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1.
Neuroscience ; 300: 307-13, 2015 Aug 06.
Article in English | MEDLINE | ID: mdl-26028470

ABSTRACT

Central GABA(A) receptors mediate GABAergic phasic and tonic inhibition. While synaptic αßγ GABA(A) receptors primarily mediate phasic inhibition, extrasynaptic αßδ receptors play an important role in mediating tonic inhibition. Etomidate is a general anesthetic that produces its effects by enhancing GABA(A) receptor activity. We previously showed that etomidate modulates the gating of oocyte-expressed αßγ and αßδ receptors with similar overall allosteric impact, but different pharmacological patterns. In αßγ receptors, etomidate enhances apparent GABA sensitivity (reduces GABA EC50), modestly increases maximal GABA efficacy, and slows current deactivation without affecting desensitization (Zhong et al., 2008). In αßδ receptors characterized by low GABA efficacy, etomidate dramatically increases responses to both low and maximal GABA. The effects of etomidate on desensitization and deactivation of αßδ receptors are unknown. To investigate the kinetic effects of etomidate on α1ß3δ receptors of defined subunit arrangement, we expressed concatenated trimer (ß3-α1-δ) and dimer (ß3-α1) GABA(A) receptor subunit assemblies in human embryonic kidney (HEK)293T cells and recorded whole-cell voltage-clamp currents during rapid external solution exchanges. As expected, etomidate substantially increased maximal GABA-induced currents and prolonged deactivation. Moreover, desensitization was significantly decreased by etomidate. During prolonged GABA applications, etomidate enhanced steady-state currents more than peak currents. Thus, etomidate enhances tonic GABAergic inhibition through extrasynaptic αßδ receptors by both augmenting gating and reducing desensitization.


Subject(s)
Etomidate/pharmacology , GABA Agents/pharmacology , Receptors, GABA-A/metabolism , Anesthetics, Intravenous/pharmacology , HEK293 Cells , Humans , Membrane Potentials/drug effects , Patch-Clamp Techniques , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , gamma-Aminobutyric Acid/pharmacology
2.
Br J Pharmacol ; 171(3): 789-98, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24199598

ABSTRACT

BACKGROUND AND PURPOSE: Neuronal GABA(A) receptors are pentameric chloride ion channels, which include synaptic αßγ and extrasynaptic αßδ isoforms, mediating phasic and tonic inhibition respectively. Although the subunit arrangement of αßγ receptors is established as ß-α-γ-ß-α, that of αßδ receptors is uncertain and possibly variable. We compared receptors formed from free α1, ß3 and δ or γ2L subunits and concatenated ß3-α1-δ and ß3-α1 subunit assemblies (placing δ in the established γ position) by investigating the effects of R-(+)-etomidate (ETO), an allosteric modulator that selectively binds to transmembrane interfacial sites between ß3 and α1. EXPERIMENTAL APPROACH: GABA-activated receptor-mediated currents in Xenopus oocytes were measured electrophysiologically, and ETO-induced allosteric shifts were quantified using an established model. KEY RESULTS: ETO (3.2 µM) similarly enhanced maximal GABA (1 mM)-evoked currents in oocytes injected with 5 ng total mRNA and varying subunit ratios, for α1ß3(1:1), α1ß3δ(1:1:1) and α1ß3δ(1:1:3), but this potentiation by ETO was significantly greater for ß3-α1-δ/ß3-α1(1:1) receptors. Reducing the amount of α1ß3δ(1:1:3) mRNA mixture injected (0.5 ng) increased the modulatory effect of ETO, matching that seen with ß3-α1-δ/ß3-α1(1:1, 1 ng). ETO similarly reduced EC50s and enhanced maxima of GABA concentration-response curves for both α1ß3δ and ß3-α1-δ/ß3-α1 receptors. Allosteric shift parameters derived from these data depended on estimates of maximal GABA efficacy, and the calculated ranges overlap with allosteric shift values for α1ß3γ2L receptors. CONCLUSION AND IMPLICATIONS: Reducing total mRNA unexpectedly increased δ subunit incorporation into receptors on oocyte plasma membranes. Our results favour homologous locations for δ and γ2L subunits in α1ß3γ2/δ GABA(A) receptors.


Subject(s)
Etomidate/pharmacology , GABA-A Receptor Agonists/pharmacology , Models, Molecular , Receptors, GABA-A/metabolism , Allosteric Regulation/drug effects , Anesthetics, General/chemistry , Anesthetics, General/metabolism , Anesthetics, General/pharmacology , Animals , Cell Membrane/drug effects , Cell Membrane/metabolism , Drug Partial Agonism , Etomidate/chemistry , Etomidate/metabolism , Female , GABA-A Receptor Agonists/chemistry , GABA-A Receptor Agonists/metabolism , Humans , Nerve Tissue Proteins/agonists , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Oocytes/drug effects , Oocytes/metabolism , Patch-Clamp Techniques , Protein Subunits/agonists , Protein Subunits/chemistry , Protein Subunits/metabolism , RNA, Messenger/metabolism , Rats , Receptors, GABA-A/chemistry , Receptors, GABA-A/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Xenopus
3.
Cancer Gene Ther ; 18(3): 153-66, 2011 Mar.
Article in English | MEDLINE | ID: mdl-20865021

