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James R. Halpert - One of the best experts on this subject based on the ideXlab platform.
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Engineering mammalian Cytochrome P450 2B1 by directed evolution for enhanced catalytic tolerance to temperature and dimethyl sulfoxide
Protein engineering design & selection : PEDS, 2006Co-Authors: Santosh Kumar, Hong Liu, Ling Sun, B. K. Muralidhara, James R. HalpertAbstract:The previously laboratory-evolved Cytochrome P450 2B1 quadruple mutant V183L/F202L/L209A/S334P (QM), which showed enhanced H(2)O(2)-mediated substrate oxidation, has now been shown to exhibit a >3.0-fold decrease in K(m,HOOH) for 7-ethoxy-4-trifluoromethylcoumarin (7-EFC) O-deethylation compared with the parental enzyme L209A. Subsequently, a streamlined random mutagenesis and a high-throughput screening method were developed using QM to screen and select mutants with enhanced tolerance of catalytic activity to temperature and dimethyl sulfoxide (DMSO). Upon screening >3000 colonies, we identified QM/L295H and QM/K236I/D257N with enhanced catalytic tolerance to temperature and DMSO. QM/L295H exhibited higher activity than QM at a broad range of temperatures (35-55 degrees C) and maintained approximately 1.4-fold higher activity than QM at 45 degrees C for 6 h. In addition, QM/L295H showed a significant increase in T(m,app) compared with L209A. QM/L295H and QM/K236I/D257N exhibited higher activity than QM at a broad range of DMSO concentrations (2.5-15%). Furthermore, QM/K236I/D257N/L295H was constructed by combining QM/K236I/D257N with L295H using site-directed mutagenesis and exhibited a >2-fold higher activity than QM at nearly the entire range of DMSO concentrations. In conclusion, in addition to engineering mammalian Cytochromes P450 for enhanced activity, directed evolution can also be used to optimize catalytic tolerance to temperature and organic solvent.
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Directed evolution of mammalian Cytochrome P450 2B1: mutations outside of the active site enhance the metabolism of several substrates, including the anticancer prodrugs cyclophosphamide and ifosfamide.
The Journal of biological chemistry, 2005Co-Authors: Santosh Kumar, David J. Waxman, Chong S. Chen, James R. HalpertAbstract:Cytochrome P450 2B1 has been subjected to directed evolution to investigate the role of amino acid residues outside of the active site and to engineer novel, more active P450 catalysts. A high throughput screening system was developed to measure H(2)O(2)-supported oxidation of the marker fluorogenic substrate 7-ethoxy-4-trifluoromethylcoumarin (7-EFC). Random mutagenesis by error-prone polymerase chain reaction and activity screening were optimized using the L209A mutant of P450 2B1 in an N-terminally modified construct with a C-terminal His tag (P450 2B1dH). Two rounds of mutagenesis and screening and one subcloning step yielded the P450 2B1 quadruple mutant V183L/F202L/L209A/S334P, which demonstrated a 6-fold higher k(cat) than L209A. Further random or site-directed mutagenesis did not improve the activity. When assayed in an NADPH-supported reconstituted system, V183L/L209A demonstrated lower 7-EFC oxidation than L209A. Therefore, F202L/L209A/S334P was generated, which showed a 2.5-fold higher k(cat)/K(m) for NADPH-dependent 7-EFC oxidation than L209A. F202L/L209A/S334P also showed enhanced catalytic efficiency with 7-benzyloxyresorufin, benzphetamine, and testosterone, and a 10-fold increase in stereoselectivity for testosterone 16alpha-versus 16beta-hydroxylation compared with 2B1dH. Enhanced catalytic efficiency of F202L/L209A/S334P was also retained in the full-length P450 2B1 background with 7-EFC and testosterone as substrates. Finally, the individual mutants were tested for metabolism of the anti-cancer prodrugs cyclophosphamide and ifosfamide. Several of the mutants showed increased metabolism via the therapeutically beneficial 4-hydroxylation pathway, with L209A/S334P showing 2.8-fold enhancement of k(cat)/K(m) with cyclophosphamide and V183L/L209A showing 3.5-fold enhancement with ifosfamide. Directed evolution can thus be used to enhance P450 2B1 catalytic efficiency across a panel of substrates and to identify functionally important residues distant from the active site.
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Functional role of residues in the helix B' region of Cytochrome P450 2B1.
