Evaluation of Gene Induction of Drug-Metabolizing Enzymes and Transporters in Primary Culture of Human Hepatocytes Using High-Sensitivity Real-Time Reverse Transcription PCR

Nishimura, Masuhiro; Yoshitsugu, Hiroki; Naito, Shinsaku; Hiraoka, Isao

doi:10.1248/yakushi.122.339

Cited by 78 publications

(63 citation statements)

References 18 publications

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“…14,16) Preparation for Fractionation Cultured cells were seeded into 100-mm tissue culture plates (Costar; Corning) and incubated with medium alone for 24 h. Then, cell fractionation were prepared as follows.…”

Section: Methodsmentioning

confidence: 99%

“…The TaqMan method is exquisitely sensitive and permits the quantitative evaluation of the very small amounts of mRNA, as reported previously. 15,16) Table 3 shows the ratio of target mRNA expression to b-actin mRNA expression in the three cell lines. b-actin was used as the endogenous control in the measurement of target mRNA.…”

Section: Chemosensitivity Of Nsclc Cell Lines To Anticancer Drugsmentioning

confidence: 99%

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Expression of Multidrug Resistance Proteins and Accumulation of Cisplatin in Human Non-small Cell Lung Cancer Cells

Ikuta

Takemura

Sasaki

et al. 2005

Biological & Pharmaceutical Bulletin

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Lung cancer is currently the leading cause of cancer deaths worldwide. Primary lung cancers are classified into two main histological groups: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC constitutes approximately 85% of all lung cancers and often shows intrinsic multidrug resistance (MDR), whereas SCLC almost always responds well to various anticancer agents. 1) Cisplatin is an established antitumor agent for the treatment of advanced human NSCLC and is employed in cisplatin-based adjuvant chemotherapy. 2,3) Although the development of resistance to cisplatin is one of the major obstacles in the successful treatment of NSCLC, the molecular mechanisms involved remain poorly understood. In order to overcome the problems related to drug resistance and to improve the clinical outcome of patients with NSCLC, the mechanisms of cancer chemoresistance must be more clearly elucidated.Our previous in vitro study using three NSCLC cell lines has demonstrated that cisplatin has triggered apoptosis more easily in the chemosensitive human NSCLC cell line than the chemoresistant cell line, based on biochemical and morphological findings. 4) We have also reported that an increased transcriptional level of constitutive signal transducer and activator of transcription (STAT) 3 may be related to the suppressive regulation of the apoptotic pathway in intrinsically chemoresistant NSCLC cells. 4)However, multiple factors are involved in the chemoresistance of cancer cells, suggesting that other mechanisms or factors may also contribute to the resistance of the NSCLC cell lines. Therefore, it is needed to further study other cellular mechanisms of chemoresistance, such as ATP-binding cassette transporters and lung resistance-related protein (LRP), important in drug disposition and in the development of MDR, together with measuring the concentration of anticancer agent in cancer cells.During the past decade, there have been many studies linking various transporters to MDR both in cell culture and in the clinical setting. Of these proteins, P-glycoprotein (MDR1), multidrug resistance-associated protein 1 (MRP1), and LRP have attracted considerable attention in studies of cancer chemotherapy. In tumor cell lines, MDR is often associated with overexpression of ATP-dependent drug efflux proteins belonging to the superfamily of ATP-binding cassette transporters: the 170-kDa MDR1 and the 190-kDa MRP1.5) These proteins bind to and transport various structurally unrelated compounds to maintain their intracellular concentrations below cytotoxic levels. In addition to an overall decrease in intracellular drug concentration, redistribution of the drug from the nucleus to the cytoplasm has also been implicated in MDR of cancer cells. 6) Recently, another drug resistance-related protein, referred to as LRP, has been identified.7) LRP has been found to be identical to the human major vault protein, which is the major component of vaults.8) Vaults are mainly located in the cytoplasm, and approximately 5% of cellular va...

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Section: Methodsmentioning

confidence: 99%

Section: Chemosensitivity Of Nsclc Cell Lines To Anticancer Drugsmentioning

confidence: 99%

Expression of Multidrug Resistance Proteins and Accumulation of Cisplatin in Human Non-small Cell Lung Cancer Cells

Ikuta

Takemura

Sasaki

et al. 2005

Biological & Pharmaceutical Bulletin

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“…Those were composed of 10 smokers, 1 ex-smoker and 9 non-smokers, and 11 alcohol drinkers and 9 non-drinkers according to the definitions of the M Present addresses: 8 Zoology Department, Faculty of Science, Al-Azhar University (Assiut Branch), Assiut, Egypt. 9 To whom correspondence should be addressed. E-mail: fukumoto@idac.tohoku.ac.jp Abbreviations: CYP, cytochrome P450; PBL, peripheral blood leukocytes; HCC, hepatocellular carcinoma; ICC, intrahepatic cholangiocarcinoma; BDK, bile duct cancer; RT-PCR, reverse transcription-polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.…”