ABSTRACT

The therapeutic utility of oncolytic adenoviruses controlled by a single, tumor-specific regulatory element may be limited by the intra- and inter-tumoral heterogeneity that characterizes many cancers. To address this issue, we constructed an oncolytic adenovirus that uses two distinct tumor-specific promoters (DF3/Muc1 and hTERT) to drive separate E1A expression cassettes, in combination with deletion of the viral E1B region, which confers additional tumor selectivity and increased oncolytic activity. The resultant virus, Adeno-DF3-E1A/hTERT-E1A, induced higher levels of E1A oncoprotein, enhanced oncolysis and an earlier and higher apoptotic index in infected tumor cells than following infection with Adeno-hTERT-E1A, which harbors a single hTERT promoter-driven E1A cassette. In isolated U251 human gliosarcoma cell holoclones (putative cancer stem cells), where DF3/Muc1 expression is substantially enriched and hTERT expression is decreased compared with the parental U251 cell population, E1A production and oncolysis were strongly decreased following infection with Adeno-hTERT-E1A, but not Adeno-DF3-E1A/hTERT-E1A. The strong oncolytic activity of Adeno-DF3-E1A/hTERT-E1A translated into superior anti-tumor activity over Adeno-hTERT-E1A in vivo in a U251 solid tumor xenograft model, where hTERT levels were >90% suppressed and the DF3/Muc1 to hTERT expression ratio was substantially increased compared with cultured U251 cells. The enhanced anti-tumor activity of the dual-targeted Adeno-DF3-E1A/hTERT-E1A was achieved despite premature viral host cell death and decreased production of functional viral progeny, which limited tumor cell spread of the viral infection. These findings highlight the therapeutic benefit of targeting oncolytic viruses to heterogeneous tumor cell populations.


Subject(s)
Adenoviridae/genetics , Adenovirus E1A Proteins/metabolism , Antigens, Neoplasm/genetics , Mucin-1/genetics , Oncolytic Viruses/genetics , Promoter Regions, Genetic , Telomerase/genetics , Adenoviridae/metabolism , Adenovirus E1A Proteins/genetics , Animals , Apoptosis/genetics , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Gene Order , Genetic Vectors , HEK293 Cells , Humans , Male , Mice , Mice, SCID , Neoplasms/therapy , Oncolytic Viruses/metabolism , Virus Replication/genetics , Xenograft Model Antitumor Assays
4.
Curr Cancer Drug Targets ; 9(6): 777-88, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19754361

ABSTRACT

Metronomic cyclophosphamide treatment is associated with anti-angiogenic activity and is anticipated to generate exploitable hypoxia using hypoxia-activated prodrugs. Weekly administration of tirapazamine (TPZ; 5 mg/kg body weight i.p.) failed to inhibit the growth of 9L gliosarcoma tumors grown s.c. in scid mice. However, the anti-tumor effect of weekly cyclophosphamide (CPA) treatment (140 mg/kg BW i.p.) was substantially enhanced by weekly TPZ administration. An extended tumor free period and increased frequency of tumor eradication without overt toxicity were observed when TPZ was given 3, 4 or 5 days after each weekly CPA treatment. Following the 2(nd) CPA injection, Electron Paramagnetic Resonance (EPR) Oximetry indicated significant increases in tumor pO(2), starting at 48 hr, which further increased after the 3(rd) CPA injection. pO(2) levels were, however, stable in growing untreated tumors. A strong negative correlation (-0.81) between tumor pO(2) and tumor volume during 21 days of weekly CPA chemotherapy was observed, indicating increasing tumor pO(2) with decreasing tumor volume. Furthermore, CPA treatment resulted in increased tumor uptake of activated CPA. CPA induced increases in VEGF RNA, which reached a maximum on day 1, and in PLGF RNA which was sustained throughout the treatment, while anti-angiogenic host thrombospondin-1 increased dramatically through day 7 post-CPA treatment. Weekly cyclophosphamide treatment was anticipated to generate exploitable hypoxia. However, our findings suggest that weekly CPA treatment induces a functional improvement of tumor vasculature, which is characterized by increased tumor oxygenation and drug uptake in tumors, thus counter-intuitively, benefiting intratumoral activation of TPZ and perhaps other bioreductive drugs.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Cyclophosphamide/administration & dosage , Neoplasms/drug therapy , Triazines/administration & dosage , Angiogenesis Inducing Agents/metabolism , Angiogenesis Inhibitors/administration & dosage , Angiogenesis Inhibitors/pharmacokinetics , Angiogenesis Inhibitors/pharmacology , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacokinetics , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Cell Line, Tumor , Cyclophosphamide/pharmacokinetics , Cyclophosphamide/pharmacology , Drug Administration Schedule , Humans , Hypoxia/chemically induced , Mice , Mice, SCID , NADPH-Ferrihemoprotein Reductase/metabolism , Neoplasms/metabolism , Neoplasms/pathology , Oxygen/metabolism , Time Factors , Tirapazamine , Triazines/pharmacology , Xenograft Model Antitumor Assays
5.
Cancer Gene Ther ; 14(12): 935-44, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17853921

