Dynamic Modulation of Thymidylate Synthase Gene Expression and Fluorouracil Sensitivity in Human Colorectal Cancer Cells

<div><p>Biomarkers have revolutionized cancer chemotherapy. However, many biomarker candidates are still in debate. In addition to clinical studies, <i>a priori</i> experimental approaches are needed. Thymidylate synthase (TS) expression is a long-standing candidate as a biomarker for 5-fluorouracil (5-FU) treatment of cancer patients. Using the Tet-OFF system and a human colorectal cancer cell line, DLD-1, we first constructed an <i>in vitro</i> system in which TS expression is dynamically controllable. Quantitative assays have elucidated that TS expression in the transformant was widely modulated, and that the dynamic range covered 15-fold of the basal level. 5-FU sensitivity of the transformant cells significantly increased in response to downregulated TS expression, although being not examined in the full dynamic range because of the doxycycline toxicity. Intriguingly, our <i>in vitro</i> data suggest that there is a linear relationship between TS expression and the 5-FU sensitivity in cells. Data obtained in a mouse model using transformant xenografts were highly parallel to those obtained <i>in vitro</i>. Thus, our <i>in vitro</i> and <i>in vivo</i> observations suggest that TS expression is a determinant of 5-FU sensitivity in cells, at least in this specific genetic background, and, therefore, support the possibility of TS expression as a biomarker for 5-FU-based cancer chemotherapy.</p></div

5-FU sensitivity of TFTS66 cells <i>in vitro</i>.

<p><b>A.</b> Effects of 5-FU on the cell cycle of TFTS66 and parental DLD-1 cells. Exponentially growing cells were treated with 5-FU concentrations indicated and subjected to flowcytometry. Fluorescence histograms are shown. <b>B.</b> The design of the <i>in vitro</i> colony formation assays is shown. Fifty thousand TFTS66 and TFC7 cells per dish were grown under the Dox concentrations indicated and treated with the indicated concentrations of 5-FU for 72 h. At Day 10, colonies were counted. Throughout the experiments, cells were maintained in media containing HygB and G418. Each experiment was triplicated. <b>C.</b> Survival curves of TFTS66 and TCF7 cells exposed to 5-FU. The survival fractions were calculated as a percentage of the untreated (<i>i</i>.<i>e</i>. 0 μM 5-FU) control, and the mean values are plotted against the 5-FU concentration: rectangle, TFTS66; circle, TFC7. The symbols are shaded according to the Dox concentrations. <b>D.</b> The IC50 value in each group was determined as the 5-FU concentration corresponding to 50% survival in the linearized survival curves. Standard error bars are shown at both ends of the linearized survival curves. <b>E.</b> The obtained IC50 values are plotted as a function of the TS expression level determined by immunoblotting (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123076#pone.0123076.g002" target="_blank">Fig 2B</a>): rectangle, TFTS66; circle, TFC7. The symbols are similarly shaded according to the Dox concentrations.</p

Anti-tumor effects of 5-FU in mice carrying TFTS66 cell xenografts.

<p>^a Tegafur, an oral prodrug of 5-FU, was administered as a combined formulation with CDHP and oxonate. 5-FU doses are expressed as those of tegafur.</p><p>^b Difference to the control, group A, was examined by Dunnett's test.</p><p>^c Difference between two 5-FU-administered groups (group E vs. C and F vs. D) was examined by Student's t-test.</p><p>^d Tumor growth inhibition (TGI) was calculated according to the following formula: TGI [%] = [(mean tumor weight (TW) of the control group)—(mean TW of the treated group)] / (mean TW of the control group) × 100.</p><p>^e Body weight change (BWC) on day 15 were calculated according to the following formula: BWC [%] = [(BW on day 15)—(BW on day 0)] / (BW on day 0) × 100.</p><p>Anti-tumor effects of 5-FU in mice carrying TFTS66 cell xenografts.</p

Dox effects on gene expression in TFTS66 cells.

