Quantitation of Dihydropyrimidine Dehydrogenase Expression by Real-Time Reverse Transcription Polymerase Chain Reaction

doi:10.1006/abio.1999.4461

Analytical Biochemistry

Volume 278, Issue 2, 15 February 2000, Pages 175-184

https://doi.org/10.1006/abio.1999.4461 Get rights and content

Abstract

Several recent studies have reported a correlation between intratumor dihydropyrimidine dehydrogenase (DPD) messenger RNA (mRNA) levels and sensitivity to 5-fluorouracil (5-FU). However, significant tissue requirements and labor-intensive methodology have limited the large-scale studies necessary for statistical validation. In addition, the semiquantitative results obtained by these methods further limit their application. We have developed a real-time reverse transcription-PCR (RT-PCR) assay, based on TaqMan fluorescence methodology, capable of rapid and accurate quantitation of DPD mRNA levels in biopsy-sized tissue samples. Results obtained with this approach indicate a linear dynamic range of 10⁸–10³ DPD mRNA copies, with an intra-assay variation of <5%. We evaluated the data using three different methods (absolute standard curve, relative standard curve, and comparative C_T) and show them to be equivalent. This RT-PCR assay was validated by quantitative comparison to Northern blot analysis in five tissues. In addition, analysis of 18 colorectal tumor and liver tissue specimens demonstrated a significant correlation (r² = 0.90) between DPD enzyme activity and mRNA levels. This method provides the first high-throughput, reproducible, and sensitive technique capable of determining DPD mRNA expression levels in nanogram amounts of total RNA.

References (37)

Z. Lu et al.
Purification and characterization of dihydropyrimidine dehydrogenase from human liver
J. Biol. Chem.
(1992)
A. Beck et al.
A role for dihydropyrimidine dehydrogenase and thymidylate synthase in tumour sensitivity to fluorouracil
Eur. J. Cancer
(1994)
M.R. Johnson et al.
Semi-automated radioassay for determination of dihydropyrimidine dehydrogenase (DPD) activity. Screening cancer patients for DPD deficiency, a condition associated with 5-fluorouracil toxicity
J. Chromatogr. B
(1997)
A.J. Berk et al.
Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids
Cell
(1977)
P.A. Sharp et al.
Transcription maps of adenovirus
Methods Enzymol.
(1980)
Scrip's Cancer Chemotherapy Report, 1996, Scrip World Pharmaceutical News, PJB Publications Ltd....
J.L. Grem
Fluoropyrimidines
G.C. Heggie et al.
Clinical pharmacokinetics of 5-fluorouracil and its metabolites in plasma, urine, and bile
Cancer Res.
(1987)
S. Kondo
Apoptosis by antitumor agents and other factors in relation to cell cycle checkpoints
J. Radiat. Res.
(1995)
R.B. Diasio
Sorivudine and 5-fluorouracil: A clinically significant drug–drug interaction due to inhibition of dihydropyrimidine dehydrogenase
Br. J. Clin. Pharmcol.
(1998)

G.B. Morrison et al.

Dihydropyrimidine dehydrogenase deficiency: A pharmacogenetic defect causing severe adverse reactions to 5-fluorouracil-based chemotherapy

Oncol. Nursing Forum

(1997)

E. Christensen et al.

Clinical variability in three Danish patients with dihydropyrimidine dehydrogenase deficiency all homozygous for the same mutation

J. Inherit. Metab. Dis.

(1998)

M.R. Johnson et al.

Life-threatening toxicity in a dihydropyrimidine dehydrogenase deficient patient following treatment with topical 5-fluorouracil

Clin. Cancer Res.

(1999)

X. Wei et al.

Molecular basis of the human dihydropyrimidine dehydrogenase deficiency and 5-fluorouracil toxicity

J. Clin. Invest.

(1996)

J.L. Au et al.

Clinical pharmacological studies of concurrent infusion of 5-fluorouracil and thymidine in treatment of colorectal carcinomas

Cancer Res.