ABSTRACT

The therapeutic utility of cytochrome P450-based enzyme prodrug therapy is well established by preclinical studies and in initial clinical trials. The underlying premise of this gene therapy is that intratumoral P450 expression leads to in situ activation of anticancer P450 prodrugs, such as cyclophosphamide (CPA), with intratumoral accumulation of its activated 4-OH metabolite. In mice bearing 9L gliosarcomas expressing the CPA 4-hydroxylase P450 2B6, enhanced tumor apoptosis was observed 48 h after CPA treatment; however, intratumoral 4-OH-CPA levels were indistinguishable from those of P450-deficient tumors, indicating that the bulk of activated CPA is derived from hepatic metabolism. In contrast, in 9L tumors expressing P450 2B11, a low K(m) CPA 4-hydroxylase, intratumoral 4-OH-CPA levels were higher than in blood, liver and P450-deficient tumors. Intratumoral 4-OH-CPA increased dose-dependently, without saturation at 140 mg kg(-1) CPA, suggesting restricted tumor cell permeation of the parent drug. To circumvent this problem, CPA was administered by direct intratumoral injection, which increased the maximum concentration and area under the curve of drug concentration x time (AUC) of intratumoral 4-OH-CPA by 1.8- and 2.7-fold, respectively. An overall 3.9-fold increase in intratumoral 4-OH-CPA AUC, and in antitumor activity, was obtained when CPA release to systemic circulation was delayed using the slow-release polymer poloxamer 407 as vehicle for intratumoral CPA delivery. These findings highlight the advantage of gene therapy strategies that combine low K(m) P450 prodrug activation enzymes with slow, localized release of P450 prodrug substrates.


Subject(s)
Antineoplastic Agents, Alkylating/pharmacokinetics , Aryl Hydrocarbon Hydroxylases/biosynthesis , Cyclophosphamide/pharmacokinetics , Genetic Therapy , Gliosarcoma/enzymology , Gliosarcoma/therapy , Prodrugs/pharmacokinetics , Steroid Hydroxylases/biosynthesis , Animals , Apoptosis/drug effects , Apoptosis/genetics , Aryl Hydrocarbon Hydroxylases/genetics , Cell Line, Tumor , Cytochrome P450 Family 2 , Delayed-Action Preparations/pharmacokinetics , Dose-Response Relationship, Drug , Gene Expression , Gliosarcoma/genetics , Humans , Liver/enzymology , Mice , Mice, Inbred ICR , Mice, SCID , Poloxamer/pharmacokinetics , Steroid Hydroxylases/genetics
6.
Cancer Res ; 61(11): 4437-44, 2001 Jun 01.
Article in English | MEDLINE | ID: mdl-11389073

ABSTRACT

Transduction of tumor cells with a cyclophosphamide (CPA)-activating cytochrome P-450 (P450) gene provides the capacity for localized prodrug activation and greatly sensitizes solid tumors to CPA treatment in vivo. The therapeutic impact of this P450-based cancer gene therapy strategy can be substantially enhanced by cotransduction of P450 reductase, a rate-limiting component of P450-dependent intratumoral CPA activation. The present study examined the possibility of further improving P450/P450 reductase-based gene therapy by using a novel schedule of CPA administration, involving repeated CPA injection every 6 days and previously shown to have an antiangiogenic component. 9L gliosarcoma cells transduced with the CPA-activating enzyme couple P450 2B6/P450 reductase and grown s.c. in immunodeficient severe combined immunodeficient (scid) mice were repeatedly challenged with 140 mg/kg CPA every 6 days. Full tumor regression leading to eradication of six of eight tumors was observed when the tumor size at the time of initial drug treatment was approximately 400 mm(3) (approximately 1.5% of body weight). Little or no overt toxicity of the repeated CPA treatment regimen was observed. The same CPA schedule was much less effective in inducing regression of 9L tumors that were not transduced with P450/P450 reductase. Repeated CPA treatment of mice bearing large, late-stage P450/P450 reductase-transduced tumors (approximately 9-16% of body weight) resulted in major (> or =95%) regression in 15 of 16 tumors, with tumor eradication observed in 2 cases. Although CPA resistance was found to emerge in the population of P450/P450 reductase-transduced tumors, this resistance primarily involved a loss of expression of the transduced P450 and/or P450 reductase gene, rather than development of intrinsic cellular resistance to the activated form of CPA. These findings demonstrate that repeated CPA treatment on a 6 day schedule can be highly effective when combined with P450/P450 reductase gene therapy and suggest that repeated transduction of tumors with prodrug-activation genes may be necessary to achieve tumor eradication and a sustained therapeutic response.