Archives of biochemistry and biophysics, 2005Co-Authors: Wataru Honma, Emily E. Scott, Hong Liu, James R. HalpertAbstract:Comparison of several recently determined X-ray crystal structures of mammalian Cytochrome P450 family 2 enzymes suggests considerable movement of helix B′ when ligands bind. To investigate the functional role of helix B′ in P450 2B1, residues 100–109 were substituted with alanine and phenylalanine. Kinetic properties were examined with the typical 2B substrates 7-benzyloxyresorufin, 7-ethoxy-4-trifluoromethylcoumarin, benzphetamine, and testosterone. Several mutants showed 2- to 3-fold changes in kcat values and significant differences in catalytic efficiencies among the substrates examined, consistent with structural information suggesting that the helix B′ region can adopt multiple conformations with different contact residues depending on the substrate. Homology modeling of P450 2B1 was performed based on an inhibitor-bound P450 2B4 structure, and the docking analyses were consistent with experimental results. The findings suggest that residues in the helix B′ region affect regio- and stereoselective oxidation in P450 family 2 enzymes as well as substrate entry.
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Mutagenesis and molecular dynamics suggest structural and functional roles for residues in the N-terminal portion of the Cytochrome P450 2B1 I helix.
Archives of Biochemistry and Biophysics, 2004Co-Authors: Emily E. Scott, Hong Liu, James R. HalpertAbstract:To investigate their potential roles in ligand access, binding, and subsequent metabolism, residues in the N-terminal portion of the Cytochrome P450 2B1 I helix were mutated to alanine and phenylalanine. Of the 18 mutants from E286 to S294 only 7 yielded holoprotein in an Escherichia coli expression system. Substitutions at positions 289, 290, 292, and 294 caused >/= 2-fold changes in kcat and/or Km for two or more of the 2B1 substrates examined, testosterone, 7-ethoxy-4-trifluoromethylcoumarin, 7-benzyloxyresorufin, and benzphetamine. I290 substitutions had the largest effects on steady-state parameters for three substrates and increased benzphetamine affinity. Steered molecular dynamics simulations of testosterone egress along the I helix identified hydrophobic interactions with I290, L293, and S294 and water bridges to E286 and S294. Sensitivity of holoprotein formation to substitution and effects on substrate binding and metabolism suggest structural and functional roles for residues in the N-terminus of the Cytochrome P450 2B1 I helix.
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A rational approach to Re-engineer Cytochrome P450 2B1 regioselectivity based on the crystal structure of Cytochrome P450 2C5.
The Journal of biological chemistry, 2003Co-Authors: Santosh Kumar, Emily E. Scott, Hong Liu, James R. HalpertAbstract:The regioselectivity for progesterone hydroxylation by Cytochrome P450 2B1 was re-engineered based on the x-ray crystal structure of Cytochrome P450 2C5. 2B1 is a high K(m) progesterone 16alpha-hydroxylase, whereas 2C5 is a low K(m) progesterone 21-hydroxylase. Initially, nine individual 2B1 active-site residues were changed to the corresponding 2C5 residues, and the mutants were purified from an Escherichia coli expression system and assayed for progesterone hydroxylation. At 150 microm progesterone, I114A, F297G, and V363L showed 5-15% of the 21-hydroxylase activity of 2C5, whereas F206V showed high activity for an unknown product and a 13-fold decrease in K(m). Therefore, a quadruple mutant, I114A/F206V/F297G/V363L (Q), was constructed that showed 60% of 2C5 progesterone 21-hydroxylase activity and 57% regioselectivity. Based on their 2C5-like testosterone hydroxylation profiles, S294D and I477F alone and in combination were added to the quadruple mutant. All three mutants showed enhanced regioselectivity (70%) for progesterone 21-hydroxylation, whereas only Q/I477F had a higher k(cat). Finally, the remaining three single mutants, V103I, V367L, and G478V, were added to Q/I477F and Q/S294D/I477F, yielding seven additional multiple mutants. Among these, Q/V103I/S294D/I477F showed the highest k(cat) (3-fold higher than that of 2C5) and 80% regioselectivity for progesterone 21-hydroxylation. Docking of progesterone into a three-dimensional model of this mutant indicated that 21-hydroxylation is favored. In conclusion, a systematic approach to convert P450 regioselectivity across subfamilies suggests that active-site residues are mainly responsible for regioselectivity differences between 2B1 and 2C5 and validates the reliability of 2B1 models based on the crystal structure of 2C5.
Paul F. Hollenberg - One of the best experts on this subject based on the ideXlab platform.
