BACKGROUND
thymidylate synthase  is a folate-dependent enzyme that catalyzes the reductive methylation of dump by  <dig> -methylenetetrahydrofolate to form dtmp and dihydrofolate  <cit> .
because the ts-catalyzed enzymatic reaction provides the sole intracellular de novo source of thymidylate, an essential precursor for dna biosynthesis, this enzyme has been an important target for cancer chemotherapy for the past  <dig> years  <cit> .
ts is also one of the most extensively investigated biomarkers in recent years  <cit> .
in certain cases, ts has been shown to be a significant biomarker for predicting patient responses to 5-fu based therapy.
however, in other studies, the expression level of ts alone is not sufficient for clinical prognosis.
the goal of this study is to provide global comprehensive gene profiles and networks at multiple levels of gene regulation that are affected by endogenous levels of ts protein and 5-fu administration.
this information will provide the basis to identity biomarker gene candidates that can be further validated using clinical samples for future clinical diagnosis and prognosis.
the rational for investigating genes affected by ts and 5-fu at both transcriptional and post-transcriptional levels is that ts, in addition to its critical enzymatic function, functions as a rna binding protein  <cit> .
the translation of human ts mrna is regulated by its own protein product via a negative autoregulatory mechanism whereby the binding of ts protein to at least two distinct sequences on its own ts mrna results in translational repression  <cit> .
ts is also capable of interacting with several other cellular mrnas such as p <dig> mrna and c-myc mrna  <cit> .
previous studies demonstrated that ts protein regulates p <dig> gene expression at least in part, at the translational level  <cit> .
in this case, ts may be involved in coordinating the regulation of expression and/or function of cellular growth and proliferation and it is conceivable that ts may play an essential role as a regulator of cell cycle related events.
more importantly, this study will have direct clinical relevance in that the mechanism of acute and long term 5-fu related drug resistance is distinct.
the acute induction of ts expression after 5-fu treatment was regulated at the translational level and long term resistance for 5-fu is related to transcriptional activation and gene amplification of ts  <cit> .
thus, it would be particularly important to systematically investigate other potential post-transcriptional regulated genes via ts protein.
this may be especially vital for the discovery of additional chemotherapeutic response related markers that otherwise would be missed by simply profiling steady state total mrnas  <cit> .
a recent report suggested that ts may also function as an oncogene to transform nih3t <dig> cells  <cit> .
a comprehensive gene expression profiling analysis may also gain new insights into signaling pathways that were deregulated by over-expression of ts.
in this regard, it is critical to develop more comprehensive molecular expression profiles to provide candidate genes that can potentially be used for predicting clinical outcomes for colorectal cancer.
in this study, a systems biology approach was used to investigate genes altered by the overexpression of ts at both transcriptional and post-transcriptional levels using human genome expression array in ts-depleted human colon cancer hct-c <dig> cells and hct-c <dig>  cells stably transfected with the human ts cdna expression plasmid.
in addition, both cell lines were treated with 5-fu for different time periods in an attempt to identify both acute response genes as well as delayed response genes affected by the 5-fu exposure.
a number of genes were identified by comparing the expression profiles obtained from both steady state total mrnas and polysome associated mrna transcripts isolated from 5-fu treated hct-c <dig>  and hct-c <dig>  cells.
comprehensive gene lists were generated that were relevant for both ts dependent and independent cytotoxicity by 5-fu exposure.
we also discovered genes and pathways that may be involved for the oncogenic function of ts.
RESULTS
characterization of hct-c <dig>  and hct-c <dig>  cells
the hct-c <dig>  cell line contains a missense mutation at amino acid  <dig> of the ts protein that results in a near complete inactivation of ts enzyme activity  <cit> .
hct-c <dig>  cells were stably transfected with an expression construct containing full length human ts cdna.
hct-c <dig>  cells were able to grow without thymidine supplement in the media  <cit> .
however, hct-c <dig>  cells require  <dig> μm thymidine in rpmi <dig> medium.
the doubling times of both cells were identical at  <dig> hrs.
previous study has demonstrated that the mechanism for the decreased p <dig> expression occurred at the translational level  <cit> .
we use p <dig> as a positive control gene for monitoring our high throughput expression profiling approach.
our microarray gene expression analysis indicates that there is no difference in the expression level of p <dig> based on steady state total mrna .
however, the expression level of actively translated p <dig> mrna in hct-c <dig>  cells was found to decrease by  <dig> -fold compared to the p <dig> mrna level in hct-c <dig>  cells .
this is consistent with our previous results that p <dig> was regulated at the translational level via ts  <cit> .
effect of 5-fu on the expression of ts and p53
the effect of 5-fu on the expression of both ts and p <dig> was investigated using western immunoblot analysis.
hct-c <dig>  cells were treated with  <dig> μm 5-fu for  <dig> hrs and  <dig> hrs.
ts protein expression was increased by nearly 4-fold  at  <dig> hrs and 10-fold at  <dig> hrs  .
the ts-fdump-tetrahydrofolate covalent ternary complex was clearly visible after 5-fu treatment.
