Effect of a natural mutation in the 5 ` untranslated region on the translational control of p53 mRNA

Tumor-suppressor protein p53, the `guardian of the genome', is critical in maintaining cellular homeostasis and genomic stability. Earlier, we have reported the discovery of internal ribosome entry sites (IRESs) within the p53 mRNA that regulate the translation of the full length and its N-terminal-truncated isoform, Delta N-p53. Polypyrimidine tract-binding protein (PTB) is an IRES trans-acting factor that positively regulates the IRES activities of both p53 isoforms by relocating from nucleus to the cytoplasm during stress conditions. Here we have demonstrated the putative contact points of PTB on the p53 IRES RNA. Studies on mutations that occur naturally in the 5' untranslated region (5' UTR) in p53 mRNA were lacking. We have investigated a naturally occurring C-to-T single-nucleotide polymorphism (SNP) first reported in human melanoma tumors. This SNP is at position 119 in the 5' UTR of p53 mRNA and we demonstrate that it has consequences on the translational control of p53. Introduction of this SNP has led to decrease in cap-independent translation from p53 5' UTR in bicistronic reporter assay. Further, the effects of this SNP on cap-independent translation have been studied in the context of p53 cDNA as well. Interestingly, the 5' UTR with this SNP has shown reduced binding to PTB that can be corroborated to its weaker IRES activity. Previously, it has been shown that G2-M checkpoint, DNA-damaging stress and oncogenic insult favor IRES-mediated translation. Under similar conditions, we demonstrate that this SNP interferes with the enhancement of the IRES activity of the 5' UTR. Taken together, the results demonstrate for the first time that SNP in the 5' UTR of the p53 mRNA might have a role in translational control of this critical tumor-suppressor gene.

IRES mediated translational regulation of p53 isoforms

p53 is a well known tumor suppressor protein that plays a critical role in cell cycle arrest and apoptosis. It has several isoforms which are produced by transcriptional and posttranscriptional regulatory mechanisms. p53 mRNA has been demonstrated to be translated into two isoforms, full-length p53 (FL-p53) and a truncated isoform N-p53 by the use of alternative translation initiation sites. The mechanism of translation regulation of these two isoforms was further elucidated by the discovery of IRES elements in the p53 mRNA. These two IRESs were shown to regulate the translation of p53 and N-p53 in a distinct cell-cycle phase-dependent manner. This review focuses on the current understanding of the regulation of p53 IRES mediated translation and the role of cis and trans acting factors that influence expression of p53 isoforms. (C) 2013 John Wiley & Sons, Ltd.

Plasmon-mediated emergence of collective emission and enhanced quantum efficiency in quantum dot films

We present experimental and theoretical results on monolayer colloidal cadmium selenide quantum dot films embedded with tiny gold nanoparticles. By varying the density of the embedded gold nanoparticles, we were able to engineer a plasmon-mediated crossover from emission quenching to enhancement regime at interparticle distances for which only quenching of emission is expected. This crossover and a nonmonotonic variation of photoluminescence intensity and decay rate, in experiments, is explained in terms of a model for plasmon-mediated collective emission of quantum emitters which points to the emergence of a new regime in plasmon-exciton interactions. The presented methodology to achieve enhancement in optical quantum efficiency for optimal doping of gold nanoparticles in such ultrathin high-density quantum dot films can be beneficial for new-generation displays and photodetectors.

Negative regulators of a growth factor-mediated signaling pathway in the nematode Caenorhabditis elegans

Vulval differentiation in C. elegans is mediated by an Epidermal growth factor (EGF)- EGF receptor (EGFR) signaling pathway. I have cloned unc-101, a negative regulator of vulval differentiation of the nematode C. elegans. unc-101 encodes a homolog of AP47, the medium chain of the trans-Golgi clathrin-associated protein complex. This identity was confirmed by cloning and comparing sequence of a C. elegans homolog of AP50, the medium chain of the plasma membrane clathrin-associated protein complex. I provided the first genetic evidence that the trans-Golgi clathrin-coated vesicles are involved in regulation of an EGF signaling pathway. Most of the unc-101 alleles are deletions or nonsense mutations, suggesting that these alleles severely reduce the unc-101 activity. A hybrid gene that contains parts of unc-101 and mouse AP4 7 rescued at least two phenotypes of unc-101 mutations, the Unc and the suppression of vulvaless phenotype of let-23(sy1) mutation. Therefore, the functions of AP47 are conserved between nematodes and mammals.

unc-101 mutations can cause a greater than wild-type vulval differentiation in combination with certain mutations in sli-1, another negative regulator of the vulval induction pathway. A mutation in a new gene, rok-1, causes no defect by itself, but causes a greater than wild-type vulval differentiation in the presence of a sli-1 mutation. The unc-101; rok-1; sli-1 triple mutants display a greater extent of vulval differentiation than any double mutant combinations of unc-101, rok-1 and sli-1. Therefore, rok-1 locus defines another negative regulator of the vulval induction pathway.