Subject(s)
Antineoplastic Agents, Alkylating/administration & dosage , Cyclophosphamide/administration & dosage , Cytochrome P-450 Enzyme System/genetics , Genetic Therapy/methods , NADPH-Ferrihemoprotein Reductase/genetics , Animals , Antineoplastic Agents, Alkylating/pharmacokinetics , Biotransformation , Brain Neoplasms/drug therapy , Brain Neoplasms/enzymology , Brain Neoplasms/genetics , Brain Neoplasms/therapy , Combined Modality Therapy , Cyclophosphamide/pharmacokinetics , Cytochrome P-450 Enzyme System/metabolism , Dose-Response Relationship, Drug , Drug Administration Schedule , Gliosarcoma/drug therapy , Gliosarcoma/enzymology , Gliosarcoma/genetics , Gliosarcoma/therapy , Humans , Male , Mice , Mice, Inbred ICR , NADPH-Ferrihemoprotein Reductase/metabolism , Rats , Transduction, Genetic
7.
Cancer Res ; 60(14): 3761-9, 2000 Jul 15.
Article in English | MEDLINE | ID: mdl-10919648

ABSTRACT

Tirapazamine (TPZ) is a bioreductive drug that exhibits greatly enhanced cytotoxicity in hypoxic tumor cells, which are frequently radiation-resistant and chemoresistant. TPZ exhibits particularly good activity when combined with alkylating agents such as cyclophosphamide (CPA). The present study examines the potential of combining TPZ with CPA in a cytochrome P450-based prodrug activation gene therapy strategy. Recombinant retroviruses were used to transduce 9L gliosarcoma cells with the genes encoding P450 2B6 and NADPH-P450 reductase. Intratumoral coexpression of P450 2B6 with P450 reductase sensitized 9L tumor cells to CPA equally well under normoxic (19.6% O2) and hypoxic (1% O2) conditions. The P450 2B6/P450 reductase combination also sensitized 9L tumor cells to TPZ under both culture conditions. Interestingly, bystander cytotoxic effects were observed for both CPA and TPZ under hypoxia. Furthermore, TPZ exerted a striking growth-inhibitory effect on CPA-treated 9L/2B6/P450 reductase cells under both normoxia and hypoxia, which suggests the utility of this drug combination for P450-based gene therapy. To evaluate this possibility, 9L tumor cells were transduced in culture with P450 2B6 and P450 reductase and grown as solid tumors in severe combined immune deficient mice in vivo. Although these tumors showed little response to TPZ treatment alone, tumor growth was significantly delayed, by up to approximately four doubling times, when TPZ was combined with CPA. Some toxicity from the drug combination was apparent, however, as indicated by body weight profiles. These findings suggest the potential benefit of incorporating TPZ, and perhaps other bioreductive drugs, into a P450/P450 reductase-based gene therapy strategy for cancer treatment.


Subject(s)
Antineoplastic Agents, Alkylating/administration & dosage , Antineoplastic Agents/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Cyclophosphamide/administration & dosage , Cytochrome P-450 Enzyme System/metabolism , NADH, NADPH Oxidoreductases/metabolism , Triazines/administration & dosage , Animals , Antineoplastic Combined Chemotherapy Protocols/toxicity , Body Weight/drug effects , Cell Division/drug effects , Dose-Response Relationship, Drug , Gene Transfer Techniques , Genetic Vectors , Gliosarcoma/drug therapy , Gliosarcoma/genetics , Humans , Hypoxia , Mice , Mice, SCID , NADPH-Ferrihemoprotein Reductase , Neoplasm Transplantation , Oxygen/metabolism , Prodrugs/pharmacology , Retroviridae/metabolism , Time Factors , Tirapazamine , Transduction, Genetic , Tumor Cells, Cultured
8.
Methods Mol Med ; 35: 85-94, 2000.
Article in English | MEDLINE | ID: mdl-21390801

ABSTRACT

Studies of tumor cell lines expressing individual cytochrome P450 genes are essential for evaluation of the utility of P450 prodrug activation-based cancer gene therapy (1). P450-expressing tumor cells may also be useful to identify novel P450 gene /prodrug combinations (see Chapter 5). The evaluation of candidate P450 genes for use in prodrug activation gene therapy is greatly facilitated by the availability of P450-expressing tumor cell lines, which can be prepared by the retroviral transduction methods described in this chapter. Finally, it should be noted that the methods described in this chapter are not limited to P450 gene transduction, and can readily be applied to the transduction of other genes of interest to cancer gene therapy. In vitro cytotoxicity assays using these cell lines can then be carried out as described in Chapter 7 prior to initiating more costly and labor intensive in vivo tumor studies in animal models (described in Chapter 8).

