in this study.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Stable Transfection.
MCF-7WT cells (5 x 104/well) were cultured in 35-mm dishes until 70% confluent, and Superfect (Qiagen, West Sussex, United Kingdom) was used to cotransfect with pRcCMV/neo/nuclear factor B (NFB)-p50 and pcDNA3.1/Hygro/NFB-p65, following the manufacturer’s instructions. Empty vector transfected cells were used as a negative control. The successfully transfected clones were selected in G418 (500 µg/mL; for negative control cell line) or coselected in G418 (500 µg/mL) and hygromycin (150 µg/mL; for NFB p50 + p65-cotransfected cell line). To avoid bias between individual clones, all of the selected cells were collected as a pooled population for further analysis.
Western Blot Analysis.
Total protein (100 µg/lane) was electrophoresed through a 10% NuPAGE Bis-Tris gel (Invitrogen) and transferred to a polyvinylidene difluoride membrane (Millipore, Watford, United Kingdom). The blots were stained with primary antibodies (NFB/p50 and p65, cyclin A, cyclin D3, cyclin-dependent kinase 2 (Cdk2), B-Myb, and TS, 1:250; c-FLIP, thymidine kinase (TK), and C-YES, 1:1000; 
-tubulin, 1:2000) overnight at 4°C and then with horseradish peroxidase-conjugated donkey antirabbit or mouse secondary antibodies (1:5,000) for 1 hour at room temperature. The signal was detected with an ECL Western blotting detection kit (Amersham Pharmacia Biotech, Piscataway, NJ).
Electrophoretic Mobility-Shift Assays.
Nuclear protein extraction and electrophoretic mobility-shift assays (EMSA) were carried out as described previously (8)
. Nuclear extract (5 µg) was incubated with 1 µg of poly(dI · dC) (Sigma) in binding buffer [50 mmol/L Tris (pH 7.6), 250 mmol/L KCl, 25 mmol/L DTT, 5 mmol/L EDTA, and 25% glycerol] for 10 minutes at room temperature. Approximately 20,000 cpm of 32P-labeled 22-mer double-stranded NFB DNA probe (5'-AGTTGAGGGGACTTTCCCAGGC-3') was added and incubated at room temperature for 20 minutes. Oct-1 (5'-TGTCGAATGCAAATCACTAGAA-3') was used as loading control for EMSA. The EMSA conditions for Oct-1 were the same as those for NFB. For binding specificity determination, 5 µg of nuclear extract from p50- and p65-transfected MCF-7 cells were incubated with 20 x wild-type or mutant (5'-AGTTGATATTACTTTTATAGGC-3') unlabelled NFB probe for 30 minutes before EMSA. The complexes were separated on a 6% polyacrylamide gel and exposed to autoradiographs.
Flow Cytometric Analysis of DNA Content.
Cells were cultured in 5-FU-free medium in 25 cm flasks until 70% confluent and harvested by trypsinization. After fixation in 70% ice-cold EtOH for 10 minutes and incubation with RNase A (100 µg/mL) and propidium iodide (50 µg/mL) for 30 minutes, 10,000 cells from each sample were subjected to fluorescence-activated cell sorter (FACS) Scan (Becton Dickinson, Franklin Lakes, NJ) analysis.
Gene Expression Analysis on HG-U133A Arrays.
Cells (70% confluent) cultured in 5-FU-free medium in 75 cm flasks were harvested by trypsinization. Total RNA was isolated with TRIzol reagent (Invitrogen) according to the manufacturer’s protocols. Preparation of cDNA (Invitrogen) and biotin-labeled cRNA (ENZO Diagnostics, Farmingdale NY) from 8 µg of total RNA, followed by HG-U133A GeneChip hybridization, washing, staining and scanning were carried out according to the standard protocols (Affymetrix, Santa Clara, CA).
Data Transformation and Statistical Analysis.
