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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

Dox effects on gene expression in TFTS66 cells.A. 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. The absolute values of the TYMS 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.
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pone.0123076.g003: Dox effects on gene expression in TFTS66 cells.A. 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. The absolute values of the TYMS 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.

Mentions: Before addressing the 5-FU sensitivity of TFTS66 cells, we examined alterations in gene expression caused by Dox exposure in this transformant. In order to observe genome-wide alterations, we adopted an expression microarray approach. The comparison between TFTS66 cells at Dox0 and its parental line, DLD-1 (Fig 3A) and that between TFTS66 cells at Dox0.5 and Dox0 (Fig 3B) were displayed in scatter plots. The level of RNA complimentary to the TYMS cDNA sequence was very high in TFTS66 cells at Dox0 (Fig 3A, left), but strongly suppressed in the Dox0.5 state (Fig 3A, right), confirming the above results. The TS RNA level and the TS protein quantity determined by immunoblotting were highly parallel (Fig 2B) and, intriguingly, there was a completely linear relationship between them (p = 0.999) (Fig 3B, right). Among the genes functioning in the nucleotide metabolisms, only the folate receptor 1 gene, FOLR1, was found to be significantly upregulated in TFTS66 cells. In parallel with the TS RNA, FOLR1 expression was markedly downregulated in cells at Dox0.5 (Fig 3A, right). Conversely, we found that Dox exposure induces several classes of genes of particular interest. They include ones implicated in functions related to cellular transport or apoptosis. Several representative genes, the expression levels of which were more than two-fold higher in cells at Dox0.5 compared to the Dox0 state, are listed in S1 Table.


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 effects on gene expression in TFTS66 cells.A. 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. The absolute values of the TYMS 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123076.g003: Dox effects on gene expression in TFTS66 cells.A. 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. The absolute values of the TYMS 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.
Mentions: Before addressing the 5-FU sensitivity of TFTS66 cells, we examined alterations in gene expression caused by Dox exposure in this transformant. In order to observe genome-wide alterations, we adopted an expression microarray approach. The comparison between TFTS66 cells at Dox0 and its parental line, DLD-1 (Fig 3A) and that between TFTS66 cells at Dox0.5 and Dox0 (Fig 3B) were displayed in scatter plots. The level of RNA complimentary to the TYMS cDNA sequence was very high in TFTS66 cells at Dox0 (Fig 3A, left), but strongly suppressed in the Dox0.5 state (Fig 3A, right), confirming the above results. The TS RNA level and the TS protein quantity determined by immunoblotting were highly parallel (Fig 2B) and, intriguingly, there was a completely linear relationship between them (p = 0.999) (Fig 3B, right). Among the genes functioning in the nucleotide metabolisms, only the folate receptor 1 gene, FOLR1, was found to be significantly upregulated in TFTS66 cells. In parallel with the TS RNA, FOLR1 expression was markedly downregulated in cells at Dox0.5 (Fig 3A, right). Conversely, we found that Dox exposure induces several classes of genes of particular interest. They include ones implicated in functions related to cellular transport or apoptosis. Several representative genes, the expression levels of which were more than two-fold higher in cells at Dox0.5 compared to the Dox0 state, are listed in S1 Table.

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