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

Transformation strategy.A. The original Kozak-like motif in the human TYMS cDNA was modified to the Kozak consensus sequence using partially complementary PCR primers (see Materials and methods). B. The Kozak-modified TYMS cDNA, TSCD3, was amplified by PCR and subcloned into the cDNA expression vector, pTRE2hyg. C. 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123076.g001: Transformation strategy.A. The original Kozak-like motif in the human TYMS cDNA was modified to the Kozak consensus sequence using partially complementary PCR primers (see Materials and methods). B. The Kozak-modified TYMS cDNA, TSCD3, was amplified by PCR and subcloned into the cDNA expression vector, pTRE2hyg. C. 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.

Mentions: The plasmid carrying 1.6 kilobase pairs (kb) TYMS cDNA fragment, pcHTS1, has previously reported [10]. From this plasmid, a 1.0 kb fragment was amplified by polymerase chain reaction (PCR), using mismatch primers that alter the original Kozak-like motif in the 5’ untranslated region to the Kozak consensus sequence [11] and generate new restriction sites, NheI and EcoRV (Fig 1A). This NheI-EcoRV fragment including the modified TYMS cDNA, TSCD3, was subcloned into the cDNA expression vector in the Tet system, pTRE2hyg, which is commercially provided by Clontech Laboratories Inc. (Fig 1B). The constructed vector was designated as pTRE2hyg-TS3. The vectors expressing the Tet transactivators, pTet-ON and pTet-OFF, were also obtained from Clontech Laboratories Inc.


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)

Transformation strategy.A. The original Kozak-like motif in the human TYMS cDNA was modified to the Kozak consensus sequence using partially complementary PCR primers (see Materials and methods). B. The Kozak-modified TYMS cDNA, TSCD3, was amplified by PCR and subcloned into the cDNA expression vector, pTRE2hyg. C. 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123076.g001: Transformation strategy.A. The original Kozak-like motif in the human TYMS cDNA was modified to the Kozak consensus sequence using partially complementary PCR primers (see Materials and methods). B. The Kozak-modified TYMS cDNA, TSCD3, was amplified by PCR and subcloned into the cDNA expression vector, pTRE2hyg. C. 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.
Mentions: The plasmid carrying 1.6 kilobase pairs (kb) TYMS cDNA fragment, pcHTS1, has previously reported [10]. From this plasmid, a 1.0 kb fragment was amplified by polymerase chain reaction (PCR), using mismatch primers that alter the original Kozak-like motif in the 5’ untranslated region to the Kozak consensus sequence [11] and generate new restriction sites, NheI and EcoRV (Fig 1A). This NheI-EcoRV fragment including the modified TYMS cDNA, TSCD3, was subcloned into the cDNA expression vector in the Tet system, pTRE2hyg, which is commercially provided by Clontech Laboratories Inc. (Fig 1B). The constructed vector was designated as pTRE2hyg-TS3. The vectors expressing the Tet transactivators, pTet-ON and pTet-OFF, were also obtained from Clontech Laboratories Inc.

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