9.
Biochem Biophys Res Commun ; 260(2): 377-81, 1999 Jul 05.
Article in English | MEDLINE | ID: mdl-10403778

ABSTRACT

Pregnane X Receptor (PXR) has been recently shown to regulate the inducible expression of CYP3A genes in response to xenobiotics and steroids. PXR forms a heterodimer with the retinoic acid receptor (RXR) and this complex binds to and transactivates an 18bp region containing two everted repeats TGA(A/C)CT separated by 6 nucleotides (ER6) and located at approximately -150 in the CYP3A4 promoter. In this work we have isolated and sequenced the proximal 5'-flanking region of CYP3A7 from two different human genomic libraries. In contrast to a previously reported sequence (Itoh et al., 1992), we did not observe any mutation in the 3'-half of the CYP3A7 ER6 element. Using electrophoretic mobility shift assays and cotransfection experiments we show that this element is able to bind the PXR:RXR complex and transactivates the expression of a down stream promoter in response to rifampicin, clotrimazole, and RU-486, three compounds known to specifically activate the human PXR. This is consistent with the fact that CYP3A7 mRNA is inducible in several primary cultures of human hepatocytes from different patients, as well as in two hepatocarcinoma cell lines HuH7 and HepG2, in response to these compounds. In contrast to a previous report (Blumberg et al., 1998), based on the sequence published by Itoh et al., we conclude that CYP3A7, like CYP3A4, is inducible in response to xenobiotics and presumably in a large proportion of the population.


Subject(s)
Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 Enzyme System/genetics , Promoter Regions, Genetic , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Steroid/metabolism , Adult , Base Sequence , Cell Line , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/metabolism , DNA Primers , Humans , Pregnane X Receptor , Protein Binding , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Steroid/genetics , Transcriptional Activation
10.
Drug Metab Rev ; 31(2): 503-22, 1999 May.
Article in English | MEDLINE | ID: mdl-10335450

ABSTRACT

Cytochrome P450-based cancer gene therapy is a novel prodrug activation strategy for cancer treatment that has substantial potential for improving the safety and efficacy of cancer chemotherapeutics. The primary goal of this strategy is to selectively increase tumor cell exposure to cytotoxic drug metabolites generated locally by a prodrug-activating P450 enzyme. This strategy has been exemplified for the alkylating agents cyclophosphamide and ifosfamide, which are bioactivated by select P450 enzymes whose expression is generally high in liver and deficient in tumor cells. Transduction of tumors with a prodrug-activating P450 gene, followed by prodrug treatment, greatly increases intratumoral formation of activated drug metabolites. This leads to more efficient killing of the transduced tumor cells without a significant increase in host toxicity. P450 gene therapy is accompanied by substantial bystander cytotoxicity which greatly enhances the therapeutic effect by extending it to nearby tumor cells not transduced with the therapeutic P450 gene. Although endogenous P450 reductase is not expected to be a limiting factor in prodrug activation in tumor cells that express moderate levels of an exogenous P450 gene, P450 reductase transduction has recently been found to substantially enhance intratumoral prodrug activation and its associated therapeutic effects. Using this gene combination, an overall 50- to 100-fold increase in tumor cell kill in vivo over that provided by hepatic drug activation alone has been observed. Striking improvements in therapeutic effects can thus be achieved using an established anticancer drug in an intratumoral prodrug activation strategy based on the combination of a cytochrome P450 gene with the gene encoding NADPH-P450 reductase. This strategy is readily extendable to several other widely used P450-activated cancer chemotherapeutic prodrugs, as well as to prodrugs that undergo P450 reductase-dependent bioreductive activation and which may exhibit synergy when combined with P450-activated prodrugs in a P450/P450 reductase-based cancer gene therapeutic regimen.


Subject(s)
Cytochrome P-450 Enzyme System/genetics , Genetic Therapy/trends , Neoplasms/therapy , Cytochrome P-450 Enzyme System/metabolism , Forecasting , Humans , NADH, NADPH Oxidoreductases/genetics , NADH, NADPH Oxidoreductases/metabolism , NADPH-Ferrihemoprotein Reductase , Neoplasms/genetics , Prodrugs/metabolism , Prospective Studies
11.
Cancer Res ; 58(19): 4391-401, 1998 Oct 01.
Article in English | MEDLINE | ID: mdl-9766669

ABSTRACT

Cyclophosphamide (CPA) and ifosfamide (IFA) are widely used anticancer prodrugs that are bioactivated in the liver by specific cytochrome P450 enzymes (CYPs). The therapeutic activity of these antitumor agents can be compromised by a low therapeutic index that is, in part, due to the systemic distribution of activated drug metabolites. Here, recombinant retroviruses were used to deliver six different CPA- or IFA-metabolizing human CYP genes to 9L gliosarcoma cells: 2B6, 2C8, 2C9, 2C18 (Met385 and Thr385 alleles), 2C19, and 3A4. Intratumoral cytochrome P450 expression conferred substantial sensitivity to CPA cytotoxicity, with the most dramatic effects seen with CYP2B6. Strong CPA chemosensitivity was also seen following transduction of CYP2C18-Met, despite a very low level of CYP protein expression (>60-fold lower than that of 2B6). In contrast to CPA, the cytotoxicity of IFA was greatest toward tumor cells transduced with CYP3A4, followed by CYPs 2B6 and 2C18-Met. A substantial further increase in chemosensitivity was achieved upon transduction of 2B6 or 2C18-Met-expressing tumor cells with P450 reductase, which provided for more efficient intratumoral prodrug activation and cytotoxicity at lower drug concentrations. With 2B6- plus P450 reductase-transduced tumor cells, CPA but not IFA conferred a strong cell contact-independent bystander cytotoxic effect on non-P450-expressing 9L cells. CPA treatment of tumors that were transduced with 2B6 or 2C18-Met together with P450 reductase and were grown s.c. in immunodeficient mice resulted in a large enhancement of the liver P450-dependent antitumor effect seen with control 9L tumors, with no apparent increase in host toxicity (growth delay of >25-50 days in P450-expressing tumors versus approximately 5-6 days without P450). CYP2B6 plus P450 reductase and CYP2C18-Met plus P450 reductase thus appear to be excellent gene combinations for use with CPA in P450/prodrug activation-based cancer gene therapy.