the expression of wild type p <dig> was also increased in response to 5-fu treatment .
effect of ts on steady state total mrnas expression
the global gene expression profile of hct-c <dig>  and hct-c <dig>  cells based on steady state total mrna expression was analyzed using human high density codelink oligo array .
although the growth characteristics of the two cell lines are similar in terms of doubling time, many genes are already affected by overexpressing ts protein.
our analysis revealed that over  <dig> genes were changed in their expression in response to ts expression .
the partial gene list and the clustering analysis are shown in figure  <dig> .
genes such as centb <dig> and mt <dig>  which are involved in cell proliferation and signaling, were up-regulated by ts.
casp <dig>  a gene involved in regulation of i-κb/nf-κb cascade, apoptosis and signal transduction, was decreased 22-fold by ts overexpression.
effect of ts on polysome associated mrnas
to discover novel genes post-transcriptionally regulated by ts protein, we isolated the polysome associated mrnas from both hct-c <dig>  and hct-c <dig>  cells and performed expression analysis using human high density codelink oligo array .
over  <dig> genes were effected in ts overexpressing hct-c <dig>  cells  .
genes involved in protein biosynthesis  and cell cycle control  were up-regulated at the post-transcriptional level.
the overlapping genes  between steady state total mrna and polysome associated mrna profiles are listed in table  <dig>
steady state mrnas affected by 5-fu treatment in hct-c <dig>  cells
to identify acute and delayed response genes following 5-fu treatment in hct-c <dig>  cells, gene expression profiling analysis was performed on steady state total mrnas isolated from control and 5-fu treated samples at  <dig> hrs and  <dig> hrs time points.
the expression analysis reveals that over  <dig> genes were affected by 5-fu treatment by one-way anova analysis .
the clustering analysis is shown in figure  <dig>  .
the expression analysis clearly showed a different gene expression profiles between  <dig> hrs and  <dig> hrs 5-fu exposure.
based on the comparison of the expression profiles among the hct-c <dig>  control and 5-fu treated samples, a number of genes were acutely increased in response to 5-fu.
genes  such as orcl <dig> , prps <dig> , ddx <dig> , dkk <dig> , and eif4e  are some of the important genes affected by the acute 5-fu treatment based on our model.
expression of some of the genes, such as sprr1a, ddb <dig>  and cdkn1a, was only increased in response to 5-fu at  <dig> hrs .
polysome associated mrna profiles with 5-fu treatment in hct-c <dig>  cells
to identify acute and delayed response genes that were affected at post-transcriptional level by 5-fu treatment, polysome associated mrnas were isolated from control, and 5-fu treated samples at  <dig> hrs and  <dig> hrs.
gene expression analysis via microarray revealed that over  <dig> genes were affected in response to 5-fu .
the clustering analyses are shown in figure  <dig> .
gene expression analysis revealed dynamic changes in expression at  <dig> hrs and  <dig> hrs of 5-fu treatment.
by comparing the expression profiles between control hct-c <dig>  and 5-fu treated samples, we discovered additional post-transcriptionally controlled genes that otherwise would be missed if the expression analysis was only performed using steady state total mrnas.
genes  such as nfic , srp <dig> , tsc <dig> , ireb <dig> , and cebpb  are some of the genes that were regulated at the post-transcriptional level in response to 5-fu exposure with unique expression dynamics.
genes associated with 5-fu induced ts independent cytotoxicity
to identify genes associated with ts independent cytotoxicity affected by 5-fu treatment, hct-c <dig>  cells were treated with  <dig> μm 5-fu for  <dig> hrs and  <dig> hrs in the presence of  <dig> μm thymidine.
the presence of thymidine provides the essential precursor for dna biosynthesis for the hct-c <dig>  cells thereby preventing the ts mediated cytotoxicity caused by 5-fu.
in this case, the cytotoxicity of 5-fu in hct-c <dig>  cells was a result of direct incorporation of 5-fu metabolites to dna and rna.
based on the clustering analysis of steady state total mrna profiling of control hct-c <dig>  cells and 5-fu treated samples in the presence of  <dig> μm thymidine, the gene expression profiles generated from these samples are mainly associated with ts independent toxicity in response to 5-fu by direct incorporation to rna and dna.
<dig> genes  were affected.
the clustering analysis is shown in figure  <dig> .
to further define the genes that were associated with ts independent toxicity, we compared the expression clusters  from both hct-c <dig>  and hct-c <dig>  cells treated with 5-fu using venn diagram analysis.
the overlapping gene list is shown in table  <dig>  genes such as rrm <dig> , cdkn1a , sei <dig> , ddb <dig> , sfn  were associated with ts independent cytotoxicity to 5-fu treatment.
gene expression analysis from figure  <dig> was used for the construction of the cell cycle control pathway after 5-fu treatment illustrated in figure  <dig>
discussion
in this study, a systems biology approach was used to analyze global gene expression and its networks affected by ts and 5-fu at multiple levels of control.
to the best of our knowledge, this is the first report to address genes that are affected at both transcriptional and post-transcriptional levels affected by elevated ts protein expression and 5-fu treatment.