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Covalent Modification of Thr302 in Cytochrome P450 2B1 by the Mechanism-Based Inactivator 4-tert-Butylphenylacetylene
The Journal of pharmacology and experimental therapeutics, 2010Co-Authors: Hsia Lien Lin, Haoming Zhang, Monica I. Jushchyshyn, Paul F. HollenbergAbstract:The mechanism of inactivation of Cytochrome P450 2B1 (CYP2B1) by 4-tert-butylphenylacetylene (BPA) has been characterized previously to be caused by the covalent binding of a reactive intermediate to the apoprotein rather than heme destruction (J Pharmacol Exp Ther 331:392-403, 2009). The identification of a BPA-glutathione conjugate and the increase in the mass of the BPA-adducted apoprotein have indicated that the mass of adduct is 174 Da, equivalent to the mass of BPA plus one oxygen atom. To identify the adducted residue, BPA-inactivated CYP2B1 was digested with trypsin, and the digest was then analyzed by using capillary liquid chromatography with a LTQ linear ion trap mass spectrometer as the detector. A mass shift of 174 Da was used for a SEQUEST database search. The tandem mass spectrometry fragmentation of the modified peptide and the identity of modified residue were determined. The results revealed a mass increase of 174 Da for the peptide sequence (296)FFAGTSSTTLR(308) in the I-helix of CYP2B1 and that the site of adduction formation is Thr302. Homology modeling and ligand docking studies showed that BPA binds in close proximity to both the heme iron and Thr302 with the distances being 2.96 and 3.42 A, respectively. The identification of Thr302 in the CYP2B1 active site as the site of covalent modification leading to inactivation by BPA supports previous hypotheses that this conserved Thr residue may play a crucial role for various functions in P450s.
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Effect of Conformational Dynamics on Substrate Recognition and Specificity as Probed by the Introduction of a de Novo Disulfide Bond into Cytochrome P450 2B1
The Journal of biological chemistry, 2009Co-Authors: Haoming Zhang, Cesar Kenaan, Djemel Hamdane, Gaston Hui Bon Hoa, Paul F. HollenbergAbstract:The conformational dynamics of Cytochrome P450 2B1 (CYP2B1) were investigated through the introduction of a disulfide bond to link the I- and K-helices by generation of a double Cys variant, Y309C/S360C. The consequences of the disulfide bonding were examined both experimentally and in silico by molecular dynamics simulations. Under high hydrostatic pressures, the partial inactivation volume for the Y309C/S360C variant was determined to be −21 cm3mol−1, which is more than twice as much as those of the wild type (WT) and single Cys variants (Y309C, S360C). This result indicates that the engineered disulfide bond has substantially reduced the protein plasticity of the Y309C/S360C variant. Under steady-state turnover conditions, the S360C variant catalyzed the N-demethylation of benzphetamine and O-deethylation of 7-ethoxy-trifluoromethylcoumarin as the WT did, whereas the Y309C variant retained only 39% of the N-demethylation activity and 66% of the O-deethylation activity compared with the WT. Interestingly, the Y309C/S360C variant restored the N-demethylation activity to the same level as that of the WT but decreased the O-deethylation activity to only 19% of the WT. Furthermore, the Y309C/S360C variant showed increased substrate specificity for testosterone over androstenedione. Molecular dynamics simulations revealed that the engineered disulfide bond altered substrate access channels. Taken together, these results suggest that protein dynamics play an important role in regulating substrate entry and recognition.
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The highly conserved Glu149 and Tyr190 residues contribute to peroxynitrite-mediated nitrotyrosine formation and the catalytic activity of Cytochrome P450 2B1.
Chemical research in toxicology, 2005Co-Authors: Hsia Lien Lin, Haoming Zhang, Lucy Waskell, Paul F. HollenbergAbstract:Tyr190 in Cytochrome P450 2B1 has previously been shown to be a prime target for nitration by peroxynitrite (PN) resulting in nitrotyrosine formation and the inactivation of this enzyme. Modeling s...
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Mutation of tyrosine 190 to alanine eliminates the inactivation of Cytochrome P450 2B1 by peroxynitrite.
Chemical research in toxicology, 2003Co-Authors: Hsia Lien Lin, Ute M. Kent, Haoming Zhang, Lucy Waskell, Paul F. HollenbergAbstract:We have previously reported that Cytochrome P450 2B1 was inactivated by peroxynitrite and that the decrease in the catalytic activity correlated with an increase in the nitration of tyrosine. Diges...
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Mechanistic studies of Cytochrome P450 2B1 inactivation by xanthates.