Preliminary filtering of data were done with Microarray Analysis Suite v5.0, MicroDB v3.0 and DMT v3.0 (Affymetrix). The "detection call" algorithm (Affymetrix) was used to produce a filtered gene list containing transcripts that were "present" in at least one cell line to select transcripts expressed at a level detectable on the HG-U133A arrays. The "change call" algorithm (Affymetrix) was used to further refine this gene list by selecting genes that were called "increase/marginal increase" or "decrease/marginal decrease" in all resistant cells compared with their relevant paired sensitive parent line (n = 127). In comparative analysis, the change call algorithm compares each probe pair within a set between experimental and baseline arrays. A change P value is calculated based on differences between perfect match (PM) and mismatch (MM) as well as between PM and background with Wilcoxon’s Signed Rank test. At least four of nine cell lines expressed these 127 transcripts. Most of these transcripts were expressed in all cells (n = 91), with different levels of expression between sensitive and resistant pairs. This detection and change call-filtered gene list (n = 127) was exported into GeneSpring v6.1 for further analysis.
The signal was further normalized in GeneSpring v6.1. (a) Data transformation, measurements <0.01 were adjusted to 0.01 to allow more efficient analysis of log-transformed data; (b) per chip normalization, each measurement on an individual array was normalized to the 50th percentile of all of the measurements on the array; and (c) per gene to median, each gene was normalized to its median value across all arrays in the experiment, to compare the relative change in gene expression levels across different arrays.
The normalized gene expression measurements were subjected to further threshold and probability filtering in GeneSpring v6.1 to identify genes correlated with 5-FU resistance. 5-FU resistance-associated gene sets were further analyzed with unsupervised and supervised hierarchical clustering based on the standard correlation of logarithmic transformed data. Leave-one-out cross-validation based on Fishers exact t test hypergeometric probability and K nearest neighbours was used to assess the predictive power of the filtered gene sets.
Student’s t test was used for other data analysis in this study.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Compared with the parental cell lines, the drug-resistant cell lines tolerate significantly higher concentrations of 5-FU (5.5- to 35.9-fold, P < 0.01).
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In unsupervised analysis, threshold and probability filtering were used in DMTv3.0 and GeneSpring v6.1 software to identify genes associated with 5-FU resistance in all cancer cell lines. A set of 127 genes expressed in 
4 cell lines and called as changed in expression between all resistant and paired parental lines was identified with the "detection and change call" algorithms in DMTv3.0 (see Materials and Methods). Forty-one of the 127 transcripts were statistically significantly associated with sensitivity to 5-FU in all cell lines (Welch’s t test P < 0.05 with Benjamini and Hochberg false discovery rate correction). The mean expression of these 41 transcripts was altered by at least 1.5-fold between the resistant and the sensitive groups. Using a parametric model I fixed-effects 2-way ANOVA (P < 0.05) with Benjamini and Hochberg false discovery rate correction (MCF-7FU1 was excluded from this analysis to balance the groups), we identified expression of 39 of these 41 genes as associated significantly with 5-FU sensitivity without substantial association with the primary tumor site (breast versus CRC). No interaction effect was observed between the tissue and 5-FU sensitivity phenotypic groups. This refined set of 5-FU sensitivity-associated tissue site-independent genes (n = 39) clustered samples according to 5-FU sensitivity class in unsupervised hierarchical analysis and successfully discriminated each individual cell line (data not shown).