we believe that this will provide a better understanding of complex network related to the multi-functions of ts protein as an rna binding protein or as a potential oncogene.
it also provides a technology platform to systematically discover potential post-transcriptionally controlled genes by ts and 5-fu treatment.
as our comparative model, we used a pair of human colon cancer cell lines: the mutant hct-c <dig> subline in which the ts protein had been rendered marginally active and the ts over-expressing subline, hct-c <dig>  created by stable expression of a human ts cdna plasmid.
both cell lines express wild-type p <dig> protein  <cit> .
p <dig> is one of the known genes affected by the over-expression of ts at the translational level due to its rna binding function  <cit> .
using our expression profiling approach, the levels of p <dig> mrna are not significantly changed based on steady state total mrna profile.
however, the polysome associated p <dig> mrna was decreased by  <dig> -fold in hct-c <dig>  cells compared with hct-c <dig>  cells .
these results, taken together, confirmed that our comprehensive gene profiling analysis approach is capable of discovering new post-transcriptionally regulated genes.
the gene profiling analysis based on steady state total mrna transcripts revealed over  <dig> genes that were deregulated by ts over-expression .
although this study does not provide any direct evidence for ts as a cell cycle regulator, a number of cell cycle control and apoptotic control genes were perturbed by ts over-expression.
one of the genes is inhibin/activin a  that was up-regulated by nearly 22-fold in hct-c <dig>  cells.
inhba is a growth factor that is involved in cell proliferation  <cit> .
inhba does this through type i and type ii receptor serine kinases  <cit> .
this may be a potential pathway by which ts is able to influence the cellular transformation process  <cit> .
in fact, it has been reported recently that inhba is over-expressed in stage iv colorectal cancer  <cit> .
additionally, single stranded rna interacting protein , a gene responsible for mrna splicing, was also up-regulated by 28-fold.
the gene is a member of a small family of proteins which bind single stranded dna/rna.
these proteins are characterized by the presence of two sets of ribonucleoprotein consensus sequence  that contain conserved motifs, rnp <dig> and rnp <dig>  originally described in rna binding proteins, and required for dna binding.
these proteins have been implicated in such diverse functions as dna replication, gene transcription, cell cycle progression and apoptosis  <cit> .
the encoded protein was isolated by virtue of its binding to an upstream element of the alpha <dig>  collagen promoter.
the observation that this protein localizes mostly in the cytoplasm suggests that it may be involved in a cytoplasmic function such as controlling rna metabolism, rather than transcription.
thus, rbms <dig> may play an important role as part of the post-transcriptional control mediated by ts.
conversely, genes such as caspase  <dig>  that control cell cycle arrest and apoptosis, were down-regulated some 22-fold.
hence, future studies will focus on determining whether ts protein regulates the expression of these genes directly by binding to the mrnas or indirectly through downstream mechanisms.
using our systems biology approach of polysome associated mrna comparisons, we have identified a list of genes that are over- or under-expressed due to ts protein over-expression .
many of them play key roles in cell-cell signalling, protein biosynthesis, rna processing, dna repair, cell cycle control and translational regulation.
some of them are previously known translational controlled genes such as egfr  <cit> , one of the key targets for the latest targeted anticancer drug development for colon cancer treatment  <cit> .
in this study, the expression of egfr was found to increase by nearly 8-fold by ts over-expression.
many other genes on this list are novel potential post-transcriptionally regulated genes.
among the down regulated genes, cyclin dependent kinase inhibitor p <dig>  was decreased by over 5-fold.
apoptosis inhibitor bak <dig> was also decreased by 6-fold in ts over-expressing cells.
it seems that over expressing ts has set the stage for cells to have many growth advantages using multiple mechanisms.
to identify genes regulated at both transcriptional and post-transcriptional level, we performed an overlapping analysis by comparing the gene lists generated from steady state total mrnas and polysome associated mrnas using venn diagram.
there are  <dig> overlapping genes between the two lists .
d <dig> , up-regulated by 5-fold in hct-c <dig>  cells, has been shown to be regulated at the translational level via eif2γ  <cit> .
we have identified an additional  <dig> genes based on polysome associated mrnas which could be easily missed if only steady state mrna profiling is performed.
this approach provides a technical platform to systematically analyzing gene expression at post-transcriptional level.
it also reveals the importance of analyze gene expression at multiple levels.
based on the clinical observation and detailed molecular investigations  <cit> , ts can be acutely induced with a short time 5-fu exposure and the mechanism of ts induction was regulated, at least in part, at the translational level  <cit> .
with this notion, we designed a strategy that allows us to capture both acute and delayed response genes in response to 5-fu exposure.
to achieve this, both hct-c <dig>  and hct-c <dig>  cells were treated with  <dig> μm 5-fu for  <dig> hrs and  <dig> hrs.
figures  <dig> and  <dig> listed genes with known functions based on one-way anova clustering analysis from steady state total mrna profiling from each cell line, respectively.
a clear and unique expression pattern revealed a dynamic response to 5-fu treatment .