Subject(s)
Antineoplastic Agents/pharmacokinetics , Cyclophosphamide/pharmacokinetics , Cytochrome P-450 Enzyme System/biosynthesis , Cytochrome P-450 Enzyme System/genetics , Gene Transfer Techniques , Gliosarcoma/drug therapy , Ifosfamide/pharmacokinetics , Animals , Antineoplastic Agents/therapeutic use , Clone Cells , Cyclophosphamide/therapeutic use , Cytochrome P-450 CYP2B1/metabolism , Cytochrome P-450 Enzyme System/metabolism , Genetic Vectors , Gliosarcoma/pathology , Humans , Ifosfamide/therapeutic use , Male , Mice , Mice, Inbred ICR , Mice, SCID , Retroviridae , Reverse Transcriptase Polymerase Chain Reaction , Transplantation, Heterologous , Tumor Cells, Cultured
12.
J Hepatol ; 26 Suppl 2: 54-62, 1997.
Article in English | MEDLINE | ID: mdl-9204410

ABSTRACT

In the human fetal liver, CYP3A7 is expressed as early as the 13th week of gestation. This continues to the perinatal period when it is sharply repressed prior to birth. Concomitantly, the expression of CYP3A4, not detectable in the fetus, sharply increases in the perinatal period to remain elevated throughout adulthood. The mechanisms controlling these developmental patterns of expression have not yet been elucidated at the molecular level. The aim of the present work was to make a functional analysis of the 5'-flanking regions of CYP3A4 and CYP3A7 in different cell lines, including CHO, HepG2, WRL68 and Caco-2 TC7, after cotransfection with two hepato-specific transcription factors, C/EBP alpha and DBP. Six deletions of different length of the 5'-flanking region of each gene, spanning from -1240 to +11 for CYP3A4 and from -1157 to +13 for CYP3A7, were analysed by reporter gene assay. With the CYP3A4 constructs, C/EBP alpha stimulated the transcriptional activity in CHO cells in a way that suggested the presence of at least two C/EBP alpha-responsive elements, one downstream of -55 and one upstream of this position. In CYP3A7, the proximal element exhibited comparable stimulation to the corresponding one in CYP3A4, although the more distal one appeared to respond to a much smaller extent. CYP3A4 and CYP3A7 constructs also responded to C/EBP alpha in HepG2 and WRL68. However, only CYP3A4 and not CYP3A7 was transactivated by this factor in the Caco-2-TC7 cell line. In CHO cells, only the shortest proximal promoter deletion of CYP3A4 (downstream of -57) responded to DBP, while neither the longer constructs nor the CYP3A7 deletions were transactivated. Although preliminary, our results suggest that C/EBP alpha, and possibly other members of the C/EBP family, play a prominent part in the expression of the CYP3A family in man, and that the two genes respond differently to C/EBP alpha and DBP, two factors that exhibit a strict proliferation-dependent pattern of expression in the liver.


Subject(s)
Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 Enzyme System/genetics , DNA-Binding Proteins/physiology , Mixed Function Oxygenases/genetics , Nuclear Proteins/physiology , Transcription Factors/physiology , Animals , CCAAT-Enhancer-Binding Proteins , CHO Cells , Cricetinae , Cytochrome P-450 CYP3A , Humans , Transcriptional Activation
13.
Environ Mol Mutagen ; 30(1): 56-64, 1997.
Article in English | MEDLINE | ID: mdl-9258330