I analyzed a second gene encoding an AP47 homolog in C. elegans. This gene, CEAP47, encodes a protein 72% identical to both unc-101 and mammalian AP47. A hybrid gene containing parts of unc-101 and CEAP47 sequences can rescue phenotypes of unc-101 mutants, indicating that UNC- 101 and CEAP47 proteins can be redundant if expressed in the same set of cells.

Enhancement of GABAA Receptor-Mediated Inhibitory Postsynaptic Currents Induced by "Partial Oxygen-Glucose Deprivation

下载PDF阅读器"氧糖剥夺"模型作为研究脑缺血的离体模型被广泛使用,该模型模拟了局灶性脑缺血的主要病理变化.然而在缺血病灶核心区与正常脑组织之间称为缺血半暗带的区域,脑血流也有程度不一的降低.为了模拟这种病理变化,发展了一种"不完全氧糖剥夺"的离体脑片模型,该模型满足两个条件,灌流液里氧气部分剥夺而葡萄糖含量降低;"氧糖剥夺"可以导致谷氨酸介导的兴奋性毒性,从而引起神经细胞的坏死.而A型γ-氨基丁酸受体(GABAAR)介导的神经元抑制性活动可以对抗谷氨酸引起的兴奋性毒性,因此近年来引起广泛的研究兴趣.而谷氨酸受体和γ-氨基丁酸受体功能在缺血半暗带是否有改变尚不得而知.因此本文采用海马脑片全细胞膜片钳的记录方法,研究"不完全氧糖剥夺"对海马CA1区神经元的A型γ-氨基丁酸受体介导的抑制性突触后膜电流(IPSCs)的影响.研究发现"不完全氧糖剥夺"使GABAAR介导的IPSCs的峰值增加而衰减时程延长.进一步研究发现该电流的峰值增加是由于GABAAR-氯离子通道的电导增加所致,而与氯离子的反转电位变化无关.这些发现提示在脑缺血的缺血半暗带区域GABAAR介导的神经元抑制性活动可能是增强的,这可能是神经元面对缺血状态产生自我保护的一种内稳态机制.

C1q-like inhibits p53-mediated apoptosis and controls normal hernatopoiesis during zebrafish embryogenesis

Except for the complement C1q, the immunological functions of other C1q family members have remained unclear. Here we describe zebrafish C1q-like, whose transcription and translation display a uniform distribution in early embryos, and are restricted to mid-hind brain and eye in later embryos. In vitro studies showed that C1q-like could inhibit the apoptosis induced by ActD and CHX in EPC cells, through repressing caspase 3/9 activities. Moreover, its physiological roles were studied by morpholino-mediated knockdown in zebrafish embryogenesis. In comparison with control embryos, the C1q-like knockdown embryos display obvious defects in the head and cramofacial development mediated through p53-induced apoptosis, which was confirmed by the in vitro transcribed C1q-like mRNA or p53 MO co-injection. TUNEL assays revealed extensive cell death, and caspase 3/9 activity measurement also revealed about two folds increase in C1q-like morphant embryos, which was inhibited by p53 MO co-injection. Real-time quantitative PCR showed the up-regulation expression of several apoptosis regulators such as p53, mdm2, p21, Box and caspase 3, and down-regulation expression of hbae1 in the C1q-like morphant embryos. Knockdown of C1q-like in zebrafish embryos decreased hemoglobin production and impaired the organization of mesencephalic vein and other brain blood vessels. Interestingly, exposure of zebrafish embryos to UV resulted in an increase in mRNA expression of C1q-like, whereas over-expression of C1q-like was not enough resist to the damage. Furthermore, C1q-like transcription was up-regulated in response to pathogen Aeromonas hydrophila, and embryo survival significantly decreased in the C1q-like morphants after exposure to the bacteria. The data suggested that C1q-like might play an antiapoptotic and protective role in inhibiting p53-dependent and caspase 3/9-mediated apoptosis during embryogenesis, especially in the brain development, and C1q-like should be a novel regulator of cell survival during zebrafish embryogenesis. (c) 2008 Elsevier Inc. All rights reserved.