Archives of biochemistry and biophysics, 2000Co-Authors: Stanislav Yanev, Ute M. Kent, Elizabeth S. Roberts, David P. Ballou, Paul F. HollenbergAbstract:Abstract Xanthates have previously been shown to inactivate the phenobarbital-inducible rat Cytochrome P450 2B1 as well as its human homologue P450 2B6. The inactivation was mechanism-based and the loss in enzymatic activity was due to covalent binding of a reactive xanthate intermediate to the P450 2B1 apoprotein. In this report, we investigated various mechanistic events to elucidate the individual step(s) in the P450 catalytic cycle that are compromised due to the inactivation by xanthates. Different xanthates displayed typical type I binding spectra and the spectral binding constants were in the low-millimolar range. A dramatic loss in 7-ethoxy-4-(trifluoromethyl)coumarin activity was observed when P450 2B1 was incubated with five different xanthates in the presence of NADPH. With the exception of the C14 xanthate, virtually no loss of absorbance at 418 or 450 nm in the reduced-CO complex was observed. Long-chain xanthates were able to affect the rate of the first electron transfer in the P450 catalytic cycle by stabilizing the heme in its low-spin state. n -Octyl xanthate (C8) metabolism led to very little observable oxy–ferro intermediate complex formation. The alternate oxidant tert -butyl hydroperoxide was able to support the inactivation reaction of C8 in the absence of reductase or NADPH. The rates of reduction of native, C8-exposed, and C8-inactivated P450 2B1 were measured. The C8-inactivated P450 had a 62% lower rate of reduction in the absence or presence of benzphetamine compared to the native enzyme. Product formation of the three enzyme preparations was quantified with benzphetamine as the substrate. The C8-inactivated P450 2B1 exhibited a much lower rate of NADPH consumption and formation of formaldehyde. However, the ratio of H 2 O 2 to formaldehyde production increased from 1:1 for the native enzyme to 2.8:1 for the inactivated P450. Together these observations indicate that the covalent modification of P450 2B1 by a reactive intermediate of xanthates reduces the rate of the first electron transfer by the reductase and also leads to uncoupling of electron transfer from product formation by diverting a greater proportion of the electrons to H 2 O 2 formation.
E.a. Chiocca - One of the best experts on this subject based on the ideXlab platform.
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Multimodal Cancer Treatment Mediated by a Replicating Oncolytic Virus That Delivers the Oxazaphosphorine/Rat Cytochrome P450 2B1 and Ganciclovir/Herpes Simplex Virus Thymidine Kinase Gene Therapies
Cancer research, 1999Co-Authors: Manish K. Aghi, Xandra O. Breakefield, Ting Chao Chou, K. Suling, E.a. ChioccaAbstract:Multimodal therapy is generally more effective than single-agent treatment for cancer. rRP450 is an engineered herpes simplex viral mutant that replicates in and kills tumor cells in a relatively selective fashion. It also expresses, in infected cells, the cyclophosphamide (CPA)-sensitive rat Cytochrome P450 2B1 (CYP2B1) and the ganciclovir (GCV)-sensitive herpes simplex virus thymidine kinase (HSV-TK) transgenes. We show that cultured rat 9L and human U87ΔEGFR glioma cells, infected and lysed by rRP450, also exhibit supra-additive sensitivity to both CPA and GCV, as determined by Chou-Talalay synergy analysis. DNA cross-linking, assayed by ethidium bromide fluorescence, was significantly inhibited in the presence of GCV, suggesting that interactions between the CPA/CYP2B1 and GCV/HSV-TK gene therapies occurred at the level of DNA repair. In vivo, regression of 9L s.c. tumor volumes in athymic mice was achieved only by the multimodal treatment allowed by rRP450 viral oncolysis combined with CPA/CYP2B1 and GCV/HSV-TK gene therapies, whereas all other treatment combinations produced only tumor growth retardation.
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multimodal cancer treatment mediated by a replicating oncolytic virus that delivers the oxazaphosphorine rat Cytochrome P450 2B1 and ganciclovir herpes simplex virus thymidine kinase gene therapies
Cancer Research, 1999Co-Authors: Manish K. Aghi, Xandra O. Breakefield, Ting Chao Chou, K. Suling, E.a. ChioccaAbstract:Multimodal therapy is generally more effective than single-agent treatment for cancer. rRP450 is an engineered herpes simplex viral mutant that replicates in and kills tumor cells in a relatively selective fashion. It also expresses, in infected cells, the cyclophosphamide (CPA)-sensitive rat Cytochrome P450 2B1 (CYP2B1) and the ganciclovir (GCV)-sensitive herpes simplex virus thymidine kinase (HSV-TK) transgenes. We show that cultured rat 9L and human U87ΔEGFR glioma cells, infected and lysed by rRP450, also exhibit supra-additive sensitivity to both CPA and GCV, as determined by Chou-Talalay synergy analysis. DNA cross-linking, assayed by ethidium bromide fluorescence, was significantly inhibited in the presence of GCV, suggesting that interactions between the CPA/CYP2B1 and GCV/HSV-TK gene therapies occurred at the level of DNA repair. In vivo, regression of 9L s.c. tumor volumes in athymic mice was achieved only by the multimodal treatment allowed by rRP450 viral oncolysis combined with CPA/CYP2B1 and GCV/HSV-TK gene therapies, whereas all other treatment combinations produced only tumor growth retardation.