there are  <dig> genes that were proved to be significant factors for acute 5-fu treatment .
they include cxcr <dig>  flj <dig>  rsp <dig>  akap <dig>  orc6l, prps <dig>  ddx <dig>  dkk <dig>  and eif4e.
dkk <dig> functions as an agonist in the wnt signaling pathway to stimulate cell proliferation during brain development  <cit> .
however, little is known about the function of dkk <dig> in cancer.
this is the first report to show that dkk <dig> may play an important role in colorectal cancer possibly via the wnt signaling pathway  <cit> .
eif4e, one of the key translational initiation factors, has been shown to play key roles in cell cycle control and is an important marker for determining chemosensitivity  <cit> .
we speculate that the acute induction of eif4e expression by 5-fu may be contributing to the resistance mechanism.
following  <dig> hr 5-fu exposure, nearly all of the acutely up-regulated genes had returned to baseline levels.
however, additional genes such as sprr1a, ddb <dig>  and cdkn1a were only up-regulated after  <dig> hrs.
these differential gene expression profiles suggest that single time point microarray data from clinical samples should be viewed with caution and not over-interpreted.
interestingly, we discovered an additional  <dig> genes associated with for 5-fu treatment based on profiling of polysome associated mrna transcripts .
the induction of p <dig> will contribute a significant part for cell cycle arrest and apoptosis based on previous reports  <cit> .
in addition, we also found genes such as, g-protein coupled receptor  <dig> , colon cancer antigen  <dig> , tsc <dig>  and p <dig> , were perturbed with 5-fu exposure.
sei <dig> is a ckd4-binding protein to regulate cdk <dig> activity  <cit>  and was up-regulated following 5-fu exposure.
g2a and sdccag <dig> were acutely up-regulated by 5-fu treatment whereas tsc <dig> was a delayed response gene affected by 5-fu.
it has been recently shown that tsc <dig> is regulated at the translational level by hypoxia  <cit> .
tsc <dig> is also involved in mtor signaling pathway to regulate cell growth, proliferation, and cell death  <cit> .
mtor is one of the key pathways for translational regulation via eif4e  <cit> .
these gene expression profiles may be helpful in elucidating additional resistance mechanisms related to 5-fu treatment.
we also determined the marker genes that were associated with ts independent cytotoxicity using hct-c <dig>  cells treated with 5-fu in the presence of  <dig> μm thymidine.
the main cytotoxicity caused by 5-fu was the inhibition of ts to block the sole de novo synthesis of thymidylate, an essential precursor for dna biosynthesis.
however, in the presence of  <dig> μm thymidine, the cytotoxicity caused by 5-fu in hct-c <dig>  cells was mainly by the direct incorporation of 5-fu metabolite into dna and rna.
the genes involved in ts independent cellular toxicity have not been systematically investigated in the past.
in this study, we discovered a number of genes that were associated with ts independent cytotoxicity of 5-fu.
genes involved in dna replication , dna repair , dna metabolism , rna catabolism , rna processing , cell signaling , and apoptosis  are on this list .
it's not surprising to see these genes on the list because direct incorporation of 5-fu metabolite to rna and dna is the major part of cytotoxicity via ts independent mechanism  <cit> .
it has been shown that incorporation of 5-fu into rna affects pre-mrna splicing process  <cit> .
this study provides the molecular targets and regulatory network that are potentially responsible for future enhancement of such cellular toxicity to tumors.
genes associated with ts independent cytotoxicity caused by 5-fu were listed in table  <dig>  this list includes genes such as rrm <dig>  sei <dig>  gpibb, ddb <dig>  and cdkn1a.
a recent report showed that rrm <dig>  was regulated at the translational level by the upstream augs  <cit> .
this finding provides further validation to our approach as a systematic discovery platform for novel translational regulated genes.
it has been shown recently that decreasing rrm <dig> level in hct- <dig>  cells sensitize cells to dna damaging agents and ribonucleotide reductase inhibitors  <cit> .
it is clear that multiple genes will be responsible for determining the sensitivity in response to 5-fu other than ts itself  <cit> .
these marker genes are important for us to understand the complex network regulated by ts and 5-fu.
to go a step further, we also attempted to analyze them in the context of the regulatory network.
as an example, we try to put these genes into a biological context via analyzing the cell cycle related genes affected by ts over-expression using pathway analysis, and the results are shown in figure  <dig>  it's quite clear that ts over-expression decreases p <dig> level, thereby affecting its downstream genes such as p <dig> and the rest of the cell cycle related genes.
this approach also helps to visualize the inter-relationship between ts and cell cycle related genes.
we believe this is a starting point to show the power of systems biology approach for dissecting the mechanism and network of gene regulation.
preliminary results using colon cancer patient samples demonstrated that some of these genes can be used as predictors for 5-fu based therapy .