The prediction strength and accuracy of the filtered gene list was estimated with leave-one-out cross-validation based on Fisher’s exact test hypergeometric probability of each gene and K nearest neighbours (K = 3). A prediction rule built from the gene expression patterns of the 33 transcripts representing 29 unique genes, with the highest predictive strength (prediction strength [–ln P value] of each gene = 4.83; Table 2
), discriminated 5-FU sensitivity phenotype with 100% accuracy and high predictive strength (K nearest neighbours P value ratio of neighborhood samples of each phenotype <0.08). These genes were able to separate cell lines according to 5-FU sensitivity phenotype with standard correlation of logarithmic-transformed gene expression data in unsupervised hierarchical clustering (Fig. 1A)
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B, apoptotic, cell death, cell cycle, pyrimidine metabolic, DNA replication, oncogenes, tumor suppressor genes, drug resistance, and signal transduction pathways; n = 8347). Threshold (1.5-fold) and probability filtering (Welch’s t test, P < 0.05) of these ontological groups (GeneSpring v6.1) identified 100 transcripts, representing 91 unique genes, the mean expression of which was consistently and significantly altered between the sensitive and resistant groups and which were called as changed in expression in at least four resistant cell lines compared with the paired parental line (change call algorithm, DMTv3.0; see Materials and Methods; Table 3
and supplementary data). Gene expression levels of these 100 transcripts separated hierarchical clustering samples into two primary groups based on 5-FU sensitivity phenotype (Fig. 1B)
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, genes involved in 5-FU metabolism were significantly down-regulated. The products of a number of these genes (TK, orotate phosphoribosyltransferase, pyrimidine nucleoside phosphorylase, and uridine monophosphate kinase) are directly or indirectly involved in the conversion of 5-FU into its cytotoxic metabolites. TS and c-yes genes are commonly coamplified in TS inhibitor-resistant cancer cell lines. Overexpression of TS mRNA was only detected by microarray analysis in two CRC cell lines. Overexpression of a neighboring gene, C-YES was detected in all cell lines (all "Increased," change call algorithm, DMTv3.0), although it did not reach statistical significance (Table 3)
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Another intriguing group is DNA replication licensing factors and cell cycle related genes. The expression of many genes governing DNA replication (e.g., DNA polymerase , MCM2, and MCM7) and those involved in G1-S (B-Myb, cyclin D3, and Cdk2) or S-phase (cyclin A) transition was significantly down-regulated in resistant compared with sensitive lines (Table 3)
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NFB-related Genes and 5-FU Resistance.
NFB is a transcription factor that has been reported to antagonize apoptosis induced by specific cytokines and anticancer drugs (8, 9, 10)
. The mean expression of NFB p65 mRNA was increased by over 1.5-fold in the resistant compared with the sensitive groups, although it did not reach statistical significance, and variable changes were observed in paired samples (Table 3)
. Because of the reported role of NFB in chemoresistance, protein expression levels of NFB p50 and p65 were analyzed in more detail. An increase in p65 protein was detected in all 5-FU-resistant cell lines compared with the sensitive parent line (Fig. 2A)
. In contrast to p65, p50 protein overexpression was only detected in one 5-FU-resistant CRC cell line (Fig. 2A)
. Consistent with our previous data, strongly enhanced NFB DNA binding (Fig. 2B
; ref. 11
) and transcriptional activities (data not shown) were detected in all 5-FU-resistant cell lines. To determine the effect of NFB on 5-FU resistance, the human breast cancer cell line MCF-7 was cotransfected with NFB p50 and p65. The transfected cells showed high expression of NFB p50 and p65 proteins (Fig. 3A)
and stronger NFB DNA binding (Fig. 3B)
and transcriptional (data not shown) activities. The NFB-transfected cells (IC50, 22.5 µmol/L) are more resistant to 5-FU-induced cytotoxicity than the control cells (IC50, 3.75 µmol/L; Fig. 3C
).
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, c-FLIPL protein was overexpressed in resistant compared with the sensitive parental lines (Fig. 2A)
. Overexpression of c-FLIPL protein was more consistent and much stronger than that of c-FLIPS in resistant cell lines (Fig. 2A)
.
Down-Regulation of G1 Checkpoint-related Genes and Delayed G1-S Transition in 5-FU-resistant Cell Lines.
Statistically significant down-regulation of cell cycle-related genes (including cyclin A, cyclin D3, Cdk2, and B-Myb) was detected in 5-FU-resistant cells by microarray analysis (Table 3)
. Consistent with the microarray data, cyclin A, cyclin D3, and Cdk2 proteins were down-regulated in resistant-cancer cell lines (Fig. 4A)
. Although microarray analysis showed significant down-regulation of b-Myb mRNA in all resistant cell lines, b-Myb protein was not differentially expressed between the resistant and parental cell lines.