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diffusible cytotoxic metabolites contribute to the in vitro bystander effect associated with the cyclophosphamide Cytochrome P450 2B1 cancer gene therapy paradigm
Clinical Cancer Research, 1995Co-Authors: Ming X. Wei, Takashi Tamiya, Xandra O. Breakefield, R J Rhee, E.a. ChioccaAbstract:Tumor cells become sensitive to the inert prodrug cyclophosphamide (CPA) after transfer of the gene encoding Cytochrome P450 2B1. This enzyme activates CPA into 4-hydroxycyclophosphamide, which ultimately degrades into acrolein and phosphoramide mustard, the anticancer and DNA-alkylating metabolite. It is imperative that any prodrug-activating gene therapy strategy against cancer possess the capacity to affect the proliferation of tumor cells even when they do not express the transgene (bystander effect), because current methodologies cannot achieve gene transduction in all tumor cells. Prodrug-activating gene therapy schemes described to date exhibit a bystander effect that is not mediated by conditioned medium in culture and may depend on cell contact. In contrast, we find that CPA-sensitized, P450-expressing C6 glioma cells (C6-P450) transfer cytotoxicity to nonexpressing cells by releasing diffusible metabolites through the medium. A 3-h exposure to the prodrug is necessary and sufficient to achieve killing of the transfected cells, and medium conditioned by these cells can kill untransfected cells with similar potency. This bystander effect occurs in the presence of CPA even when only 10% of cells in culture express the P450 2B1 gene, and it is not reproduced by cells that have been irradiated. In an animal model of intracerebral brain tumors, expression of the P450 2B1 gene within the neoplastic cells enhanced significantly the antitumor effect of CPA, even when it was administered systemically. This study shows that CPA/P450 2B1 gene therapy represents a novel tumor-killing strategy that displays an expanded range of cytotoxic action both spatially and temporally within tumor cells and significantly potentiates the anticancer action of CPA when administered i.v.
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Diffusible cytotoxic metabolites contribute to the in vitro bystander effect associated with the cyclophosphamide/Cytochrome P450 2B1 cancer gene therapy paradigm.
Clinical cancer research : an official journal of the American Association for Cancer Research, 1995Co-Authors: Ming X. Wei, Takashi Tamiya, Xandra O. Breakefield, R J Rhee, E.a. ChioccaAbstract:Tumor cells become sensitive to the inert prodrug cyclophosphamide (CPA) after transfer of the gene encoding Cytochrome P450 2B1. This enzyme activates CPA into 4-hydroxycyclophosphamide, which ultimately degrades into acrolein and phosphoramide mustard, the anticancer and DNA-alkylating metabolite. It is imperative that any prodrug-activating gene therapy strategy against cancer possess the capacity to affect the proliferation of tumor cells even when they do not express the transgene (bystander effect), because current methodologies cannot achieve gene transduction in all tumor cells. Prodrug-activating gene therapy schemes described to date exhibit a bystander effect that is not mediated by conditioned medium in culture and may depend on cell contact. In contrast, we find that CPA-sensitized, P450-expressing C6 glioma cells (C6-P450) transfer cytotoxicity to nonexpressing cells by releasing diffusible metabolites through the medium. A 3-h exposure to the prodrug is necessary and sufficient to achieve killing of the transfected cells, and medium conditioned by these cells can kill untransfected cells with similar potency. This bystander effect occurs in the presence of CPA even when only 10% of cells in culture express the P450 2B1 gene, and it is not reproduced by cells that have been irradiated. In an animal model of intracerebral brain tumors, expression of the P450 2B1 gene within the neoplastic cells enhanced significantly the antitumor effect of CPA, even when it was administered systemically. This study shows that CPA/P450 2B1 gene therapy represents a novel tumor-killing strategy that displays an expanded range of cytotoxic action both spatially and temporally within tumor cells and significantly potentiates the anticancer action of CPA when administered i.v.
Xandra O. Breakefield - One of the best experts on this subject based on the ideXlab platform.
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multimodal cancer treatment mediated by a replicating oncolytic virus that delivers the oxazaphosphorine rat Cytochrome P450 2B1 and ganciclovir herpes simplex virus thymidine kinase gene therapies
Cancer Research, 1999Co-Authors: Manish K. Aghi, Xandra O. Breakefield, Ting Chao Chou, K. Suling, E.a. ChioccaAbstract:Multimodal therapy is generally more effective than single-agent treatment for cancer. rRP450 is an engineered herpes simplex viral mutant that replicates in and kills tumor cells in a relatively selective fashion. It also expresses, in infected cells, the cyclophosphamide (CPA)-sensitive rat Cytochrome P450 2B1 (CYP2B1) and the ganciclovir (GCV)-sensitive herpes simplex virus thymidine kinase (HSV-TK) transgenes. We show that cultured rat 9L and human U87ΔEGFR glioma cells, infected and lysed by rRP450, also exhibit supra-additive sensitivity to both CPA and GCV, as determined by Chou-Talalay synergy analysis. DNA cross-linking, assayed by ethidium bromide fluorescence, was significantly inhibited in the presence of GCV, suggesting that interactions between the CPA/CYP2B1 and GCV/HSV-TK gene therapies occurred at the level of DNA repair. In vivo, regression of 9L s.c. tumor volumes in athymic mice was achieved only by the multimodal treatment allowed by rRP450 viral oncolysis combined with CPA/CYP2B1 and GCV/HSV-TK gene therapies, whereas all other treatment combinations produced only tumor growth retardation.
