Limits...
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

Dox-dependent TS expression in TFTS66 cells.A. 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. 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. C. The quantity of TS protein (TStotal, see Materials and methods) 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. D. 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).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4400010&req=5

pone.0123076.g002: Dox-dependent TS expression in TFTS66 cells.A. 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. 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. C. The quantity of TS protein (TStotal, see Materials and methods) 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. D. 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).

Mentions: Cells were washed twice with 0.02% EDTA in phosphate-buffered saline (PBS), pelleted and kept at –80°C until use. Cell pellets were lysed in 2X Laemmli's sodium dodecyl sulphate (SDS) sample buffer [12], sonicated and then cooled on ice. After centrifugation at14,000 x g for 30 min at 4°C, supernatants were collected. Lysates corresponding to 50 μg were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and separated proteins were electrotransferred onto nitrocellulose membranes, BA83 (GE Healthcare Bio-Science Corp., Piscataway, NJ, USA), using Transblot SD (Bio-Rad Laboratories, Hercules, CA, USA). After blocking with 1X TBS (10 mM Tris-HCl; pH 7.4, 0.9% NaCl) solution including 5% bovine serum albumin (BSA) and 0.05% Tween 20 at 52°C for 1 h, the membranes were reacted with appropriately diluted primary antibody solutions at 4°C overnight. The membranes were then incubated with a horseradish peroxidase-conjugated protein A (GE Healthcare Bio-Science Corp.) or anti-rabbit immunoglobulin G secondary antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), and the resulting bands were visualized using the ECL Advance kit (GE Healthcare Bio-Science Corp.) and scanned by a CCD camera in the Chemi Doc system (Bio-Rad Laboratories). The signal intensity of each band was quantified on digitized images using Molecular Analyst software (Bio-Rad Laboratories). In each assay, in addition to experiments, 75, 50, 20, 10 and 0 μg of standard cell lysates were run on a same membrane and probed as a standard curve for detection. When the detection characteristics obtained from cell lysate titrations are highly linear, antigens are detected quantitatively. Using linear detection characteristics, the expression level corresponding to each experiment can be estimated by interpolation and relatively quantified (see Fig 2A). Mouse monoclonal antibody raised against recombinant human TS has previously been described elsewhere [13] and was obtained from Immuno-Biological Laboratories Co., Ltd. (Gunma, Japan).


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)

Dox-dependent TS expression in TFTS66 cells.A. 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. 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. C. The quantity of TS protein (TStotal, see Materials and methods) 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. D. 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).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123076.g002: Dox-dependent TS expression in TFTS66 cells.A. 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. 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. C. The quantity of TS protein (TStotal, see Materials and methods) 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. D. 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).
Mentions: Cells were washed twice with 0.02% EDTA in phosphate-buffered saline (PBS), pelleted and kept at –80°C until use. Cell pellets were lysed in 2X Laemmli's sodium dodecyl sulphate (SDS) sample buffer [12], sonicated and then cooled on ice. After centrifugation at14,000 x g for 30 min at 4°C, supernatants were collected. Lysates corresponding to 50 μg were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and separated proteins were electrotransferred onto nitrocellulose membranes, BA83 (GE Healthcare Bio-Science Corp., Piscataway, NJ, USA), using Transblot SD (Bio-Rad Laboratories, Hercules, CA, USA). After blocking with 1X TBS (10 mM Tris-HCl; pH 7.4, 0.9% NaCl) solution including 5% bovine serum albumin (BSA) and 0.05% Tween 20 at 52°C for 1 h, the membranes were reacted with appropriately diluted primary antibody solutions at 4°C overnight. The membranes were then incubated with a horseradish peroxidase-conjugated protein A (GE Healthcare Bio-Science Corp.) or anti-rabbit immunoglobulin G secondary antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), and the resulting bands were visualized using the ECL Advance kit (GE Healthcare Bio-Science Corp.) and scanned by a CCD camera in the Chemi Doc system (Bio-Rad Laboratories). The signal intensity of each band was quantified on digitized images using Molecular Analyst software (Bio-Rad Laboratories). In each assay, in addition to experiments, 75, 50, 20, 10 and 0 μg of standard cell lysates were run on a same membrane and probed as a standard curve for detection. When the detection characteristics obtained from cell lysate titrations are highly linear, antigens are detected quantitatively. Using linear detection characteristics, the expression level corresponding to each experiment can be estimated by interpolation and relatively quantified (see Fig 2A). Mouse monoclonal antibody raised against recombinant human TS has previously been described elsewhere [13] and was obtained from Immuno-Biological Laboratories Co., Ltd. (Gunma, Japan).

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