ABSTRACT

The nuclease P1 modification of the 32P-postlabeling technique was used to study the biological activity of 7H-dibenzo[c,g]carbazole (DBC) and some of its derivatives, including N-methyldibenzo[c,g]carbazole (N-MeDBC), 5,9-dimethyldibenzo[c,g]carbazole (5,9-diMeDBC), 5,9,N-trimethyldibenzo[c,g]carbazole (5,9,N-triMeDBC), 6-methoxydibenzo[c,g]carbazole (6-McODBC), N-acetyldibenzo[c,g]carbazole (N-AcDBC), N-hydroxymethyldibenzo[c,g]carbazole (N-HMeDBC) in primary mouse embryo cells. A very good correlation was found between carcinogenic specificity in vivo of these N-heterocyclic aromatic hydrocarbons and their DNA-adduction in vitro. Primary mouse embryo cells were able to metabolize and detect tissue-specific sarcomagens N-MeDBC and 6-MeODBC as well as derivatives with both sarcomagenic and hepatocarcinogenic activity, DBC, N-AcDBC, and N-HMeDBC. The strong specific hepatocarcinogen 5,9-diMeDBC in vivo, did not induce any DNA-adducts in the embryo cells, which suggests that the enzymatic composition of the target tissue probably is the determining factor in the organ specificity of this derivative. 5,9,N-triMeDBC, derivative without any carcinogenic activity in vivo, did not induce any DNA-adducts in primary mouse embryo cells. Pretreatment of cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) apparently stimulated DNA-adduct formation in the cells exposed to DBC, 6-MeODBC, and N-MeDBC. No or a very slight effect of TCDD on DNA-adduct formation was found in cells exposed to N-HMeDBC and N-AcDBC. Preliminary results have shown that TCDD slightly induced cytochrome P4501A1-linked ethoxyresorufin O-deethylase (EROD) activity in primary mouse embryo cells. These data suggest the role of cytochrome P4501A1 in the metabolism of DBC derivatives with sarcomagenic activity.


Subject(s)
Carbazoles/toxicity , Carcinogens/toxicity , DNA Adducts/metabolism , Embryo, Mammalian/drug effects , Embryo, Mammalian/metabolism , Animals , Cells, Cultured , Embryo, Mammalian/cytology , Mice , Organ Specificity/drug effects , Polychlorinated Dibenzodioxins/pharmacology
14.
Eur J Biochem ; 237(3): 642-52, 1996 May 01.
Article in English | MEDLINE | ID: mdl-8647108

ABSTRACT

The Caco-2 cell line, derived from a human colon adenocarcinoma, is unique in its property of spontaneously differentiating into a mature enterocyte cell type during its growth in culture. In this work, we compared the response of the CYP1A1 gene with the benzimidazole derivatives omeprazole and lansoprazole, and with the classical inducer beta-naphthoflavone in the Caco-2 cells at various culture stages. In addition, we characterized the Caco-2 aryl-hydrocarbon receptor. The protein-synthesis inhibitor cycloheximide led to a derepression of the CYP1A1 gene transcription, and to a superinduction when combined with either beta-naphthoflavone or benzimidazoles. Taking advantage of the spontaneous differentiation of Caco-2 cells in long-term cultures, we observed a difference in behavior between the classical inducer beta-naphthoflavone and the atypical inducer omeprazole. In the poorly differentiated cells, both compounds elicited comparable dose/response and rate of induction of CYP1A1 gene expression. In the fully differentiated cells, in contrast, the induction by omeprazole was only transient, whereas the response to beta-naphthoflavone was long lasting. The Caco-2 aryl-hydrocarbon receptor exhibited binding characteristics similar to those determined for human liver and other tissues. The induction of CYP1A1 transcription by benzimidazole derivatives in Caco-2 cells occurred with no direct binding of benzimidazole derivatives to the aryl-hydrocarbon receptor, as in human hepatocytes. However, transient transfection experiments clearly showed that the xenobiotic-responsive element enhancer, with which the activated aryl-hydrocarbon receptor interacts, could drive the induction of a heterologous promoter in the presence of benzimidazoles. Finally the presence of the activated aryl-hydrocarbon receptor in the nuclei of the Caco-2 cells exposed to these molecules was clearly demonstrated by gel-retardation experiments. These results question about the mechanism of ligand-independent activation of the aryl-hydrocarbon receptor and intracellular signaling, initiated by benzimidazole derivatives.


Subject(s)
Benzimidazoles/pharmacology , Cytochrome P-450 Enzyme System/genetics , Receptors, Aryl Hydrocarbon/drug effects , Receptors, Aryl Hydrocarbon/metabolism , 2-Pyridinylmethylsulfinylbenzimidazoles , Anti-Ulcer Agents/pharmacology , Base Sequence , Benzoflavones/pharmacology , Binding Sites , Caco-2 Cells , Cell Differentiation , Cimetidine/pharmacology , Gene Expression Regulation/drug effects , Humans , Kinetics , Lansoprazole , Molecular Sequence Data , Oligonucleotide Probes/genetics , Omeprazole/analogs & derivatives , Omeprazole/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , beta-Naphthoflavone
15.
Biochem Biophys Res Commun ; 221(2): 466-70, 1996 Apr 16.
Article in English | MEDLINE | ID: mdl-8619878

ABSTRACT

In liver samples from 19 Caucasian subjects, the CYP3A5 protein was detected in 74% of individuals (14/19), while the messenger was shown to be expressed in 100% of individuals, assessed by the RT-PCR method. In order to characterise the putative mutation(s) in the messenger, accounting for the absence of protein accumulation, the full coding region of the CYP3A5 cDNA was sequenced for two unrelated individuals, one expressing the protein at a high level and one being defective. A point mutation in exon 11 at position 1280 (C-->A) was found to cosegregate with the absence of protein accumulation in 2 of the 5 defective individuals. This mutation produces a change in the amino acid at position 398 (Thr-->Asn). Whether this mutation affects the stability of the protein or whether it is in linkage desequilibrium with other mutation(s) in the non-coding region of the messenger is not known. The C-->A change at 1280 generates a Tsp509 I site which can be used for routine evaluation of the frequency of this mutation in population studies.


