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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

TFTS66 cell xenografts in nude mice.A. The design of the in vivo assays is shown. Seven-week old nude mice were randomly assigned to six groups. TFTS66 cells were subcutaneously inoculated in each animal. Dox and 5-FU were administered to the animals, intraperitoneally and orally, respectively. 5-FU doses are expressed as those of tegafur. TS binding assay and immunohistochemistry were done at day 4 (n = 4, group A and B) and day 8 (n = 8). At day 15, the weight of the tumor xenografts was measured. B. Body weight was measured twice a week, and the changes are shown as means with standard errors. C. The quantity of TS protein (TSfree, see Materials and methods) in the xenograft tissues was determined by TS binding assay. Means with standard errors are shown, and significant differences are indicated with asterisks: *, p<0.05; **, p<0.01. D. Immunohistochemistry using antibody against TS. TS antigens were visualized as brown staining. Representative results are shown (scale bar: 400 μm). 5-FU doses are expressed as those of tegafur.
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pone.0123076.g005: TFTS66 cell xenografts in nude mice.A. The design of the in vivo assays is shown. Seven-week old nude mice were randomly assigned to six groups. TFTS66 cells were subcutaneously inoculated in each animal. Dox and 5-FU were administered to the animals, intraperitoneally and orally, respectively. 5-FU doses are expressed as those of tegafur. TS binding assay and immunohistochemistry were done at day 4 (n = 4, group A and B) and day 8 (n = 8). At day 15, the weight of the tumor xenografts was measured. B. Body weight was measured twice a week, and the changes are shown as means with standard errors. C. The quantity of TS protein (TSfree, see Materials and methods) in the xenograft tissues was determined by TS binding assay. Means with standard errors are shown, and significant differences are indicated with asterisks: *, p<0.05; **, p<0.01. D. Immunohistochemistry using antibody against TS. TS antigens were visualized as brown staining. Representative results are shown (scale bar: 400 μm). 5-FU doses are expressed as those of tegafur.

Mentions: The antitumor effects of 5-FU were thus compared in TFTS66 cell xenografts that expressed TS at a very high level and those with an extremely low TS expression (Fig 5A). 5-FU was orally administered as a combined formulation of tegafur (an oral prodrug of 5-FU), CDHP and oxonate, which is now known as ‘S-1’ [9] and widely used for treatment of various human cancers. The two 5-FU doses (expressed as those of tegafur), 8.3 and 10.0 mg/kg/day, were chosen. Eight or twelve animals were assigned to each of the six groups. Although the experiments were well tolerated by most of the animals, weight loss was evident, particularly in those administered with Dox (Fig 5B), and, in those administered with Dox and 5-FU 10.0 mg/kg/day, three animals succumbed (group F, Table 1). TS expression, assessed by TS binding assay (Fig 5C), was well under control throughout the experiments. TS expression was suppressed to a very low level in the animals administered with Dox, whereas being maintained at relatively high levels in those without the Dox treatment, although the variance was not small in the latter groups at Day 15 (Fig 5C). The results of immunohistochemistry confirmed these findings, and intratumoral heterogeneity was observed (Fig 5D). Finally, the tumor weight in the animals of each group was measured and compared (Table 1). Importantly, Dox itself exhibited no significant growth inhibitory effects on tumor xenografts (compare Group A and B in Table 1), which is highly parallel to our in vitro data (compare Dox0 and 0.1/TFC7 in Fig 4). The effects of 5-FU was evident in Group E comprising animals administered with Dox and 5-FU 8.3 mg/kg/day. A significant inhibition of tumor growth was observed in the animals of this group, compared to those administered with only 8.3 mg/kg/day of 5-FU (Group C) (p = 0.008). Tumor growth was markedly inhibited also in Group F, which was, however, statistically not significant (p = 0.28), presumably due to the limited number of animals. Thus, using TSTF66 cell xenografts, we confirmed that cells expressing TS at a high level are more resistant to 5-FU than those with low TS expression also in vivo. These in vitro and in vivo observations are highly parallel and clearly suggest that TS expression is a determinant of 5-FU sensitivity in cells, at least in this specific 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)