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. Compared with parental cell lines, the 5-FU-resistant cells consisted a significantly higher percentage of cells in G1-G0-phase and a lower percentage in S phase (P < 0.01; Table 4
; Fig. 4C
). These data indicate that DNA synthesis in 5-FU-resistant cells is delayed, which may be caused by deregulation of key cell cycle-related genes. BrdUrd and PI traced cell cycle analysis showed that the resistant cells were delayed in G1 (breast cancer cell lines) or at the G1-S boundary (H630-R10; data not shown).
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B-induced Arrest at G1 Checkpoint.B-transfected and control MCF-7 cells. The NFB-transfected cells grew much slower than the control cells (Fig. 5A)
. FACS analysis indicated that a higher percentage of NFB-transfected cells were in G1-G0 phase with a reduced population in S-phase (Fig. 5B)
. Down-regulation of key G1-S transition-related proteins (cyclin D3, Cdk2, and B-Myb) was also detected in the transfected cells (Fig. 5C)
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, down-regulation of TK protein was observed in 5-FU-resistant H630 and T47D cancer cell lines (Fig. 6)
. However, down-regulated expression of TK mRNA in the resistant MCF-7 cells (Table 3)
was not reflected at the protein level (Fig. 6)
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indicating that the post-transcriptional mechanisms are involved in TS protein overexpression. C-yes is an oncogene located adjacent to TS. We have reported previously that c-yes and TS genes are coamplified and overexpressed in TS inhibitor-resistant cancer cell lines (4)
. In this study, overexpression of C-YES protein (Fig. 6)
and mRNA (Table 3)
was consistently detected in 5-FU-resistant cell lines. However, the weakest up-regulation of c-yes mRNA, which was observed in the MCF7FU1-resistant cells, was not detected at the protein level. Another adjacent gene, which regulates TS expression (rTS), was overexpressed in three resistant cell lines compared with the paired sensitive parent (Table 3)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Maxwell et al. (14) detected 5-FU-inducible genes (SSAT, annexin II, thymosin-ß-10, MAT-8 and chaperonin-10) in 5-FU-treated MCF-7 cells, and enhanced basal expression of these genes in resistant H630-R10 cells. Enhanced basal expression of 5-FU-inducible genes in resistant cells is not associated with increased cell cycle arrest or apoptosis (14) . Overexpression of most of these genes (Annexin II, MAT-8, and thymosin 10) was also detected in H630-R10 cells in our study (data not shown). The predictive markers may not necessarily represent molecules critical to the acquisition or maintenance of the resistant phenotype (4 , 14) . Some or all of these genes may be markers of the phenotype that have no role in resistance (e.g., coamplified genes) or they may represent the amplified end points of critical cellular pathways, in which the upstream mediators of the resistant phenotype undergo small but critical expression changes.
Supervised analysis of the microarray data using gene ontologies was used to examine pathways and factors shown previously or hypothesized to play a role in 5-FU metabolism or resistance. This analysis identified 100 transcripts representing 91 unique human genes the mean expression of which was statistically significantly altered in 5-FU-resistant cell lines compared with sensitive parental cell lines by 1.5-fold, and the expression of which was either increased or decreased in at least four of the resistant cell lines compared with the paired parental line.
We verified the protein expression levels of five of these genes using Western blot. The protein and mRNA expression patterns from all except one (b-myb) of the tested genes were either correlated in all cell lines (cyclin A, cyclin D3, and cdk2) or correlated in both the CRC but only one breast cancer cell line (TK). For the gene where expression of mRNA and protein was not correlated (b-Myb), post-transcriptional and/or post-translational mechanisms of regulation are likely to exist. The protein expression levels of an additional five genes, the expression of which was altered in some or all sensitive/resistant pairs (change call algorithm, DMTv3.0), but the mean expression of which was not statistically significantly different between the resistant and sensitive groups, were also evaluated. The expression of three of these genes was correlated at the mRNA and protein level in 40% (p65 and TS) to 80% (c-yes and c-FlipL) of the cell lines, indicating post-transcriptional mechanisms of regulation exist in some cell lines. The change in expression of c-FlipS mRNA was correlated with protein expression changes in all cell lines. Thus, there was a strong correlation between microarray and protein data for at least 70% of the genes evaluated. The importance of a strong correlation existing between the mRNA and protein expression levels depends on both the cellular function of the factor under investigation as well as the question being addressed. To gain insight into the molecular mechanisms of the phenotype, the status of the protein product is critical.