Site-directed gene integration in transgenic zebrafish mediated by cre recombinase using a combination of mutant Lox sites

With current gene-transfer techniques in fish, insertion of DNA into the genome occurs randomly and in many instances at multiple sites. Associated position effects, copy number differences, and multiple gene interactions make gene expression experiments difficult to interpret and fish phenotype less predictable. To meet different fish engineering needs, we describe here a gene targeting model in zebrafish. At first, four target zebrafish lines, each harboring a single genomic lox71 target site, were generated by zebrafish transgenesis. The zygotes of transgenic zebrafish lines were coinjected with capped Cre mRNA and a knockin vector pZklox66RFP. Site-specific integration event happened from one target zebrafish line. In this line two integrant zebrafish were obtained from more than 80,000 targeted embryos (integrating efficiency about 10(-4) to 10(-5)) and confirmed to have a sole copy of the integrating DNA at the target genome site. Genomic polymerase chain reaction analysis and DNA sequencing verified the correct gene target events where lox71 and lox66 have accurately recombined into double mutant lox72 and wild-type loxP. Each integrant zebrafish chosen for analysis harbored the transgene rfp at the designated egfp concatenates. Although the Cre-mediated recombination is site specific, it is dependent on a randomly placed target site. That is, a genomic target cannot be preselected for integration based solely on its sequence. Conclusively, an rfp reporter gene was successfully inserted into the egfp target locus of zebrafish genome by Cre-lox-mediated recombination. This site-directed knockin system using the lox71/lox66 combination should be a promising gene-targeting platform serving various purposes in fish genetic engineering.

Adenovirus-mediated wild-type P53 Transfer radiosensitizes H1299 cells to subclinical-dose carbon-ion irradiation through the restoration of p53 function

To determine whether adenovirus-mediated wild-type p53 transfer after radiotherapy could radiosensitize non-small-cell lung cancer (NSCLC) cells to subclinical-dose carbon-ion beam (C-beam), H1299 cells were exposed to a C-beam or -ray and then infected with 5 MOI of AdCMV-p53 or GFP (C-beam or -ray with p53 or GFP).Cell cycle was detected by flow cytometric analysis. The apoptosis was examined by a fluorescent microscope with DAPI staining. DNA fragmentation was monitored by the TUNEL assay. P53 mRNA was detected by reverse-transcriptase polymerase chain reaction. The expression of p53, MDM2, and p21 was monitored by Western blot. Survival fractions were determined by colony-forming assay. The percentages of G1-phase cells in C-beam with p53 increased by 8.2%–16.0%, 5.2%–7.0%, and 5.8%–18.9%, respectively, compared with C-beam only, -ray with p53, or p53 only. The accumulation of G2-phase cells in C-beam with p53 increased by 5.7%–8.9% and 8.8%–14.8%, compared with those in -ray with p53 or p53 only, respectively. The percentage of apoptosis for C-beam with p53 increased by 7.4%–19.1%, 5.8%–11.7%, and 5.2%–19.2%, respectively, compared with C-beam only, -ray with p53, or p53 only. The level of p53 mRNA in C-beam with p53 was significantly higher than that in p53 only. The expression level of p53 and p21 in C-beam with p53 was significantly higher than that in both C-beam with GFP and p53 only. The survival fractions for C-beam with p53 were significantly less than those for the other groups (p 0.05). The data suggested that AdCMV-p53 transfer could more efficiently radiosensitize H1299 cells to subclinical-dose C-beam irradiation through the restoration of p53 function.

Experimental inhibition of porcupine-mediated Wnt O-acylation attenuates kidney fibrosis.

