High Levels of Recombinant CYP3A4 Expression in Chinese Hamster Ovary Cells Are Modulated by Coexpressed Human P450 Reductase and Hemin Supplementation

Ding, Shaohong; Yao, Denggao; Burchell, Brian; Wolf, Charles; Friedberg, Thomas

doi:10.1006/abbi.1997.0405

Cited by 33 publications

(52 citation statements)

References 30 publications

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“…The stable CYP3A4 transfectant was found to lose CYP activity within a few weeks -a phenomenon not uncommon with cells stably transfected with CYPs. 22 We do not know the mechanism by which this activity was lost, but other evidence in our laboratory suggests that it is not a mechanism involving gene regulation, rather that enzyme activity is decreased. The loss of CYP activity in the stable transfectant made it difficult to perform in vitro studies; this was not perceived to be a problem in vivo because CYPs activity is stable in vivo.…”

Section: Cancer Gene Therapymentioning

confidence: 94%

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Bioreductive GDEPT using cytochrome P450 3A4 in combination with AQ4N

et al. 2002

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The bioreductive drug, AQ4N, is metabolized under hypoxic conditions and has been shown to enhance the antitumor effects of radiation and chemotherapy drugs. We have investigated the role of cytochrome P450 3A4 ( CYP3A4 ) in increasing the metabolism of AQ4N using a gene -directed enzyme prodrug therapy ( GDEPT ) strategy. RIF -1 murine tumor cells were transfected with a mammalian expression vector containing CYP3A4 cDNA. In vitro AQ4N metabolism, DNA damage, and clonogenic cell kill were assessed following exposure of transfected and parental control cells to AQ4N. The presence of exogenous CYP3A4 increased the metabolism of AQ4N and significantly enhanced the ability of the drug to cause DNA strand breaks and clonogenic cell death. Cotransfection of CYP reductase with CYP3A4 showed a small enhancement of the effect in the DNA damage assay only. A single injection of CYP3A4 into established RIF -1 murine tumors increased the metabolism of AQ4N, and when used in combination with radiation, three of nine tumors were locally controlled for >60 days. This is the first demonstration that CYPs alone can be used in a GDEPT strategy for bioreduction of the cytotoxic prodrug, AQ4N. AQ4N is the only CYP -activated bioreductive agent in clinical trials. Combination with a GDEPT strategy may offer a further opportunity for targeting radiation -resistant and chemo -resistant hypoxic tumor cells.

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Section: Cancer Gene Therapymentioning

confidence: 94%

“…In addition, the role of P450 reductase was assessed, as it has previously been described as a rate -limiting component of P450-dependent drug activation. 21,22 Materials and methods…”

mentioning

confidence: 99%

Bioreductive GDEPT using cytochrome P450 3A4 in combination with AQ4N

et al. 2002

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“…Chinese hamster ovary cells (CHO)-wild type (CHO-WT), stably transfected CHO-HR and CHO-HR-3A4 cell lineswere kindly provided by Thomas FRIEDBERG and C Roland WOLF from the Biomedical Research Centre at the University of Dundee, Scotland, UK [23] . The CHO-WT and CHO-HR cell lines were maintained in monolayer culture at 37 ºC in a humidified 5% CO 2 atmosphere in high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, 100 µg/mL streptomycin and HAT Supplement (100 μmol/L hypoxanthine, 0.4 μmol/L aminopterin and 16 μmol/L thymidine).…”

Section: Cell Culturementioning

confidence: 99%

“…To prepare cell extracts for CYP3A4 immunoblotting, CHO-WT, CHO-HR (as controls) and CHO-HR-3A4 cells were plated in 100-mm dishes and, on the next day, treated with C-1311 for up to 120 h. After the treatment, cells were harvested by trypsin and resuspended in 0.2 mL of 10 mmol/L sodium phosphate buffer (pH 8.0) containing 2 mmol/L MgCl 2 , 2 mmol/L dithiothreitol and 1 mmol/L EDTA [23] . Cells were lysed by homogenization on ice for 3 min.…”

Section: Preparation Of Cell Extracts and Immunoblotting For Cyp3a4mentioning

confidence: 99%

CYP3A4 overexpression enhances apoptosis induced by anticancer agent imidazoacridinone C-1311, but does not change the metabolism of C-1311 in CHO cells