NFB p65 mRNA and protein overexpression were detected in the 5-FU-resistant cancer cell lines. The resistant cell lines had higher NFB DNA binding and transcriptional activity. Inhibition of NFB activity by IB can enhance the cytotoxicity of some anticancer drugs in vitro (8)
and in vivo (9
, 15)
. We have reported previously that TS inhibitor (5-FU and TDX)-resistant cancer cell lines possess high NFB nuclear activity (11)
. Disulfiram, an antialcoholism drug, inhibits NFB activity and sensitizes cancer cells to 5-FU-induced apoptosis (16)
. The most common NFB dimer is a p50-p65 heterodimer. To elucidate the role of NFB in 5-FU resistance, MCF-7 cells were cotransfected with NFB p50 and p65. The IC50 of 5-FU in NFB-transfected cells was 5.5-fold higher than the control cells. These data indicate that NFB can induce 5-FU resistance.
In this study, altered expression of key antiapoptotic genes (e.g., c-IAPs, XIAP, A1/Bfl-1, and IEX-1L) downstream of NFB (17, 18, 19)
was evaluated. Expression of c-IAP-1 was increased in all except one (T47D) resistant line (1.5–2.1 fold). This change did not reach statistical significance in the supervised analysis. There was a large increase in c-IAP-2 mRNA expression in resistant CRC cells lines (H630-R10, 11.0-fold and H630-R10-B 6.6-fold) but not in breast cells. Because of the nature of the probe design, the two isoforms of IEX-1 (antiapoptotic IEX-1L and proapoptotic IEX-1S) are represented by a single probe set on the HG-U133A array, making interpretation of the data difficult. IEX-1 was decreased in all three resistant breast cancer cell lines (–1.6- to –2.3-fold), and its expression was either slightly increased (H630-R10 1.4-fold) or unaltered (H630B-R10) in the resistant CRC cell lines. Bfl-1/A1 and XIAP mRNA were not expressed in any of the cells under investigation.
c-FLIP is another antiapoptotic gene, the mRNA and protein of which were overexpressed in some 5-FU-resistant cell lines (Fig. 2A
; Table 3
). c-FLIP encodes short (c-FLIPS) and long (c-FLIPL) isoforms (12)
. The c-FLIPL protein was more significantly and consistently up-regulated in all resistant cancer cell lines (Fig. 2A)
. c-FLIP inhibits cleavage and activation of caspase 8, the initiator caspase of the extrinsic apoptotic pathway (20)
. It has been reported that 5-FU transcriptionally induces CD95 expression, which triggers caspase 8 activation and apoptosis via the extrinsic apoptotic pathway (21
, 22)
. 5-FU also inhibits c-FLIP expression in cancer cell lines (23)
. High c-FLIP expression in 5-FU-resistant cell lines may block the extrinsic apoptotic pathway and inhibit the cytotoxicity of 5-FU in the resistant cells. c-FLIP can be up-regulated by NFB and c-FLIP protein overexpression can replace NFB to block the tumor necrosis factor -induced extrinsic apoptotic pathway activation in human cancer cell lines (24
, 25)
. C-Flip gene expression is 5-FU inducible (14)
. We are currently investigating the relationship between NFB and c-FLIP in 5-FU resistance.
In cancer cells, 5-FU is converted to several active metabolites intracellularly (fluorodeoxyuridine monophosphate, fluorodeoxyuridine triphosphate, and fluorouridine triphosphate) that are cytotoxic (1) . There have been several reports demonstrating the relationship of disturbance of individual enzymes involved in 5-FU metabolism with 5-FU resistance (26 , 27) . In supervised analysis of the microarray data, down-regulation of several key enzymes involved in 5-FU activation (TK, orotate phosphoribosyltransferase, uridine monophosphate kinase, and pyrimidine nucleoside phosphorylase) was identified in 5-FU-resistant cell lines. Down-regulation of these pyrimidine metabolic enzymes may represent one of the pivotal self-protective mechanisms sheltering resistant cells from the cytotoxic effects of 5-FU.