Activated Wnt signaling is critical in the pathogenesis of renal fibrosis, a final common pathway for most forms of chronic kidney disease. Therapeutic intervention by inhibition of individual Wnts or downstream Wnt/β-catenin signaling has been proposed, but these approaches do not interrupt the functions of all Wnts nor block non-canonical Wnt signaling pathways. Alternatively, an orally bioavailable small molecule, Wnt-C59, blocks the catalytic activity of the Wnt-acyl transferase porcupine, and thereby prevents secretion of all Wnt isoforms. We found that inhibiting porcupine dramatically attenuates kidney fibrosis in the murine unilateral ureteral obstruction model. Wnt-C59 treatment similarly blunts collagen mRNA expression in the obstructed kidney. Consistent with its actions to broadly arrest Wnt signaling, porcupine inhibition reduces expression of Wnt target genes and bolsters nuclear exclusion of β-catenin in the kidney following ureteral obstruction. Importantly, prevention of Wnt secretion by Wnt-C59 blunts expression of inflammatory cytokines in the obstructed kidney that otherwise provoke a positive feedback loop of Wnt expression in collagen-producing fibroblasts and epithelial cells. Thus, therapeutic targeting of porcupine abrogates kidney fibrosis not only by overcoming the redundancy of individual Wnt isoforms but also by preventing upstream cytokine-induced Wnt generation. These findings reveal a novel therapeutic maneuver to protect the kidney from fibrosis by interrupting a pathogenic crosstalk loop between locally generated inflammatory cytokines and the Wnt/β-catenin signaling pathway.

BRCA1 Suppresses Osteopontin-mediated Breast Cancer

BRCA1 is a well described breast cancer susceptibility gene thought to be involved primarily in DNA repair. However, mutation within the BRCA1 transcriptional domain is also implicated in neoplastic transformation of mammary epithelium, but responsible mechanisms are unclear. Here we show in a rat mammary model system that wild type (WT) BRCA1 specifically represses the expression of osteopontin (OPN), a multifunctional estrogen-responsive gene implicated in oncogenic transformation, particularly that of the breast. WT.BRCA1 selectively binds OPN-activating transcription factors estrogen receptor alpha, AP-1, and PEA3, inhibits OPN promoter transactivation, and suppresses OPN mRNA and protein both from an endogenous gene and a relevant model inducible gene. WT.BRCA1 also inhibits OPN-mediated neoplastic transformation characterized by morphology change, anchorage-independent growth, adhesion to fibronectin, and invasion through Matrigel. A mutant BRCA1 allele (Mut.BRCA1) associated with familial breast cancer lacks OPN suppressor effects, binds to WT.BRCA1, and impedes WT.BRCA1 suppression of OPN. Stable transfection of rat breast tumor cell lines with Mut.BRCA1 dramatically up-regulates OPN protein and induces anchorage independent growth. In human primary breast cancer, BRCA1 mutation is significantly associated with OPN overexpression. Taken together, these data suggest that BRCA1 mutation may confer increased tissue-specific cancer risk, in part by disruption of BRCA1 suppression of OPN gene transcription.

Chemotherapy and TRAIL-mediated colon cancer cell death: the roles of p53, TRAIL receptors, and c-FLIP.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has recently attracted attention as a potential therapeutic agent in the treatment of cancer. We assessed the roles of p53, TRAIL receptors, and cellular Fas-associated death domain-like interleukin-1beta-converting enzyme inhibitory protein (c-FLIP) in regulating the cytotoxic effects of recombinant TRAIL (rTRAIL) alone and in combination with chemotherapy [5-fluorouracil (5-FU), oxaliplatin, and irinotecan] in a panel of colon cancer cell lines. Using clonogenic survival and flow cytometric analyses, we showed that chemotherapy sensitized p53 wild-type, mutant, and null cell lines to TRAIL-mediated apoptosis. Although chemotherapy treatment did not modulate mRNA or cell surface expression of the TRAIL receptors death receptor 4, death receptor 5, decoy receptor 1, or decoy receptor 2, it was found to down-regulate expression of the caspase-8 inhibitor, c-FLIP. Stable overexpression of the long c-FLIP splice form but not the short form was found to inhibit chemotherapy/rTRAIL-induced apoptosis. Furthermore, siRNA-mediated down-regulation of c-FLIP, particularly the long form, was found to sensitize colon cancer cells to rTRAIL-induced apoptosis. In addition, treatment of a 5-FU-resistant cell line with 5-FU down-regulated c-FLIP expression and sensitized the chemotherapy-resistant cell line to rTRAIL. We conclude that TRAIL-targeted therapies may be used to enhance conventional chemotherapy regimens in colon cancer regardless of tumor p53 status. Furthermore, inhibition of c-FLIP may be a vital accessory strategy for the optimal use of TRAIL-targeted therapies.