CONCLUSIONS
in conclusion, the studies presented here provide a comprehensive expression profile of transcriptional and post-transcriptional levels affected by over-expression of ts protein and 5-fu treatment.
this work expands our current understanding of the complex networks regulated by ts protein.
in addition, our study also discovered candidate genes associated with ts independent cellular cytotoxicity.
in particular, the newly discovered post-transcriptionally regulated genes by ts and 5-fu will be good candidates for further investigating the molecular and cellular mechanism of such regulation.
this study further demonstrates the importance of understanding translational control in a global context in response to genotoxic stresses such as exposure to chemotherapeutic agents.

 thymidylate synthase is a folate-dependent enzyme that catalyzes the reductive methylation of dump by -methylenetetrahydrofolate to form dtmp and dihydrofolate. because the ts-catalyzed enzymatic reaction provides the sole intracellular de novo source of thymidylate, an essential precursor for dna biosynthesis, this enzyme has been an important target for cancer chemotherapy for the past years. ts is also one of the most extensively investigated biomarkers in recent years. in certain cases, ts has been shown to be a significant biomarker for predicting patient responses to 5-fu based therapy. however, in other studies, the expression level of ts alone is not sufficient for clinical prognosis. the goal of this study is to provide global comprehensive gene profiles and networks at multiple levels of gene regulation that are affected by endogenous levels of ts protein and 5-fu administration. this information will provide the basis to identity biomarker gene candidates that can be further validated using clinical samples for future clinical diagnosis and prognosis. the rational for investigating genes affected by ts and 5-fu at both transcriptional and post-transcriptional levels is that ts, in addition to its critical enzymatic function, functions as a rna binding protein. the translation of human ts mrna is regulated by its own protein product via a negative autoregulatory mechanism whereby the binding of ts protein to at least two distinct sequences on its own ts mrna results in translational repression. ts is also capable of interacting with several other cellular mrnas such as p mrna and c-myc mrna. previous studies demonstrated that ts protein regulates p gene expression at least in part, at the translational level. in this case, ts may be involved in coordinating the regulation of expression and/or function of cellular growth and proliferation and it is conceivable that ts may play an essential role as a regulator of cell cycle related events. more importantly, this study will have direct clinical relevance in that the mechanism of acute and long term 5-fu related drug resistance is distinct. the acute induction of ts expression after 5-fu treatment was regulated at the translational level and long term resistance for 5-fu is related to transcriptional activation and gene amplification of ts. thus, it would be particularly important to systematically investigate other potential post-transcriptional regulated genes via ts protein. this may be especially vital for the discovery of additional chemotherapeutic response related markers that otherwise would be missed by simply profiling steady state total mrnas. a recent report suggested that ts may also function as an oncogene to transform nih3t cells. a comprehensive gene expression profiling analysis may also gain new insights into signaling pathways that were deregulated by over-expression of ts. in this regard, it is critical to develop more comprehensive molecular expression profiles to provide candidate genes that can potentially be used for predicting clinical outcomes for colorectal cancer. in this study, a systems biology approach was used to investigate genes altered by the overexpression of ts at both transcriptional and post-transcriptional levels using human genome expression array in ts-depleted human colon cancer hct-c cells and hct-c cells stably transfected with the human ts cdna expression plasmid. in addition, both cell lines were treated with 5-fu for different time periods in an attempt to identify both acute response genes as well as delayed response genes affected by the 5-fu exposure. a number of genes were identified by comparing the expression profiles obtained from both steady state total mrnas and polysome associated mrna transcripts isolated from 5-fu treated hct-c and hct-c cells. comprehensive gene lists were generated that were relevant for both ts dependent and independent cytotoxicity by 5-fu exposure. we also discovered genes and pathways that may be involved for the oncogenic function of ts.  characterization of hct-c and hct-c cells the hct-c cell line contains a missense mutation at amino acid of the ts protein that results in a near complete inactivation of ts enzyme activity. hct-c cells were stably transfected with an expression construct containing full length human ts cdna. hct-c cells were able to grow without thymidine supplement in the media. however, hct-c cells require μm thymidine in rpmi medium. the doubling times of both cells were identical at hrs. previous study has demonstrated that the mechanism for the decreased p expression occurred at the translational level. we use p as a positive control gene for monitoring our high throughput expression profiling approach. our microarray gene expression analysis indicates that there is no difference in the expression level of p based on steady state total mrna. however, the expression level of actively translated p mrna in hct-c cells was found to decrease by -fold compared to the p mrna level in hct-c cells. this is consistent with our previous results that p was regulated at the translational level via ts. effect of 5-fu on the expression of ts and p53 the effect of 5-fu on the expression of both ts and p was investigated using western immunoblot analysis. hct-c cells were treated with μm 5-fu for hrs and hrs. ts protein expression was increased by nearly 4-fold at hrs and 10-fold at hrs. the ts-fdump-tetrahydrofolate covalent ternary complex was clearly visible after 5-fu treatment. the expression of wild type p was also increased in response to 5-fu treatment. effect of ts on steady state total