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Multimodal Cancer Treatment Mediated by a Replicating Oncolytic Virus That Delivers the Oxazaphosphorine/Rat Cytochrome P450 2B1 and Ganciclovir/Herpes Simplex Virus Thymidine Kinase Gene Therapies
Cancer research, 1999Co-Authors: Manish K. Aghi, Xandra O. Breakefield, Ting Chao Chou, K. Suling, E.a. ChioccaAbstract:Multimodal therapy is generally more effective than single-agent treatment for cancer. rRP450 is an engineered herpes simplex viral mutant that replicates in and kills tumor cells in a relatively selective fashion. It also expresses, in infected cells, the cyclophosphamide (CPA)-sensitive rat Cytochrome P450 2B1 (CYP2B1) and the ganciclovir (GCV)-sensitive herpes simplex virus thymidine kinase (HSV-TK) transgenes. We show that cultured rat 9L and human U87ΔEGFR glioma cells, infected and lysed by rRP450, also exhibit supra-additive sensitivity to both CPA and GCV, as determined by Chou-Talalay synergy analysis. DNA cross-linking, assayed by ethidium bromide fluorescence, was significantly inhibited in the presence of GCV, suggesting that interactions between the CPA/CYP2B1 and GCV/HSV-TK gene therapies occurred at the level of DNA repair. In vivo, regression of 9L s.c. tumor volumes in athymic mice was achieved only by the multimodal treatment allowed by rRP450 viral oncolysis combined with CPA/CYP2B1 and GCV/HSV-TK gene therapies, whereas all other treatment combinations produced only tumor growth retardation.
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diffusible cytotoxic metabolites contribute to the in vitro bystander effect associated with the cyclophosphamide Cytochrome P450 2B1 cancer gene therapy paradigm
Clinical Cancer Research, 1995Co-Authors: Ming X. Wei, Takashi Tamiya, Xandra O. Breakefield, R J Rhee, E.a. ChioccaAbstract:Tumor cells become sensitive to the inert prodrug cyclophosphamide (CPA) after transfer of the gene encoding Cytochrome P450 2B1. This enzyme activates CPA into 4-hydroxycyclophosphamide, which ultimately degrades into acrolein and phosphoramide mustard, the anticancer and DNA-alkylating metabolite. It is imperative that any prodrug-activating gene therapy strategy against cancer possess the capacity to affect the proliferation of tumor cells even when they do not express the transgene (bystander effect), because current methodologies cannot achieve gene transduction in all tumor cells. Prodrug-activating gene therapy schemes described to date exhibit a bystander effect that is not mediated by conditioned medium in culture and may depend on cell contact. In contrast, we find that CPA-sensitized, P450-expressing C6 glioma cells (C6-P450) transfer cytotoxicity to nonexpressing cells by releasing diffusible metabolites through the medium. A 3-h exposure to the prodrug is necessary and sufficient to achieve killing of the transfected cells, and medium conditioned by these cells can kill untransfected cells with similar potency. This bystander effect occurs in the presence of CPA even when only 10% of cells in culture express the P450 2B1 gene, and it is not reproduced by cells that have been irradiated. In an animal model of intracerebral brain tumors, expression of the P450 2B1 gene within the neoplastic cells enhanced significantly the antitumor effect of CPA, even when it was administered systemically. This study shows that CPA/P450 2B1 gene therapy represents a novel tumor-killing strategy that displays an expanded range of cytotoxic action both spatially and temporally within tumor cells and significantly potentiates the anticancer action of CPA when administered i.v.
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Diffusible cytotoxic metabolites contribute to the in vitro bystander effect associated with the cyclophosphamide/Cytochrome P450 2B1 cancer gene therapy paradigm.