Section: Methodsmentioning

confidence: 99%

“…Design of the primers and TaqMan probes and the conditions of RT-PCR performed in an ABI PRISM 7700 sequence detector system (Applied Biosystems, CA) were the same as described previously. 9) The sequences of primers and probes are presented in Table 1. Gene expression levels are presented as the ratio of target mRNA to β-actin mRNA.…”

Section: )mentioning

confidence: 99%

Cytochrome P450 gene expression levels in peripheral blood mononuclear cells in comparison with the liver

et al. 2004

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Cytochromes P450 (CYPs) compose a superfamily of similar proteins involved in detoxification and elimination, as well as activation of a wide variety of compounds. Most CYP family members are localized in the liver. In order to assess whether peripheral blood leukocytes (PBL) are available as a surrogate for the determination of CYP gene expression levels in the liver, we compared CYP gene expression levels in PBL with those in liver tissues from patients with hepatocellular carcinoma (HCC). We measured CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 2F1, 2J2, 3A4, 3A5, etabolism of foreign compounds in the body to polar, hydrophilic metabolites is an important prerequisite for detoxification and elimination of xenobiotics from the body. The cytochromes P450 (CYPs) are a superfamily of similar heme-containing proteins that are involved in the oxidative metabolism of xenobiotics. CYPs also catalyze the bioactivation and inactivation of a wide variety of endogenous compounds, including steroid hormones and eicosanoids. Most of the CYP family members are located in the liver.1-3) Many environmental factors, stress, drugs and diseases influence the expression levels of CYP family members, which may lead to alterations of hepatic drug metabolism in man. Studies on such alterations generally require systemic administration of a drug or probe substance and the application of standard pharmacokinetic approaches. 4,5) Although small amounts of needle biopsy material from the liver can be used for assessment of gene expression, the process of biopsy is accompanied by practical and ethical problems. Some CYPs are also expressed in extra-hepatic tissues such as the lung, the kidney and the small intestine. Although xenobiotics are transported via the blood and peripheral blood is obtained for routine medical examination, little is known about CYP expression in peripheral blood cells. So far, CYP1A1, 1B1, 2D6, 2E1 and 3A genes have been shown to be expressed in peripheral blood.6, 7) In order to determine whether peripheral blood leukocytes (PBL) are applicable as a surrogate for assessment of CYP gene expression in the liver, we measured CYP gene expression levels in PBL and the liver, and studied the intra-individual correlation between them. Approximately 70% of human liver CYPs is accounted for by CYP1A2, 2A6, B6, 2C, 2D6, 2E1 and 3A.8) Here, we measured the expression levels of 19 CYP genes, i.e., CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 2F1, 2J2, 3A4, 3A5, 3A7, 4A11, 4B1 and CYP27, by real-time reverse-transcription (RT)-PCR. Furthermore, we compared the CYP gene expression levels in tumor and non-tumor tissues from liver cancers to assess whether carcinogenesis is associated with specific changes in CYP gene expression levels or not. Materials and MethodsWe investigated 18 patients with hepatocellular carcinoma (HCC), 2 patients with intrahepatic cholangiocarcinoma (ICC), 2 with bile duct cancer (BDK) and 1 with colon cancer metastasized to the liver (META) who underwent surgical r...

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“…20) The pair of forward and reverse primers and the TaqMan probe for TPST1 used for RT-PCR analysis also employed sequences that have been reported previously.…”

Section: Methodsmentioning

confidence: 99%

Tissue-Specific mRNA Expression Profiles of Human Carbohydrate Sulfotransferase and Tyrosylprotein Sulfotransferase

Nishimura

Naito

2007

Biological & Pharmaceutical Bulletin

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Pairs of forward and reverse primers and TaqMan probes specific to each of 15 human sulfotransferases were prepared. The mRNA expression level of each target enzyme was analyzed in total RNA from single and pooled specimens of various human tissues (adrenal gland, bone marrow, brain, colon, heart, kidney, liver, lung, pancreas, peripheral leukocytes, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thymus, thyroid gland, trachea, and uterus) by real-time reverse transcription PCR using an ABI PRISM 7700 Sequence Detection System. The mRNA expression profiles of the sulfotransferases in these 23 different human tissues were used to identify the tissues exhibiting high transcriptional activity for these enzymes. These results provide valuable information for studies concerning the human carbohydrate sulfotransferase and tyrosylprotein sulfotransferase genes in various tissues.

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Evaluation of Gene Induction of Drug-Metabolizing Enzymes and Transporters in Primary Culture of Human Hepatocytes Using High-Sensitivity Real-Time Reverse Transcription PCR

Cited by 78 publications

References 18 publications

Expression of Multidrug Resistance Proteins and Accumulation of Cisplatin in Human Non-small Cell Lung Cancer Cells

Expression of Multidrug Resistance Proteins and Accumulation of Cisplatin in Human Non-small Cell Lung Cancer Cells

Cytochrome P450 gene expression levels in peripheral blood mononuclear cells in comparison with the liver

Tissue-Specific mRNA Expression Profiles of Human Carbohydrate Sulfotransferase and Tyrosylprotein Sulfotransferase

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