(1982)

C. Katona et al.

Putative role of dihydropyrimidine dehydrogenase in the toxic side effect of 5-fluorouracil in colorectal cancer patients

Oncology

(1998)

R. Zhang et al.

Relationship between circadian-dependent toxicity of 5-fluorodeoxyuridine and circadian rhythm of pyrimidine enzymes: Possible relevance to fluoropyrimidine chemotherapy

Cancer Res.

(1993)

R.A. Fleming et al.

Correlation between dihydropyrimidine dehydrogenase activity in peripheral mononuclear cells and systemic clearance of fluorouracil in cancer patients

Cancer Res.

(1992)

Cited by (324)

Analysis of tumour markers in esophageal carcinoma with different age groups
2022, Journal of King Saud University - Science
Esophageal cancer has been reported in various countries, including Saudi Arabia, India and United States. Many biomarkers have been used to detect cancers at an early stage to improve the survival rate of esophageal cancer.
The present study investigated the biomarkers, biglycan, myeloperoxidase, cytokine (TNF-α, IFN-γ, IL-10 and IL-12) and carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCC) and carbohydrate antigen L1 cell adhesion molecule from the tumour and non-tumour samples.
The increased amount of circulatory biglycan level was detected using the ELISA method in tumour cases compared to control samples (p < 0.001). The expression of biglycan was high in certain tumour cases and was statistically significant (p < 0.01). The variation of myeloperoxidase markers in control and experimental subjects were statistically significant (p < 0.01). Among cancer patients, elevated levels of carcinoembryonic antigen (28.3 ± 1.2 ng/ml and 30.3 ± 1.1 ng/ml), squamous cell carcinoma antigen (5.3 ± 1.1 ng/ml and 4.9 ± 0.5) and carbohydrate antigen 19–9 (18.3 ± 1.1 ng/ml and 15.1 ± 2.6 ng/ml) were detected in male and female cases (p < 0.001). In the normal tissues, the expression of L1 cell adhesion molecule was high and was expressed in low amount in tumour tissues in both male and female individuals (<0.001).
These biomarkers are useful for the early diagnosis of esophageal cancer.
High-level expression of a novel α-amylase from Thermomyces dupontii in Pichia pastoris and its application in maltose syrup production
2019, International Journal of Biological Macromolecules
A novel α-amylase gene (TdAmyA) with an open reading frame of 1431 bp, deducing 476 amino acids, was cloned from the thermophilic fungus Thermomyces dupontii L18. The recombinant α-amylase was successfully over-expressed in Pichia pastoris. The highest α-amylase activity of 38,314 U/mL was obtained with protein content of 28.7 mg/mL after 168 h high-cell density fermentation. Molecular mass of purified TdAmyA was 61.2 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 59.2 kDa by gel filtration. TdAmyA was a glycoprotein with 5.3% (w/w) of carbohydrate. TdAmyA exhibited maximal activity at 60 °C and pH 6.5, and was thermostable up to 55 °C within pH 4.5–10.0. It was more active towards linear starchy substrates than branched ones. The hydrolysis products were mainly comprised of maltose and maltotriose. TdAmyA produced the highest maltose content of 51.8% after 8 h hydrolysis. Thus, TdAmyA might be a candidate α-amylase for maltose syrup production.
Kahweol inhibits proliferation and induces apoptosis by suppressing fatty acid synthase in HER2-overexpressing cancer cells
2018, Food and Chemical Toxicology
Kahweol is a coffee-specific diterpene found in the beans of Coffea arabica and has been reported to demonstrate various biological activities, including anti-inflammatory, antioxidant, and apoptotic properties. In the present study, we examined the molecular mechanism of kahweol in human epidermal growth factor receptor-2 (HER2)-overexpressing breast cancer cells. Kahweol preferentially inhibited cell proliferation and induced cell death through the induction of a caspase 3-dependent pathway in HER2-overexpression breast cancer cell lines. Kahweol treatment substantially reduced the levels of HER2 protein, mRNA, and transcriptional activity in SKBR3 cells. Kahweol exerts its potent anticancer efficacy by the upregulation of polyomavirus enhancer activator 3 (PEA3) and downregulation of activator protein 2 (AP-2) to inhibit aberrantly activated HER2 signaling. Fatty acid synthase (FASN) expression and sterol regulatory element-binding protein-1c (SREBP-1c) activity were downregulated by kahweol. In addition, kahweol lowered the levels of phosphorylated Akt and its downstream targets mammalian target of rapamycin (mTOR) and cyclin D1. Furthermore, we found that blocking Akt signaling through kahweol treatment significantly reduced FASN expression and subsequently suppressed cell proliferation in HER2-overexpressing cancer cells. Overall, this study suggests that kahweol could be a useful adjuvant therapeutic agent in the treatment of HER2-overexpressing breast cancer.