Effect of p53 Status and STAT1 on Chemotherapy-Induced, Fas-Mediated Apoptosis in Colorectal Cancer

We investigated the role of p53 and the signal transducer and activator of transcription 1 (STAT1) in regulating Fas-mediated apoptosis in response to chemotherapies used to treat colorectal cancer. We found that 5-fluorouracil (5-FU) and oxaliplatin only sensitized p53 wild-type (WT) colorectal cancer cell lines to Fas-mediated apoptosis. In contrast, irinotecan (CPT-11) and tomudex sensitized p53 WT, mutant, and null cells to Fas-mediated cell death. Furthermore, CPT-11 and tomudex, but not 5-FU or oxaliplatin, up-regulated Fas cell surface expression in a p53-independent manner. In addition, increased Fas cell surface expression in p53 mutant and null cell lines in response to CPT-11 and tomudex was accompanied by only a slight increase in total Fas mRNA and protein expression, suggesting that these agents trigger p53-independent trafficking of Fas to the plasma membrane. Treatment with CPT-11 or tomudex induced STAT1 phosphorylation (Ser727) in the p53-null HCT116 cell line but not the p53 WT cell line. Furthermore, STAT1-targeted small interfering RNA (siRNA) inhibited up-regulation of Fas cell surface expression in response to CPT-11 and tomudex in these cells. However, we found no evidence of altered Fas gene expression following siRNA-mediated down-regulation of STAT1 in drug-treated cells. This suggests that STAT1 regulates expression of gene(s) involved in cell surface trafficking of Fas in response to CPT-11 or tomudex. We conclude that CPT-11 and tomudex may be more effective than 5-FU and oxaliplatin in the treatment of p53 mutant colorectal cancer tumors by sensitizing them to Fas-mediated apoptosis in a STAT1-dependent manner.

HIF-1 and NF-kappaB-mediated upregulation of CXCR1 and CXCR2 expression promotes cell survival in hypoxic prostate cancer cells

Hypoxic cancer cells are resistant to treatment, leading to the selection of cells with a more malignant phenotype. The expression of interleukin-8 (IL-8) plays an important role in the tumorigenesis and metastasis of solid tumors including prostate cancer. Recently, we detected elevated expression of IL-8 and IL-8 receptors in human prostate cancer tissue. The objective of the current study was to determine whether hypoxia increases IL-8 and IL-8 receptor expression in prostate cancer cells and whether this contributes to a survival advantage in hypoxic cells. IL-8, CXCR1 and CXCR2 messenger RNA (mRNA) expression in PC3 cells was upregulated in response to hypoxia in a time-dependent manner. Elevated IL-8 secretion following hypoxia was detected by enzyme-linked immunosorbent assay, while immunoblotting confirmed elevated receptor expression. Attenuation of hypoxia-inducible factor (HIF-1) and nuclear factor-kappaB (NF-kappaB) transcriptional activity using small interfering RNA (siRNA), a HIF-1 dominant-negative and pharmacological inhibitors, abrogated hypoxia-induced transcription of CXCR1 and CXCR2 in PC3 cells. Furthermore, chromatin-IP analysis demonstrated binding of HIF-1 and NF-kappaB to CXCR1. Finally, inhibition of IL-8 signaling potentiated etoposide-induced cell death in hypoxic PC3 cells. These results suggest that IL-8 signaling confers a survival advantage to hypoxic prostate cancer cells, and therefore, strategies to inhibit IL-8 signaling may sensitize hypoxic tumor cells to conventional treatments.

Short interfering RNA-mediated gene silencing in Globodera pallida and Meloidogyne incognita infective stage juveniles

