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Dynamic modulation of thymidylate synthase gene expression and fluorouracil sensitivity in human colorectal cancer cells.

Wakasa K, Kawabata R, Nakao S, Hattori H, Taguchi K, Uchida J, Yamanaka T, Maehara Y, Fukushima M, Oda S - PLoS ONE (2015)

Bottom Line: 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 in vitro 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 in vitro.

View Article: PubMed Central - PubMed

Affiliation: Clinical Research Institute, National Kyushu Cancer Center, Fukuoka, Japan.

ABSTRACT
Biomarkers have revolutionized cancer chemotherapy. However, many biomarker candidates are still in debate. In addition to clinical studies, a priori 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 in vitro 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 in vitro 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 in vitro. Thus, our in vitro and in vivo 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.

No MeSH data available.


Related in: MedlinePlus

5-FU sensitivity of TFTS66 cells in vitro.A. 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. The design of the in vitro 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. C. Survival curves of TFTS66 and TCF7 cells exposed to 5-FU. The survival fractions were calculated as a percentage of the untreated (i.e. 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. D. 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. E. The obtained IC50 values are plotted as a function of the TS expression level determined by immunoblotting (see Fig 2B): rectangle, TFTS66; circle, TFC7. The symbols are similarly shaded according to the Dox concentrations.
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pone.0123076.g004: 5-FU sensitivity of TFTS66 cells in vitro.A. 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. The design of the in vitro 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. C. Survival curves of TFTS66 and TCF7 cells exposed to 5-FU. The survival fractions were calculated as a percentage of the untreated (i.e. 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. D. 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. E. The obtained IC50 values are plotted as a function of the TS expression level determined by immunoblotting (see Fig 2B): rectangle, TFTS66; circle, TFC7. The symbols are similarly shaded according to the Dox concentrations.

Mentions: The sensitivity of TFTS66 cells to 5-FU was first examined using flowcytometry. It has been known that 5-FU treatment causes a marked accumulation of S-phase cells in cell populations sensitive to this agent [17]. We therefore treated TSTF66 and parental DLD-1 cells with different concentrations of 5-FU and analyzed them (Fig 4A). In DLD-1 cells, S phase cells accumulated according to the 5-FU concentrations, as expected. On the other hand, this phenomenon was not evident in TFTS66 cells, and the flowcytometric profiles were not largely different among the concentrations used, which indirectly suggests that TFTS66 cells are more resistant to 5-FU than DLD-1 cells. Next, we assessed the 5-FU sensitivity of TFTS66 cells by colony formation assays. TFTS66 and TCF7 cells, exposed to different concentrations of Dox, were treated with various concentrations of 5-FU for 72 h and plated (Fig 4B). Four days after the removal of 5-FU from the media, formed colonies were counted. Survival curves were plotted using mean values (Fig 4C). Each data point had a relatively small standard error (see Fig 4D). The survival of TFTS66 cells was apparently better than that of TFC7 cells, and, best in the Dox0 state. The survival of TFC7 cells did not differ, irrespective of the Dox concentration, which does imply that there were no synergistic effects between Dox and 5-FU. Remarkably, in the range above 0.1 ng/ml of Dox, the survival curves of TFTS66 cells were not largely different, although there was an evident difference between Dox0 and Dox0.05. Indeed, the change in TS expression is most dynamic between Dox0 and Dox0.05. However, the difference between Dox0.1 and Dox1 is also evident (see Fig 2B). Despite of the dynamic changes in TS expression, the survival of TFTS66 cells exposed to the high concentrations of Dox was invariable, which may be caused by the Dox-induced drug resistance suggested above. The effects of 5-FU might have been attenuated due to the induction of genes activating cellular transport, detoxification or those inhibiting cell death (see S1 Table). Therefore, we further examined the data of TFTS66 cells at Dox0, 0.05 and 0.1 and those of TFC7 cells. The IC50, the 5-FU concentration that corresponds to 50% survival, was determined from the linearized survival curves crossing 50% survival (Fig 4D), and plotted in a two-dimensional diagram of IC50 versus TS expression (Fig 4E). Intriguingly, the three data points, TFTS66 at Dox 0 and Dox0.05 and TFC7 at Dox0/0.1 (averaged), were on a straight line (R2 = 0.998). In other words, there was a highly linear relationship between TS expression and IC50 for 5-FU. The data point for TFTS66 cells at Dox0.1 was also plotted on the same diagram. However, because IC50 was at the same level between Dox0.1 and Dox0.05, this data point was not on the line, which again confirms that the survival of TFTS66 cells is invariable at the concentrations above 0.1ng/ml of Dox. It has thus been shown that in the TFTS66 transformant cellular sensitivity to 5-FU changes accordingly when TS expression level is modulated, although 5-FU sensitivity in TFTS66 cells expressing low levels of TS was not fully evaluated due to the limits of the system. These in vitro findings overall suggest that TS expression is a determinant of 5-FU sensitivity, at least in this particular genetic background.