Ameliorative effects of Juniperus rigida fruit on oxazolone- and 2,4-dinitrochlorobenzene-induced atopic dermatitis in mice
2018, Journal of Ethnopharmacology
The fruits of Juniperus rigida have been used in Korean traditional medicine for the treatment of inflammatory diseases in humans such as rheumatoid arthritis.
This study aimed to investigate the anti-atopic properties of J. rigida fruit in in vivo murine atopic dermatitis (AD) models.
BALB/c mouse ears ad SKH-1 hairless mice stimulated with oxazolone (4 weeks) and DNCB (3 weeks), respectively, were treated with the 1% Juniperus rigida fruit EtOH extract (JFE). The JFE improved AD symptoms in both oxazolone- and DNCB-induced AD mice by accelerating skin barrier recovery function and suppressing the overproduction of serum immunoglobulin E (IgE) and interleukin 4 (IL-4). The JFE was found to contain isoscutellarein-7-O-β-xylopyranoside, cupressuflavone, podocarpusflavone A, and hinokiflavone as major components based on phytochemical analysis. Eight flavonoids were isolated from JFE, and of those, cupressuflavone and isoscutellarein-7-O-β-xylopyranoside strongly down-regulated IL-4 expression and β-hexosaminidase release in RBL-2H3 cells.
Therapeutic attempts with J. rigida fruit and its active components might be useful in treating AD and related skin inflammatory diseases.
Sox2 regulates Müller glia reprogramming and proliferation in the regenerating zebrafish retina via Lin28 and Ascl1a
2017, Experimental Eye Research
Sox2 is a well-established neuronal stem cell-associated transcription factor that regulates neural development and adult neurogenesis in vertebrates, and is one of the critical genes used to reprogram differentiated cells into induced pluripotent stem cells. We examined if Sox2 was involved in the early reprogramming-like events that Müller glia undergo as they upregulate many pluripotency- and neural stem cell-associated genes required for proliferation in light-damaged adult zebrafish retinas. In the undamaged adult zebrafish retina, Sox2 is expressed in Müller glia and a subset of amacrine cells, similar to other vertebrates. Following 31 h of light damage, Sox2 expression significantly increased in proliferating Müller glia. Morpholino-mediated knockdown of Sox2 expression resulted in decreased numbers of proliferating Müller glia, while induced overexpression of Sox2 stimulated Müller glia proliferation in the absence of retinal damage. Thus, Sox2 is necessary and sufficient for Müller glia proliferation. We investigated the role of Wnt/β-catenin signaling, which is a known regulator of sox2 expression during vertebrate retinal development. While β-catenin 2, but not β-catenin 1, was necessary for Müller glia proliferation, neither β-catenin paralog was required for sox2 expression following retinal damage. Sox2 expression was also necessary for ascl1a (neurogenic) and lin28a (reprogramming) expression, but not stat3 expression following retinal damage. Furthermore, Sox2 was required for Müller glial-derived neuronal progenitor cell amplification and expression of the pro-neural marker Tg(atoh7:EGFP). Finally, loss of Sox2 expression prevented complete regeneration of cone photoreceptors. This study is the first to identify a functional role for Sox2 during Müller glial-based regeneration of the vertebrate retina.
Leptin induces CREB-dependent aromatase activation through COX-2 expression in breast cancer cells
2017, Food and Chemical Toxicology
Leptin plays a key role in the control of adipocyte formation, as well as in the associated regulation of energy intake and expenditure. The goal of this study was to determine if leptin-induced aromatase enhances estrogen production and induces tumor cell growth stimulation. To this end, breast cancer cells were incubated with leptin in the absence or presence of inhibitor pretreatment, and changes in aromatase and cyclooxygenase-2 (COX-2) expression were evaluated at the mRNA and protein levels. Transient transfection assays were performed to examine the aromatase and COX-2 gene promoter activities and immunoblot analysis was used to examine protein expression. Leptin induced aromatase expression, estradiol production, and promoter activity in breast cancer cells. Protein levels of phospho-STAT3, PKA, Akt, ERK, and JNK were increased by leptin. Leptin also significantly increased cAMP levels, cAMP response element (CRE) activation, and CREB phosphorylation. In addition, leptin induced COX-2 expression, promoter activity, and increased the production of prostaglandin E₂. Finally, a COX-2 inhibitor and aromatase inhibitor suppressed leptin-induced cell proliferation in MCF-7 breast cancer cells. Together, our data show that leptin increased aromatase expression in breast cancer cells, which was correlated with COX-2 upregulation, mediated through CRE activation and cooperation among multiple signaling pathways.