Subject(s)
Alleles , Cytochrome P-450 Enzyme System/genetics , Mixed Function Oxygenases/genetics , Polymorphism, Genetic , Adult , Base Sequence , Blotting, Western , Cytochrome P-450 CYP2E1 , DNA, Complementary , Humans , Luminescent Measurements , Microsomes, Liver/enzymology , Molecular Sequence Data , Polymerase Chain Reaction
16.
Biochem Biophys Res Commun ; 205(3): 1741-7, 1994 Dec 30.
Article in English | MEDLINE | ID: mdl-7811260

ABSTRACT

Two genomic clones containing the 3'-end of CYP3A7, the whole 5'-flanking region plus the first exon of CYP3A5 were isolated from two different human genomic banks and sequenced. The 5'-flanking region of CYP3A5, from nucleotide -1 to -1434, was 60% and 59% similar with the corresponding regions of CYP3A4 and CYP3A7, respectively. However, the similarity increased to 74% when regions from nucleotide -1 to -700 were compared and dropped to less than 42% for regions upstream from -700. The CYP3A5 promoter contains a CATA box instead of the typical TATA box and a basic transcription element (BTE). Other consensus sequences previously found in CYP3A4 and CYP3A7 were also identified in the 5'-flanking of CYP3A5 from -1 to -700. Both CYP3A5 and CYP3A7 genes appear to be tandemly associated and transcribed in the same direction on chromosome 7.


Subject(s)
Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 Enzyme System/genetics , Oxidoreductases, N-Demethylating/genetics , Base Sequence , Chromosome Mapping , Chromosomes, Human, Pair 7 , Cloning, Molecular , Cytochrome P-450 CYP3A , DNA, Complementary/genetics , Humans , Molecular Sequence Data , Multigene Family , Restriction Mapping , Sequence Homology, Nucleic Acid
17.
Eur J Biochem ; 219(3): 791-8, 1994 Feb 01.
Article in English | MEDLINE | ID: mdl-8112330

ABSTRACT

Chemical hepatocarcinogenesis in female mice, induced by 5,9-dimethyl(7H)dibenzo[c,g]carbazole, leads to the overexpression of a cytochrome P-450 of the 2a family. This protein was identified as Cyp2a-5, by the use of immunoblots obtained from isoelectric focusing gels. This method allowed the distinction of Cyp2a-5 from Cyp2a-4, another mouse liver cytochrome P-450, by taking advantage of their slightly different pI values. The theoretical pI values, determined from the amino acid sequence, were pI 9.91 for Cyp2a-4 and pI 10.01 for Cyp2a-5. Other structurally related forms were not detected. In hepatomas from female mice, only the Cyp2a-5 form was overexpressed (2-3 fold). Male mice showed a weak expression of Cyp2a-4 and Cyp2a-5 in control liver samples and in hepatomas. The expression of both forms was increased more than fivefold upon castration. Pyrazole induces specifically the Cyp2a-5 form. The Cyp2a-5 overexpression was correlated with enhanced microsomal coumarin-7-hydroxylase and testosterone-15 alpha-hydroxylase activities. An immunohistochemical study showed that Cyp2a-4 and Cyp2a-5 are expressed uniformly in female livers, but centrilobularly in male livers. In hepatomas, this localisation is perturbed; in females we observed a focal cell localisation, and the Cyp2a-containing cells were often hypertrophic and polyploid. In hepatomas from male mice, the Cyp2a-containing cells became dispersed. From a comparison with other studies, the Cyp2a-5 overexpression appears to be a general feature of hepatocarcinogenesis in mice.


Subject(s)
Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 Enzyme System/biosynthesis , Liver Neoplasms, Experimental/enzymology , Microsomes, Liver/enzymology , Mixed Function Oxygenases/biosynthesis , Animals , Carbazoles , Carcinogens , Cytochrome P-450 CYP2A6 , Cytochrome P-450 Enzyme System/chemistry , Cytochrome P-450 Enzyme System/isolation & purification , Cytochrome P-450 Enzyme System/metabolism , Female , Humans , Immunoblotting , Immunohistochemistry , Isoelectric Focusing , Liver Neoplasms, Experimental/chemically induced , Liver Neoplasms, Experimental/pathology , Male , Mice , Mice, Inbred C57BL , Mixed Function Oxygenases/chemistry , Mixed Function Oxygenases/isolation & purification , Mixed Function Oxygenases/metabolism , Sex Factors , Steroid Hydroxylases/metabolism
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