mrnas expression the global gene expression profile of hct-c and hct-c cells based on steady state total mrna expression was analyzed using human high density codelink oligo array. although the growth characteristics of the two cell lines are similar in terms of doubling time, many genes are already affected by overexpressing ts protein. our analysis revealed that over genes were changed in their expression in response to ts expression. the partial gene list and the clustering analysis are shown in figure. genes such as centb and mt which are involved in cell proliferation and signaling, were up-regulated by ts. casp a gene involved in regulation of i-κb/nf-κb cascade, apoptosis and signal transduction, was decreased 22-fold by ts overexpression. effect of ts on polysome associated mrnas to discover novel genes post-transcriptionally regulated by ts protein, we isolated the polysome associated mrnas from both hct-c and hct-c cells and performed expression analysis using human high density codelink oligo array. over genes were effected in ts overexpressing hct-c cells. genes involved in protein biosynthesis and cell cycle control were up-regulated at the post-transcriptional level. the overlapping genes between steady state total mrna and polysome associated mrna profiles are listed in table steady state mrnas affected by 5-fu treatment in hct-c cells to identify acute and delayed response genes following 5-fu treatment in hct-c cells, gene expression profiling analysis was performed on steady state total mrnas isolated from control and 5-fu treated samples at hrs and hrs time points. the expression analysis reveals that over genes were affected by 5-fu treatment by one-way anova analysis. the clustering analysis is shown in figure. the expression analysis clearly showed a different gene expression profiles between hrs and hrs 5-fu exposure. based on the comparison of the expression profiles among the hct-c control and 5-fu treated samples, a number of genes were acutely increased in response to 5-fu. genes such as orcl, prps, ddx, dkk, and eif4e are some of the important genes affected by the acute 5-fu treatment based on our model. expression of some of the genes, such as sprr1a, ddb and cdkn1a, was only increased in response to 5-fu at hrs. polysome associated mrna profiles with 5-fu treatment in hct-c cells to identify acute and delayed response genes that were affected at post-transcriptional level by 5-fu treatment, polysome associated mrnas were isolated from control, and 5-fu treated samples at hrs and hrs. gene expression analysis via microarray revealed that over genes were affected in response to 5-fu. the clustering analyses are shown in figure. gene expression analysis revealed dynamic changes in expression at hrs and hrs of 5-fu treatment. by comparing the expression profiles between control hct-c and 5-fu treated samples, we discovered additional post-transcriptionally controlled genes that otherwise would be missed if the expression analysis was only performed using steady state total mrnas. genes such as nfic, srp, tsc, ireb, and cebpb are some of the genes that were regulated at the post-transcriptional level in response to 5-fu exposure with unique expression dynamics. genes associated with 5-fu induced ts independent cytotoxicity to identify genes associated with ts independent cytotoxicity affected by 5-fu treatment, hct-c cells were treated with μm 5-fu for hrs and hrs in the presence of μm thymidine. the presence of thymidine provides the essential precursor for dna biosynthesis for the hct-c cells thereby preventing the ts mediated cytotoxicity caused by 5-fu. in this case, the cytotoxicity of 5-fu in hct-c cells was a result of direct incorporation of 5-fu metabolites to dna and rna. based on the clustering analysis of steady state total mrna profiling of control hct-c cells and 5-fu treated samples in the presence of μm thymidine, the gene expression profiles generated from these samples are mainly associated with ts independent toxicity in response to 5-fu by direct incorporation to rna and dna. genes were affected. the clustering analysis is shown in figure. to further define the genes that were associated with ts independent toxicity, we compared the expression clusters from both hct-c and hct-c cells treated with 5-fu using venn diagram analysis. the overlapping gene list is shown in table genes such as rrm, cdkn1a, sei, ddb, sfn were associated with ts independent cytotoxicity to 5-fu treatment. gene expression analysis from figure was used for the construction of the cell cycle control pathway after 5-fu treatment illustrated in figure discussion in this study, a systems biology approach was used to analyze global gene expression and its networks affected by ts and 5-fu at multiple levels of control. to the best of our knowledge, this is the first report to address genes that are affected at both transcriptional and post-transcriptional levels affected by elevated ts protein expression and 5-fu treatment. we believe that this will provide a better understanding of complex network related to the multi-functions of ts protein as an rna binding protein or as a potential oncogene. it also provides a technology platform to systematically discover potential post-transcriptionally controlled genes by ts and 5-fu treatment. as our comparative model, we used a pair of human colon cancer cell lines: the mutant hct-c subline in which the ts protein had been rendered marginally active and the ts over-expressing subline, hct-c created by stable expression of a human ts cdna plasmid. both cell lines express wild-type p protein. p is one of the known genes affected by the over-expression of ts at the translational level due to its rna binding function. using our expression profiling approach, the levels of p mrna are not significantly changed based on steady state total mrna profile. however, the polysome associated p mrna was decreased by -fold in hct-c cells compared with hct-c cells. these results, taken together, confirmed that our comprehensive gene profiling analysis approach is capable of discovering new post-transcriptionally regulated genes. the gene profiling analysis based on steady state total mrna transcripts revealed over genes that were deregulated by ts over-expression. although this study does not provide any direct evidence for ts as a cell cycle regulator, a number of cell cycle control