TFTS66 cell xenografts in nude mice.A. The design of the in vivo assays is shown. Seven-week old nude mice were randomly assigned to six groups. TFTS66 cells were subcutaneously inoculated in each animal. Dox and 5-FU were administered to the animals, intraperitoneally and orally, respectively. 5-FU doses are expressed as those of tegafur. TS binding assay and immunohistochemistry were done at day 4 (n = 4, group A and B) and day 8 (n = 8). At day 15, the weight of the tumor xenografts was measured. B. Body weight was measured twice a week, and the changes are shown as means with standard errors. C. The quantity of TS protein (TSfree, see Materials and methods) in the xenograft tissues was determined by TS binding assay. Means with standard errors are shown, and significant differences are indicated with asterisks: *, p<0.05; **, p<0.01. D. Immunohistochemistry using antibody against TS. TS antigens were visualized as brown staining. Representative results are shown (scale bar: 400 μm). 5-FU doses are expressed as those of tegafur.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4400010&req=5

pone.0123076.g005: TFTS66 cell xenografts in nude mice.A. The design of the in vivo assays is shown. Seven-week old nude mice were randomly assigned to six groups. TFTS66 cells were subcutaneously inoculated in each animal. Dox and 5-FU were administered to the animals, intraperitoneally and orally, respectively. 5-FU doses are expressed as those of tegafur. TS binding assay and immunohistochemistry were done at day 4 (n = 4, group A and B) and day 8 (n = 8). At day 15, the weight of the tumor xenografts was measured. B. Body weight was measured twice a week, and the changes are shown as means with standard errors. C. The quantity of TS protein (TSfree, see Materials and methods) in the xenograft tissues was determined by TS binding assay. Means with standard errors are shown, and significant differences are indicated with asterisks: *, p<0.05; **, p<0.01. D. Immunohistochemistry using antibody against TS. TS antigens were visualized as brown staining. Representative results are shown (scale bar: 400 μm). 5-FU doses are expressed as those of tegafur.
Mentions: The antitumor effects of 5-FU were thus compared in TFTS66 cell xenografts that expressed TS at a very high level and those with an extremely low TS expression (Fig 5A). 5-FU was orally administered as a combined formulation of tegafur (an oral prodrug of 5-FU), CDHP and oxonate, which is now known as ‘S-1’ [9] and widely used for treatment of various human cancers. The two 5-FU doses (expressed as those of tegafur), 8.3 and 10.0 mg/kg/day, were chosen. Eight or twelve animals were assigned to each of the six groups. Although the experiments were well tolerated by most of the animals, weight loss was evident, particularly in those administered with Dox (Fig 5B), and, in those administered with Dox and 5-FU 10.0 mg/kg/day, three animals succumbed (group F, Table 1). TS expression, assessed by TS binding assay (Fig 5C), was well under control throughout the experiments. TS expression was suppressed to a very low level in the animals administered with Dox, whereas being maintained at relatively high levels in those without the Dox treatment, although the variance was not small in the latter groups at Day 15 (Fig 5C). The results of immunohistochemistry confirmed these findings, and intratumoral heterogeneity was observed (Fig 5D). Finally, the tumor weight in the animals of each group was measured and compared (Table 1). Importantly, Dox itself exhibited no significant growth inhibitory effects on tumor xenografts (compare Group A and B in Table 1), which is highly parallel to our in vitro data (compare Dox0 and 0.1/TFC7 in Fig 4). The effects of 5-FU was evident in Group E comprising animals administered with Dox and 5-FU 8.3 mg/kg/day. A significant inhibition of tumor growth was observed in the animals of this group, compared to those administered with only 8.3 mg/kg/day of 5-FU (Group C) (p = 0.008). Tumor growth was markedly inhibited also in Group F, which was, however, statistically not significant (p = 0.28), presumably due to the limited number of animals. Thus, using TSTF66 cell xenografts, we confirmed that cells expressing TS at a high level are more resistant to 5-FU than those with low TS expression also in vivo. These in vitro and in vivo observations are highly parallel and clearly suggest that TS expression is a determinant of 5-FU sensitivity in cells, at least in this specific 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