Dynamic modulation of thymidylate synthase gene expression and fluorouracil sensitivity in human colorectal cancer cells.

Wakasa K, Kawabata R, Nakao S, Hattori H, Taguchi K, Uchida J, Yamanaka T, Maehara Y, Fukushima M, Oda S - PLoS ONE (2015)

5-FU sensitivity of TFTS66 cells in vitro.A. 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. The design of the in vitro 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. C. Survival curves of TFTS66 and TCF7 cells exposed to 5-FU. The survival fractions were calculated as a percentage of the untreated (i.e. 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. D. 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. E. The obtained IC50 values are plotted as a function of the TS expression level determined by immunoblotting (see Fig 2B): rectangle, TFTS66; circle, TFC7. The symbols are similarly shaded according to the Dox concentrations.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4400010&req=5

pone.0123076.g004: 5-FU sensitivity of TFTS66 cells in vitro.A. 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. The design of the in vitro 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. C. Survival curves of TFTS66 and TCF7 cells exposed to 5-FU. The survival fractions were calculated as a percentage of the untreated (i.e. 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. D. 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. E. The obtained IC50 values are plotted as a function of the TS expression level determined by immunoblotting (see Fig 2B): rectangle, TFTS66; circle, TFC7. The symbols are similarly shaded according to the Dox concentrations.
Mentions: The sensitivity of TFTS66 cells to 5-FU was first examined using flowcytometry. It has been known that 5-FU treatment causes a marked accumulation of S-phase cells in cell populations sensitive to this agent [17]. We therefore treated TSTF66 and parental DLD-1 cells with different concentrations of 5-FU and analyzed them (Fig 4A). In DLD-1 cells, S phase cells accumulated according to the 5-FU concentrations, as expected. On the other hand, this phenomenon was not evident in TFTS66 cells, and the flowcytometric profiles were not largely different among the concentrations used, which indirectly suggests that TFTS66 cells are more resistant to 5-FU than DLD-1 cells. Next, we assessed the 5-FU sensitivity of TFTS66 cells by colony formation assays. TFTS66 and TCF7 cells, exposed to different concentrations of Dox, were treated with various concentrations of 5-FU for 72 h and plated (Fig 4B). Four days after the removal of 5-FU from the media, formed colonies were counted. Survival curves were plotted using mean values (Fig 4C). Each data point had a relatively small standard error (see Fig 4D). The survival of TFTS66 cells was apparently better than that of TFC7 cells, and, best in the Dox0 state. The survival of TFC7 cells did not differ, irrespective of the Dox concentration, which does imply that there were no synergistic effects between Dox and 5-FU. Remarkably, in the range above 0.1 ng/ml of Dox, the survival curves of TFTS66 cells were not largely different, although there was an evident difference between Dox0 and Dox0.05. Indeed, the change in TS expression is most dynamic between Dox0 and Dox0.05. However, the difference between Dox0.1 and Dox1 is also evident (see Fig 2B). Despite of the dynamic changes in TS expression, the survival of TFTS66 cells exposed to the high concentrations of Dox was invariable, which may be caused by the Dox-induced drug resistance suggested above. The effects of 5-FU might have been attenuated due to the induction of genes activating cellular transport, detoxification or those inhibiting cell death (see S1 Table). Therefore, we further examined the data of TFTS66 cells at Dox0, 0.05 and 0.1 and those of TFC7 cells. The IC50, the 5-FU concentration that corresponds to 50% survival, was determined from the linearized survival curves crossing 50% survival (Fig 4D), and plotted in a two-dimensional diagram of IC50 versus TS expression (Fig 4E). Intriguingly, the three data points, TFTS66 at Dox 0 and Dox0.05 and TFC7 at Dox0/0.1 (averaged), were on a straight line (R2 = 0.998). In other words, there was a highly linear relationship between TS expression and IC50 for 5-FU. The data point for TFTS66 cells at Dox0.1 was also plotted on the same diagram. However, because IC50 was at the same level between Dox0.1 and Dox0.05, this data point was not on the line, which again confirms that the survival of TFTS66 cells is invariable at the concentrations above 0.1ng/ml of Dox. It has thus been shown that in the TFTS66 transformant cellular sensitivity to 5-FU changes accordingly when TS expression level is modulated, although 5-FU sensitivity in TFTS66 cells expressing low levels of TS was not fully evaluated due to the limits of the system. These in vitro findings overall suggest that TS expression is a determinant of 5-FU sensitivity, at least in this particular genetic background.

Bottom Line: 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 in vitro 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 in vitro.

View Article: PubMed Central - PubMed

Affiliation: Clinical Research Institute, National Kyushu Cancer Center, Fukuoka, Japan.

ABSTRACT
Biomarkers have revolutionized cancer chemotherapy. However, many biomarker candidates are still in debate. In addition to clinical studies, a priori 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 in vitro 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 in vitro 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 in vitro. Thus, our in vitro and in vivo 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.

No MeSH data available.


Related in: MedlinePlus