and apoptotic control genes were perturbed by ts over-expression. one of the genes is inhibin/activin a that was up-regulated by nearly 22-fold in hct-c cells. inhba is a growth factor that is involved in cell proliferation. inhba does this through type i and type ii receptor serine kinases. this may be a potential pathway by which ts is able to influence the cellular transformation process. in fact, it has been reported recently that inhba is over-expressed in stage iv colorectal cancer. additionally, single stranded rna interacting protein, a gene responsible for mrna splicing, was also up-regulated by 28-fold. the gene is a member of a small family of proteins which bind single stranded dna/rna. these proteins are characterized by the presence of two sets of ribonucleoprotein consensus sequence that contain conserved motifs, rnp and rnp originally described in rna binding proteins, and required for dna binding. these proteins have been implicated in such diverse functions as dna replication, gene transcription, cell cycle progression and apoptosis. the encoded protein was isolated by virtue of its binding to an upstream element of the alpha collagen promoter. the observation that this protein localizes mostly in the cytoplasm suggests that it may be involved in a cytoplasmic function such as controlling rna metabolism, rather than transcription. thus, rbms may play an important role as part of the post-transcriptional control mediated by ts. conversely, genes such as caspase that control cell cycle arrest and apoptosis, were down-regulated some 22-fold. hence, future studies will focus on determining whether ts protein regulates the expression of these genes directly by binding to the mrnas or indirectly through downstream mechanisms. using our systems biology approach of polysome associated mrna comparisons, we have identified a list of genes that are over- or under-expressed due to ts protein over-expression. many of them play key roles in cell-cell signalling, protein biosynthesis, rna processing, dna repair, cell cycle control and translational regulation. some of them are previously known translational controlled genes such as egfr, one of the key targets for the latest targeted anticancer drug development for colon cancer treatment. in this study, the expression of egfr was found to increase by nearly 8-fold by ts over-expression. many other genes on this list are novel potential post-transcriptionally regulated genes. among the down regulated genes, cyclin dependent kinase inhibitor p was decreased by over 5-fold. apoptosis inhibitor bak was also decreased by 6-fold in ts over-expressing cells. it seems that over expressing ts has set the stage for cells to have many growth advantages using multiple mechanisms. to identify genes regulated at both transcriptional and post-transcriptional level, we performed an overlapping analysis by comparing the gene lists generated from steady state total mrnas and polysome associated mrnas using venn diagram. there are overlapping genes between the two lists. d, up-regulated by 5-fold in hct-c cells, has been shown to be regulated at the translational level via eif2γ. we have identified an additional genes based on polysome associated mrnas which could be easily missed if only steady state mrna profiling is performed. this approach provides a technical platform to systematically analyzing gene expression at post-transcriptional level. it also reveals the importance of analyze gene expression at multiple levels. based on the clinical observation and detailed molecular investigations, ts can be acutely induced with a short time 5-fu exposure and the mechanism of ts induction was regulated, at least in part, at the translational level. with this notion, we designed a strategy that allows us to capture both acute and delayed response genes in response to 5-fu exposure. to achieve this, both hct-c and hct-c cells were treated with μm 5-fu for hrs and hrs. figures and listed genes with known functions based on one-way anova clustering analysis from steady state total mrna profiling from each cell line, respectively. a clear and unique expression pattern revealed a dynamic response to 5-fu treatment. there are genes that were proved to be significant factors for acute 5-fu treatment. they include cxcr flj rsp akap orc6l, prps ddx dkk and eif4e. dkk functions as an agonist in the wnt signaling pathway to stimulate cell proliferation during brain development. however, little is known about the function of dkk in cancer. this is the first report to show that dkk may play an important role in colorectal cancer possibly via the wnt signaling pathway. eif4e, one of the key translational initiation factors, has been shown to play key roles in cell cycle control and is an important marker for determining chemosensitivity. we speculate that the acute induction of eif4e expression by 5-fu may be contributing to the resistance mechanism. following hr 5-fu exposure, nearly all of the acutely up-regulated genes had returned to baseline levels. however, additional genes such as sprr1a, ddb and cdkn1a were only up-regulated after hrs. these differential gene expression profiles suggest that single time point microarray data from clinical samples should be viewed with caution and not over-interpreted. interestingly, we discovered an additional genes associated with for 5-fu treatment based on profiling of polysome associated mrna transcripts. the induction of p will contribute a significant part for cell cycle arrest and apoptosis based on previous reports. in addition, we also found genes such as, g-protein coupled receptor, colon cancer antigen, tsc and p, were perturbed with 5-fu exposure. sei is a ckd4-binding protein to regulate cdk activity and was up-regulated following 5-fu exposure. g2a and sdccag were acutely up-regulated by 5-fu treatment whereas tsc was a delayed response gene affected by 5-fu. it has been recently shown that tsc is regulated at the translational level by hypoxia. tsc is also involved in mtor signaling pathway to regulate cell growth, proliferation, and cell death. mtor is one of the key pathways for translational regulation via eif4e. these gene expression profiles may be helpful in elucidating additional resistance mechanisms related to 5-fu treatment. we also determined the marker genes that were associated with ts independent cytotoxicity using hct-c cells treated