Clinical cancer research : an official journal of the American Association for Cancer Research, 1995Co-Authors: Ming X. Wei, Takashi Tamiya, Xandra O. Breakefield, R J Rhee, E.a. ChioccaAbstract:Tumor cells become sensitive to the inert prodrug cyclophosphamide (CPA) after transfer of the gene encoding Cytochrome P450 2B1. This enzyme activates CPA into 4-hydroxycyclophosphamide, which ultimately degrades into acrolein and phosphoramide mustard, the anticancer and DNA-alkylating metabolite. It is imperative that any prodrug-activating gene therapy strategy against cancer possess the capacity to affect the proliferation of tumor cells even when they do not express the transgene (bystander effect), because current methodologies cannot achieve gene transduction in all tumor cells. Prodrug-activating gene therapy schemes described to date exhibit a bystander effect that is not mediated by conditioned medium in culture and may depend on cell contact. In contrast, we find that CPA-sensitized, P450-expressing C6 glioma cells (C6-P450) transfer cytotoxicity to nonexpressing cells by releasing diffusible metabolites through the medium. A 3-h exposure to the prodrug is necessary and sufficient to achieve killing of the transfected cells, and medium conditioned by these cells can kill untransfected cells with similar potency. This bystander effect occurs in the presence of CPA even when only 10% of cells in culture express the P450 2B1 gene, and it is not reproduced by cells that have been irradiated. In an animal model of intracerebral brain tumors, expression of the P450 2B1 gene within the neoplastic cells enhanced significantly the antitumor effect of CPA, even when it was administered systemically. This study shows that CPA/P450 2B1 gene therapy represents a novel tumor-killing strategy that displays an expanded range of cytotoxic action both spatially and temporally within tumor cells and significantly potentiates the anticancer action of CPA when administered i.v.
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Experimental Tumor Therapy in Mice Using the Cyclophosphamide-Activating Cytochrome P450 2B1 Gene
Human gene therapy, 1994Co-Authors: Ming X. Wei, Takashi Tamiya, Maureen Chase, Efstathios Boviatsis, Thomas K. H. Chang, Neil W. Kowall, Fred H. Hochberg, David J. Waxman, Xandra O. Breakefield, E. Antonio ChioccaAbstract:Most malignant tumors of the central nervous system do not respond well to chemotherapy. The anticancer drug cyclophosphamide (CPA) is largely ineffective against these neoplasms as its conversion to DNA-alkylating, cytotoxic metabolites is restricted primarily to the liver and these metabolites do not readily cross the blood-brain barrier. Here, we show that brain tumor cells can be sensitized to the cytotoxic effects of CPA, both in culture and in vivo, by introduction of the hepatic enzyme responsible for the activation of CPA, Cytochrome P450 2B1. Stable transfection of rat C6 glioma cells with the P450 2B1 gene rendered the cultured tumor cells sensitive to CPA. Further, C6 cells bearing this gene were more sensitive than parental cells to the cytotoxic action of CPA when grown subcutaneously in the flanks of athymic mice. Murine fibroblasts producing a retrovirus vector encoding P450 2B1 and expressing this enzyme were then prepared and grafted into the brains of athymic mice seeded with rat C6 gliomas. Intrathecal administration of CPA prevented the development of meningeal neoplasia and led to partial regression of the parenchymal tumor mass. By contrast, C6 glioma-bearing mice receiving fibroblasts expressing the Escherichia coli lacZ gene and CPA exhibited extensive meningeal tumors and parenchymal solid brain tumors. The in situ activation of CPA by Cytochrome P450 2B1 provides a novel approach not only for brain tumor gene therapy, but also for negative, drug-conditional selection of other defined cell populations.
Manish K. Aghi - One of the best experts on this subject based on the ideXlab platform.
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Thymidine Kinase Gene Therapies Cytochrome P450 2B1 and Ganciclovir/Herpes Simplex Virus Oncolytic Virus That Delivers the Oxazaphosphorine/Rat Multimodal Cancer Treatment Mediated by a Replicating
2013Co-Authors: Manish K. Aghi, Ting Chao Chou, K. SulingAbstract:Multimodal therapy is generally more effective than single-agent treatment for cancer. rRP450 is an engineered herpes simplex viral mutant that replicates in and kills tumor cells in a relatively selective fashion. It also expresses, in infected cells, the cyclophosphamide (CPA)-sensitive rat Cytochrome P450 2B1 (CYP2B1) and the ganciclovir (GCV)-sensitive herpes simplex virus thymidine kinase (HSV-TK) transgenes. We show that cultured rat 9L and human U87DEGFR glioma cells, infected and lysed by rRP450, also exhibit supra-additive sensitivity to both CPA and GCV, as determined by Chou-Talalay synergy analysis. DNA cross-linking, assayed by ethidium bromide fluorescence, was significantly inhibited in the presence of GCV, suggesting that interactions between the CPA/CYP2B1 and GCV/HSV-TK gene therapies occurred at the level of DNA repair. In vivo, regression of 9L s.c. tumor volumes in athymic mice was achieved only by the multimodal treatment allowed by rRP450 viral oncolysis combined with CPA/CYP2B1 and GCV/HSV-TK gene therapies, whereas all other treatment combinations produced only tumor growth retardation.