2013

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Aim: To examine whether CYP3A4 overexpression influences the metabolism of anticancer agent imidazoacridinone C-1311 in CHO cells and the responses of the cells to C-1311. Methods: Wild type CHO cells (CHO-WT), CHO cells overexpressing cytochrome P450 reductase (CPR) [CHO-HR] and CHO cells coexpressing CPR and CYP3A4 (CHO-HR-3A4) were used. Metabolic transformation of C-1311 and CYP3A4 activity were measured using RP-HPLC. Flow cytometry analyses were used to examine cell cycle, caspase-3 activity and cell apoptosis. The expression of pH 6.0-dependent β-galactosidase (SA-β-gal) was studied to evaluate accelerated senescence. ROS generation was analyzed with CM-H 2 DCFDA staining. Results: CYP3A4 overexpression did not change the metabolism of C-1311 in CHO cells: the levels of all metabolites of C-1311 increased with the exposure time to a similar extent, and the differences in the peak level of the main metabolite M3 were statistically insignificant among the three CHO cell lines. In CHO-HR-3A4 cells, C-1311 effectively inhibited CYP3A4 activity without affecting CYP3A4 protein level. In the presence of C-1311, CHO-WT cells underwent rather stable G 2 /M arrest, while the two types of transfected cells only transiently accumulated at this phase. C-1311-induced apoptosis and necrosis in the two types of transfected cells occurred with a significantly faster speed and to a greater extent than in CHO-WT cells. Additionally, C-1311 induced ROS generation in the two types of transfected cells, but not in CHO-WT cells. Moreover, CHO-HR-3A4 cells that did not die underwent accelerated senescence. Conclusion: CYP3A4 overexpression in CHO cells enhances apoptosis induced by C-1311, whereas the metabolism of C-1311 is minimal and does not depend on CYP3A4 expression.

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“…CYPRED was co-transfected along with CYP1A1, in one group, as it has been described as a rate-limiting component of P450-dependent drug activation. 24,25 Background levels of DNA damage (o4 mean tail moment) were detected in the control groups (i.e. parental cells7AQ4N, Lipofectamine Plus with AQ4N, empty vector þ AQ4N and CYPRED þ AQ4N).…”

Section: Determination Of Aq4n-induced Dna Damage In Rif-1 Cellsmentioning

confidence: 99%

Tumor-selective drug activation: a GDEPT approach utilizing cytochrome P450 1A1 and AQ4N

et al. 2006

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Drug metabolizing transgene products, which activate bioreductive cytotoxins, can be used to target treatment-resistant hypoxic tumors. The prodrug AQ4N is bioreduced in hypoxic cells by cytochrome P450s (CYPs) to the cytotoxin AQ4. Previously we have shown that intra-tumoral injection of CYP3A4 and CYP2B6 transgenes with AQ4N and radiation inhibits tumor growth. Here we examine the ability of other CYPs, in particular CYP1A1, to metabolize AQ4N, and to enhance radiosensitization. Metabolism of AQ4N was assessed using microsomes prepared from baculovirus-infected cells transfected with various CYP isoforms. AQ4N metabolism was most efficient with CYP1A1 (66.7 nmol/min/pmol) and 2B6 (34.4 nmol/min/pmol). Transient transfection of human CYP1A17CYP reductase (CYPRED) was investigated in hypoxic RIF-1 mouse cells in vitro using the alkaline comet assay. There was a significant increase in DNA damage following transient transfection of CYP1A1 compared to non-transfected cells; inclusion of CYPRED provided no additional effect. In vivo, a single intra-tumoral injection of a CYP1A1 construct in combination with AQ4N (100 mg/kg i.p.) and 20 Gy X-rays caused a 16-day delay in tumor regrowth compared to tumors receiving AQ4N plus radiation and empty vector (P ¼ 0.0344). The results show the efficacy of a CYP1A1-mediated GDEPT strategy for bioreduction of AQ4N.

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High Levels of Recombinant CYP3A4 Expression in Chinese Hamster Ovary Cells Are Modulated by Coexpressed Human P450 Reductase and Hemin Supplementation

Cited by 33 publications

References 30 publications

Bioreductive GDEPT using cytochrome P450 3A4 in combination with AQ4N

Bioreductive GDEPT using cytochrome P450 3A4 in combination with AQ4N

CYP3A4 overexpression enhances apoptosis induced by anticancer agent imidazoacridinone C-1311, but does not change the metabolism of C-1311 in CHO cells

Tumor-selective drug activation: a GDEPT approach utilizing cytochrome P450 1A1 and AQ4N

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