<p><b>A.</b> Microarray data. The expression profiles were compared between the steady state (Dox0) of TFTS66 versus the parental line, DLD-1 (left panel) and between Dox0.5 versus Dox0 in TFTS66 (right panel). Data are shown as scatter plots, and those corresponding to genes of particular interest are indicated by arrows. Red dashed lines represent the log2 fold change. <b>B.</b> The absolute values of the <i>TYMS</i> RNA level were extracted from the microarray data and are plotted against the Dox concentration, in parallel with the TS protein level determined by immunoblotting (left panel). The TS protein levels are then plotted as a function of the RNA level (right panel): open rectangle, Dox0; shaded rectangle, Dox0.5; closed rectangle, Dox1.0.</p

Transformation strategy.

<p><b>A.</b> The original Kozak-like motif in the human <i>TYMS</i> cDNA was modified to the Kozak consensus sequence using partially complementary PCR primers (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123076#sec002" target="_blank">Materials and methods</a>). <b>B.</b> The Kozak-modified <i>TYMS</i> cDNA, TSCD3, was amplified by PCR and subcloned into the cDNA expression vector, pTRE2hyg. <b>C.</b> pTRE2hyg-TS3 or an empty vector was co-transfected with pTet-ON/OFF vectors into a human colorectal cancer cell line, DLD-1. After serial selections, clones resistant to both HygB and G418 were isolated. TS expression in these clones was then examined by immunoblotting.</p

Dox-dependent TS expression in TFTS66 cells.

<p><b>A.</b> TS antigens in cell lysates of TFTS66 transformant were detected by immunoblotting using anti-human TS mouse monoclonal antibody. The standard cell lysates (0 ng/ml Dox) were titrated, and a standard curve for detection was obtained from the signal intensity on the digitized image (upper panel). Using the highly linear detection characteristics (p = 0.997), TS expression levels were quantified: rectangle, TFTS66; circle, the control transformant, TFC7. <b>B.</b> TS expression in TFTS66 cells exposed to various concentrations of Dox was assessed by immunoblotting and similarly quantified. The symbols are shaded according to the Dox concentrations. Some of the data points are also shown in Fig 2A. <b>C.</b> The quantity of TS protein (TS_total, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123076#sec002" target="_blank">Materials and methods</a>) and its catalytic activity were enzymatically assayed in lysates prepared from TFTS66 cells exposed to 0, 0.5 and 1.0 ng/ml Dox. The results are plotted as a function of the TS expression level determined by immunoblotting: open rectangle, Dox0; shaded rectangle, Dox0.5; closed rectangle, Dox1.0. <b>D.</b> TS expression in TFTS66 cells was observed using fluorescent immunocytochemistry. Cells grown on chamber slides were fixed and reacted with TS-specific antibody. Cellular distribution of TS antigens was visualized by red fluorescent signals. Cells were also counterstained with Hoechst 33342. Results obtained in TFTS66 (0, 0.1 and 1.0 ng/ml Dox) and its parental line, DLD-1, are shown (magnification X100).</p

(A) Disease specific survival curves of all patients (n = 102) according to the chemoradioselection.

<p>(B) Disease specific survival curves based on the CD44 v9 positivity of biopsy samples (n = 60) obtained from 30 chemoradioselected (CRS) patients and 30 non-chemoradioselected (N-CRS) patients. (C) Disease specific survival curves based on the CD44 v9 positivity of biopsy samples obtained from 30 N-CRS patients.</p

(A) Disease specific survival curves based on the CD44 v9 positivity of surgically removed samples obtained from 72 non-chemoradioselected (N-CRS) patients.

<p>(B) Diseasespecific survival curves of 30 N-CRS patients who had paired biopsy and surgically removed samples. The patients were divided into 2 groups according to their levels of CD44v9 expression before and after concurrent chemoradiotherapy.</p

Patient characteristics (<i>N</i> = 102).

<p>Patient characteristics (<i>N</i> = 102).</p

Proposed roles of CD44v9-expressing CSC and non-CSC in the chemoradioselection.

<p>(A) CD44v9-expressing non-CSCs are sensitive to CCRT. Intrinsic CD44v9-expressing CSCs (B) or CCRT-induced CD44v9-expressing CSCs (C) can survive CCRT. These CD44v9-expressing CSCs are considered to be highly invasive and metastatic. CSC, cancer stem cell; CCRT, concurrent chemoradiotherapy; CRS, chemoradioselected; and N-CRS, non-chemoradioselected.</p