View all citing articles on Scopus

^☆: Supported by USPHS Grant CA 62164.

²: To whom correspondence should be addressed at the Department of Pharmacology and Toxicology, Volker Hall Box 600, University of Alabama at Birmingham, Birmingham, AL 35294. Fax: 205-975-5650. E-mail: [email protected].

View full text

Regular ArticleQuantitation of Dihydropyrimidine Dehydrogenase Expression by Real-Time Reverse Transcription Polymerase Chain Reaction☆

Abstract

J. Biol. Chem.

Eur. J. Cancer

J. Chromatogr. B

Cell

Methods Enzymol.

Fluoropyrimidines

Clinical pharmacokinetics of 5-fluorouracil and its metabolites in plasma, urine, and bile

Cancer Res.

Apoptosis by antitumor agents and other factors in relation to cell cycle checkpoints

J. Radiat. Res.

Sorivudine and 5-fluorouracil: A clinically significant drug–drug interaction due to inhibition of dihydropyrimidine dehydrogenase

Br. J. Clin. Pharmcol.

Dihydropyrimidine dehydrogenase deficiency: A pharmacogenetic defect causing severe adverse reactions to 5-fluorouracil-based chemotherapy

Oncol. Nursing Forum

Clinical variability in three Danish patients with dihydropyrimidine dehydrogenase deficiency all homozygous for the same mutation

J. Inherit. Metab. Dis.

Life-threatening toxicity in a dihydropyrimidine dehydrogenase deficient patient following treatment with topical 5-fluorouracil

Clin. Cancer Res.

Molecular basis of the human dihydropyrimidine dehydrogenase deficiency and 5-fluorouracil toxicity

J. Clin. Invest.

Clinical pharmacological studies of concurrent infusion of 5-fluorouracil and thymidine in treatment of colorectal carcinomas

Cancer Res.

Putative role of dihydropyrimidine dehydrogenase in the toxic side effect of 5-fluorouracil in colorectal cancer patients

Oncology

Relationship between circadian-dependent toxicity of 5-fluorodeoxyuridine and circadian rhythm of pyrimidine enzymes: Possible relevance to fluoropyrimidine chemotherapy

Cancer Res.

Correlation between dihydropyrimidine dehydrogenase activity in peripheral mononuclear cells and systemic clearance of fluorouracil in cancer patients

Cancer Res.

Regular Article
Quantitation of Dihydropyrimidine Dehydrogenase Expression by Real-Time Reverse Transcription Polymerase Chain Reaction☆