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thymidine kinase gene therapies Cytochrome P450 2B1 and ganciclovir herpes simplex virus oncolytic virus that delivers the oxazaphosphorine rat multimodal cancer treatment mediated by a replicating
2013Co-Authors: Manish K. Aghi, Ting Chao Chou, K. SulingAbstract:Multimodal therapy is generally more effective than single-agent treatment for cancer. rRP450 is an engineered herpes simplex viral mutant that replicates in and kills tumor cells in a relatively selective fashion. It also expresses, in infected cells, the cyclophosphamide (CPA)-sensitive rat Cytochrome P450 2B1 (CYP2B1) and the ganciclovir (GCV)-sensitive herpes simplex virus thymidine kinase (HSV-TK) transgenes. We show that cultured rat 9L and human U87DEGFR glioma cells, infected and lysed by rRP450, also exhibit supra-additive sensitivity to both CPA and GCV, as determined by Chou-Talalay synergy analysis. DNA cross-linking, assayed by ethidium bromide fluorescence, was significantly inhibited in the presence of GCV, suggesting that interactions between the CPA/CYP2B1 and GCV/HSV-TK gene therapies occurred at the level of DNA repair. In vivo, regression of 9L s.c. tumor volumes in athymic mice was achieved only by the multimodal treatment allowed by rRP450 viral oncolysis combined with CPA/CYP2B1 and GCV/HSV-TK gene therapies, whereas all other treatment combinations produced only tumor growth retardation.
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Multimodal Cancer Treatment Mediated by a Replicating Oncolytic Virus That Delivers the Oxazaphosphorine/Rat Cytochrome P450 2B1 and Ganciclovir/Herpes Simplex Virus Thymidine Kinase Gene Therapies
Cancer research, 1999Co-Authors: Manish K. Aghi, Xandra O. Breakefield, Ting Chao Chou, K. Suling, E.a. ChioccaAbstract:Multimodal therapy is generally more effective than single-agent treatment for cancer. rRP450 is an engineered herpes simplex viral mutant that replicates in and kills tumor cells in a relatively selective fashion. It also expresses, in infected cells, the cyclophosphamide (CPA)-sensitive rat Cytochrome P450 2B1 (CYP2B1) and the ganciclovir (GCV)-sensitive herpes simplex virus thymidine kinase (HSV-TK) transgenes. We show that cultured rat 9L and human U87ΔEGFR glioma cells, infected and lysed by rRP450, also exhibit supra-additive sensitivity to both CPA and GCV, as determined by Chou-Talalay synergy analysis. DNA cross-linking, assayed by ethidium bromide fluorescence, was significantly inhibited in the presence of GCV, suggesting that interactions between the CPA/CYP2B1 and GCV/HSV-TK gene therapies occurred at the level of DNA repair. In vivo, regression of 9L s.c. tumor volumes in athymic mice was achieved only by the multimodal treatment allowed by rRP450 viral oncolysis combined with CPA/CYP2B1 and GCV/HSV-TK gene therapies, whereas all other treatment combinations produced only tumor growth retardation.
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multimodal cancer treatment mediated by a replicating oncolytic virus that delivers the oxazaphosphorine rat Cytochrome P450 2B1 and ganciclovir herpes simplex virus thymidine kinase gene therapies
Cancer Research, 1999Co-Authors: Manish K. Aghi, Xandra O. Breakefield, Ting Chao Chou, K. Suling, E.a. ChioccaAbstract:Multimodal therapy is generally more effective than single-agent treatment for cancer. rRP450 is an engineered herpes simplex viral mutant that replicates in and kills tumor cells in a relatively selective fashion. It also expresses, in infected cells, the cyclophosphamide (CPA)-sensitive rat Cytochrome P450 2B1 (CYP2B1) and the ganciclovir (GCV)-sensitive herpes simplex virus thymidine kinase (HSV-TK) transgenes. We show that cultured rat 9L and human U87ΔEGFR glioma cells, infected and lysed by rRP450, also exhibit supra-additive sensitivity to both CPA and GCV, as determined by Chou-Talalay synergy analysis. DNA cross-linking, assayed by ethidium bromide fluorescence, was significantly inhibited in the presence of GCV, suggesting that interactions between the CPA/CYP2B1 and GCV/HSV-TK gene therapies occurred at the level of DNA repair. In vivo, regression of 9L s.c. tumor volumes in athymic mice was achieved only by the multimodal treatment allowed by rRP450 viral oncolysis combined with CPA/CYP2B1 and GCV/HSV-TK gene therapies, whereas all other treatment combinations produced only tumor growth retardation.