with 5-fu in the presence of μm thymidine. the main cytotoxicity caused by 5-fu was the inhibition of ts to block the sole de novo synthesis of thymidylate, an essential precursor for dna biosynthesis. however, in the presence of μm thymidine, the cytotoxicity caused by 5-fu in hct-c cells was mainly by the direct incorporation of 5-fu metabolite into dna and rna. the genes involved in ts independent cellular toxicity have not been systematically investigated in the past. in this study, we discovered a number of genes that were associated with ts independent cytotoxicity of 5-fu. genes involved in dna replication, dna repair, dna metabolism, rna catabolism, rna processing, cell signaling, and apoptosis are on this list. it's not surprising to see these genes on the list because direct incorporation of 5-fu metabolite to rna and dna is the major part of cytotoxicity via ts independent mechanism. it has been shown that incorporation of 5-fu into rna affects pre-mrna splicing process. this study provides the molecular targets and regulatory network that are potentially responsible for future enhancement of such cellular toxicity to tumors. genes associated with ts independent cytotoxicity caused by 5-fu were listed in table this list includes genes such as rrm sei gpibb, ddb and cdkn1a. a recent report showed that rrm was regulated at the translational level by the upstream augs. this finding provides further validation to our approach as a systematic discovery platform for novel translational regulated genes. it has been shown recently that decreasing rrm level in hct- cells sensitize cells to dna damaging agents and ribonucleotide reductase inhibitors. it is clear that multiple genes will be responsible for determining the sensitivity in response to 5-fu other than ts itself. these marker genes are important for us to understand the complex network regulated by ts and 5-fu. to go a step further, we also attempted to analyze them in the context of the regulatory network. as an example, we try to put these genes into a biological context via analyzing the cell cycle related genes affected by ts over-expression using pathway analysis, and the results are shown in figure it's quite clear that ts over-expression decreases p level, thereby affecting its downstream genes such as p and the rest of the cell cycle related genes. this approach also helps to visualize the inter-relationship between ts and cell cycle related genes. we believe this is a starting point to show the power of systems biology approach for dissecting the mechanism and network of gene regulation. preliminary results using colon cancer patient samples demonstrated that some of these genes can be used as predictors for 5-fu based therapy.  in conclusion, the studies presented here provide a comprehensive expression profile of transcriptional and post-transcriptional levels affected by over-expression of ts protein and 5-fu treatment. this work expands our current understanding of the complex networks regulated by ts protein. in addition, our study also discovered candidate genes associated with ts independent cellular cytotoxicity. in particular, the newly discovered post-transcriptionally regulated genes by ts and 5-fu will be good candidates for further investigating the molecular and cellular mechanism of such regulation. this study further demonstrates the importance of understanding translational control in a global context in response to genotoxic stresses such as exposure to chemotherapeutic agents. 
in this study, a comprehensive gene expression analysis at the levels of both transcriptional and post-transcriptional regulation was conducted to identify response markers using human genome array with ts-depleted human colon cancer hct-c cells and hct-c cells stably transfected with the human ts cdna expression plasmid. thymidylate synthase is a critical target for cancer chemotherapy and is one of the most extensively studied biomarkers for fluoropyrimidine-based chemotherapy. this study will provide candidate markers that can be potentially used for predicting therapeutic outcomes for fluoropyrimidine-based cancer chemotherapy. in addition to its critical role in enzyme catalysis, ts functions as an rna binding protein to regulate the expression of its own mrna translation and other cellular mrnas, such as p at the translational level. however, based on the expression profiles of polysome associated mrna transcripts, over genes were affected by ts overexpression. a total of genes were found to be significantly affected by ts based on the expression profiles of steady state mrna transcripts. to our knowledge, this is the first time that a comprehensive gene expression profile regulated by ts and 5-fu was analyzed at the multiple steps of gene regulation.
 thymidylate synthase is a critical target for cancer chemotherapy and is one of the most extensively studied biomarkers for fluoropyrimidine-based chemotherapy. in addition to its critical role in enzyme catalysis, ts functions as an rna binding protein to regulate the expression of its own mrna translation and other cellular mrnas, such as p at the translational level. in this study, a comprehensive gene expression analysis at the levels of both transcriptional and post-transcriptional regulation was conducted to identify response markers using human genome array with ts-depleted human colon cancer hct-c cells and hct-c cells stably transfected with the human ts cdna expression plasmid.  a total of genes were found to be significantly affected by ts based on the expression profiles of steady state mrna transcripts. however, based on the expression profiles of polysome associated mrna transcripts, over genes were affected by ts overexpression. this indicates that additional post-transcriptionally controlled genes can be captured with profiling polysome associated mrna population. this unique approach provides a comprehensive overview of genes affected by ts. additional novel post-transcriptionally regulated genes affected by 5-fluorouracil treatment were also discovered via similar approach.  to our knowledge, this is the first time that a comprehensive gene expression profile regulated by ts and 5-fu was analyzed at the multiple steps of gene regulation. this study will provide candidate markers that can be potentially used for predicting therapeutic outcomes for fluoropyrimidine-based cancer chemotherapy. 