5-FU is an indirect TS inhibitor. TS gene amplification and overexpression are widely accepted as a common, although not universal, feature of 5-FU resistance (1) . In this study, TS protein overexpression was detected in all resistant cell lines but only resistant CRC cell lines expressed higher levels of TS mRNA. In line with previous findings (4 , 28) , overexpression of genes closely located to TS (rTS mRNA and C-YES mRNA and protein) was detected in resistant cell lines.
Unrestricted proliferation induced by breached G1 checkpoint is a fundamental characteristic of cancer cells (13
, 29
, 30)
. This feature is also considered an "Achilles heel" of cancer cells, allowing some S-phase-specific anticancer drugs (e.g., 5-FU and TDX) to selectively target cancer cells. In this study, 5-FU-resistant cells grew at much slower rates than the relevant parental cell lines in vitro. A higher percentage of resistant cells were retained in G0-G1 phase or at the G1-S boundary. In line with this phenotype, down-regulation of G1-S (cyclin D3 and Cdk2) and S-phase (cyclin A) transition-related genes was detected in 5-FU-resistant cells. The 5-FU-resistant cells also showed reduced Cdk2 kinase activity and hypophosphorylated Rb protein (data not shown). The cyclin D-Cdk4/6-cyclin E-Cdk2-Rb-E2F axis plays a pivotal role in G1-S transition (31, 32, 33, 34)
. Our findings indicated that 5-FU-resistant cells have an increased G1 checkpoint stringency, which significantly delays the transition of resistant cells from G1 into S-phase. Reduced expression of some DNA replication licensing factors (MCM2 and 7) and DNA replication or repair-related genes (Ki-67, DNA polymerase , TK, and CITED2) was also detected in 5-FU-resistant cell lines. 5-FU is an S-phase-specific anticancer drug. It has been reported that reduced proliferation rate is inversely correlated with 5-FU cytotoxicity and therapeutic response (35, 36, 37)
. A reduced proliferation rate may protect 5-FU-resistant cancer cells from the lethal attack of 5-FU. In addition, delayed S-phase entry and/or reduced S-phase traverse may provide resistant cells with enough time to repair 5-FU-induced damage before progressing to G2-M phase.
NFB transfection induced 5-FU resistance in MCF-7 cells. Unexpectedly, the NFB induced 5-FU-resistant cells also showed slower growth rates and increased G1 checkpoint stringency with reduced expression of G1-S and S-phase transition-related genes. This phenomenon indicates that increased G1 checkpoint stringency may be a common mechanism involved in 5-FU resistance. If this is the case, 5-FU resistance may be, at least partially, reversed by specific targeting of the G1-S checkpoint arrest in the resistant cells. Restoration of the G1 checkpoint by targeting Cdk2 and/or Cdk4 is currently one of the major strategies for anticancer drug development (30)
. Our findings indicate that within the complex genetic background of a cancer cell, G1 checkpoint restoration may produce slowly growing cancer cells which are resistant to S-phase-specific anticancer drugs.
In conclusion, global transcriptome profiling shows that 5-FU resistance is multifactorial and involves some or all of the following cellular pathways: overproduction of 5-FU targets; up-regulation of specific antiapoptotic proteins, reduced production of 5-FU-activating enzymes, and increased G1 checkpoint stringency with a reduced cell proliferation rate and reduction in DNA-synthetic machinery.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: Supplemental data for this article can be found at Cancer Research Online (http://cancerres@aacrjournals.org).
Requests for reprints: Weiguang Wang, Department of Medical Oncology, Beatson Institute for Cancer Research, Switchback Road, Glasgow G61 1BD, United Kingdom. Phone: 44-141-3304354; Fax: 44-141-3304127; E-mail: w.wang{at}beatson.gla.ac.uk
Received 3/23/04. Revised 5/17/04. Accepted 6/25/04.
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