The analysis of gene function through RNA interference (RNAi)-based reverse genetics in plant parasitic nematodes (PPNs) remains inexplicably reliant on the use of long double-stranded RNA (dsRNA) silencing triggers; a practice inherently disadvantageous due to the introduction of superfluous dsRNA sequence. increasing chances of aberrant or off-target gene silencing through interactions between nascent short interfering RNAs (siRNAs) and non-cognate mRNA targets. Recently, we have shown that non-nematode, long dsRNAs have a propensity to elicit profound impacts on the phenotype and migrational abilities of both root knot and cyst nematodes. This study presents, to our knowledge for the first time, gene-specific knockdown of FMRFamide-like peptide (flp) transcripts, using discrete 21 bp siRNAs in potato cyst nematode Globodera pallida, and root knot nematode Meloidogyne incognita infective (J2) stage juveniles. Both knockdown at the transcript level through quantitative (q)PCR analysis and functional data derived from migration assay, indicate that siRNAs targeting certain areas of the FMRFamide-like peptide (FLP) transcripts are potent and specific in the silencing of gene function. In addition, we present a method of manipulating siRNA activity through the management of strand thermodynamics. Initial evaluation of strand thermodynamics as a determinant of RNA-induced Silencing Complex (RISC) strand selection (inferred from knockdown efficacy) in the siRNAs presented here suggested that the purported influence of 5' stand stability on guide incorporation may be somewhat promiscuous. However, we have found that on strategically incorporating base mismatches in the sense strand of a G. pallida-specific siRNA we could specifically increase or decrease the knockdown of its target (specific to the antisense strand), presumably through creating more favourable thermodynamic profiles for incorporation of either the sense (non-target-specific) or antisense (target-specific) strand into a cleavage-competent RISC. Whilst the efficacy of similar approaches to siRNA modification has been demonstrated in the context of Drosophila whole-cell lysate preparations and in mammalian cell cultures, it remained to be seen how these sense strand mismatches may impact on gene silencing in vivo, in relation to different targets and in different sequence contexts. This work presents the first application of such an approach in a whole organism; initial results show promise. (C) 2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Antitumour prodrug development using cytochrome P450 (CYP) mediated activation

An ideal cancer chemotherapeutic prodrug is completely inactive until metabolized by a tumour-specific enzyme, or by an enzyme that is only metabolically competent towards the prodrug under physiological conditions unique to the tumour. Human cancers, including colon, breast, lung, liver, kidney and prostate, are known to express cytochrome P450 (CYP) isoforms including 3A and 1A subfamily members. This raises the possibility that tumour CYP isoforms could be a focus for tumour-specific prodrug activation. Several approaches are reviewed, including identification of prodrugs activated by tumour-specific polymorphic CYPs, use of CYP-gene directed enzyme prodrug therapy and CYPs acting as reductases in hypoxic tumour regions. The last approach is best exemplified by AQ4N, a chemotherapeutic prodrug that is bioreductively activated by CYP3A. This study shows that freshly isolated murine T50/80 mammary carcinoma and RIF-1 fibrosarcoma 4-electron reduces AQ4N to its cytotoxic metabolite, AQ4 (T50/80 K-m = 26.7 mu M, V-max = 0.43 mu M/mg protein/min; RIF-1 K-m = 33.5 mu M, V-max = 0.42 mu M/mg protein/min) via AQM, a mono-N-oxide intermediate (T50/80 K-m = 37.5 mu M; V-max = 1.4 mu M/mg protein/min; RIF-1 K-m = 37.5 mu M; V-max = 1.2 mu M/mg protein/min). The prodrug conversion was dependent on NADPH and inhibited by air or carbon monoxide. Cyp3A mRNA and protein were both present in T50/80 carcinoma grown in vivo (RIF-1 not measured). Exposure of isolated tumour cells to anoxia (2 h) immediately after tumour excision increased cyp3A protein 2-3-fold over a 12 h period, after which time the cyp protein levels returned to the level found under aerobic conditions. Conversely, cyp3A mRNA expression showed an initial 3-fold decrease under both oxic and anoxic conditions; this returned to near basal levels after 8-24 h. These results suggest that cyp3A protein is stabilized in the absence of air, despite a decrease in cyp3A mRNA. Such a 'stabilization factor' may decrease cyp3A protein turnover without affecting the translation efficiency of cyp3A mRNA. Confirmation of the CYP activation of AQ4N bioreduction was shown with human lymphoblastoid cell microsomes transfected with CYP3A4, but not those transfected with CYP2B6 or cytochrome P450 reductase. AQ4N is also reduced to AQ4 in NADPH-fortified human renal cell carcinoma (K-m = 4 mu M, V-max = 3.5 pmol/mg protein/min) and normal kidney (K-m = 4 mu M, V-max = 4.0 pmol/mg protein/min), both previously shown to express CYP3A. Germane to the clinical potential of AQ4N is that although both normal and tumour cells are capable of reducing AQ4N to its cytotoxic species, the process requires low oxygen conditions. Hence, AQ4N metabolism should be restricted to hypoxic tumour cells. The isoform selectivity of AQ4N reduction, in addition to its air sensitivity, indicates that AQ4N haem coordination and subsequent oxygen atom transfer from the active-site-bound AQ4N is the likely mechanism of N-oxide reduction. The apparent increase in CYP3A expression under hypoxia makes this a particularly interesting application of CYPs for tumour-specific prodrug activation.