4-Ipomeanol and 2-aminoanthracene cytotoxicity in C3H/10T12 cells expressing rabbit cytochrome P450 4B1

Pb, Smith; Hf, Tiano; Nesnow, Stephen; Mr, Boyd; Rm, Philpot; Langenbach, Robert

doi:10.1016/0006-2952(95)02029-2

Cited by 29 publications

(19 citation statements)

References 29 publications

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“…Compounds 1-11 were chosen because fatty acids with chain length C7-C12 are known to be processed by r-P422 . Among other known substrates for CYP4B1 enzymes are several pro-toxins, including 4-IPO 12 (Verschoyle et al, 1993;Smith et al, 1995), 3-MI 18 (Ramakanth et al, 1994;Thornton-Manning et al, 1996), 2-AF 19 (Robertson et al, 1983;Vanderslice et al, 1985) and 2-AA 20 (Robertson et al, 1983;Smith et al, 1995). The furan analogs 13-17 were chosen because of their structural similarity to 4-IPO 12.…”

Section: Resultsmentioning

confidence: 99%

Ligand characterization of CYP4B1 isoforms modified for high-level expression inEscherichia coliand HepG2 cells

Roellecke

Jäger

Gyurov

et al. 2017

Protein Engineering, Design and Selection

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Human CYP4B1, a cytochrome P450 monooxygenase predominantly expressed in the lung, inefficiently metabolizes classical CYP4B1 substrates, such as the naturally occurring furan pro-toxin 4-ipomeanol (4-IPO). Highly active animal forms of the enzyme convert 4-IPO to reactive alkylating metabolite(s) that bind(s) to cellular macromolecules. By substitution of 13 amino acids, we restored the enzymatic activity of human CYP4B1 toward 4-IPO and this modified cDNA is potentially valuable as a suicide gene for adoptive T-cell therapies. In order to find novel pro-toxins, we tested numerous furan analogs in in vitro cell culture cytotoxicity assays by expressing the wildtype rabbit and variants of human CYP4B1 in human liver-derived HepG2 cells. To evaluate the CYP4B1 substrate specificities and furan analog catalysis, we optimized the N-terminal sequence of the CYP4B1 variants by modification/truncation and established their heterologous expression in Escherichia coli (yielding 70 and 800 nmol·l −1 of recombinant human and rabbit enzyme, respectively). Finally, spectral binding affinities and oxidative metabolism of the furan analogs by the purified recombinant CYP4B1 variants were analyzed: the naturally occurring perilla ketone was found to be the tightest binder to CYP4B1, but also the analog that was most extensively metabolized by oxidative processes to numerous non-reactive reaction products.

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Section: Resultsmentioning

confidence: 99%

Ligand characterization of CYP4B1 isoforms modified for high-level expression inEscherichia coliand HepG2 cells

Roellecke

Jäger

Gyurov

et al. 2017

Protein Engineering, Design and Selection

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“…For both activated drugs, DNA alkylation seems to be the main mechanism of toxicity. 13 Rainov et al 8 demonstrated that the cyp4B1 /4-IM or cyp4B1/ 2-AA pharmacogene therapy systems can generate a strong cytotoxicity for brain tumor cells in vitro and in vivo. The presence of a bystander effect was only demonstrated for cyp4B1 /2 -AA.…”

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confidence: 99%

Differential cytotoxicity and bystander effect of the rabbit cytochrome P450 4B1 enzyme gene by two different prodrugs: Implications for pharmacogene therapy

et al. 2002

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The time course of cytotoxicity induction and the bystander effect of the rabbit cytochrome P450 4B1 ( cyp4B1 ) / 4 -ipomeanol ( 4 -IM ) or 2 -aminoanthracene ( 2 -AA ) pharmacogene therapy systems were investigated and compared with the herpes simplex virus type 1 thymidine kinase / ganciclovir ( HSV -tk / GCV ) system. Experiments were performed in rat 9L gliosarcoma cells stably expressing cyp4B1 ( 9L -4B1 ), HSV -tk ( 9L -tk ), or their egfp ( enhanced green fluorescent protein ) fusion genes. Cyp4B1 -mediated activation of 2 -AA showed a high cell killing efficiency within only 48 hours with an onset after already 15 minutes of prodrug exposure. Residual 9L -4B1 cells were mostly damaged sublethally upon 2 -AA treatment showing an S phase arrest by cell cycle analysis. 4 -IM treatment of 9L -4B1 cells generated an overall weaker cell killing, especially after prodrug exposure times of less than 48 hours. Residual cells surviving 4 -IM treatment showed a G2 / M arrest and restarted proliferation after prodrug treatment was stopped. HSV -tk / GCV pharmacogene therapy resulted in a slower cytotoxicity induction than cyp4B1 / 2 -AA treatment with a significantly lower cell killing efficiency after 24 and 48 hours. HSV -tk / GCV -mediated cytotoxicity was widely similar to the cytotoxicity induced by cyp4B1 / 4 -IM with the exception of a continuous 48 -hour prodrug exposure where 4 -IM treatment showed a significantly higher cell killing rate. Cells surviving HSV -tk / GCV suicide gene therapy were not viable and showed an S -phase arrest. Whereas HSV -tk / GCV induced a strong bystander effect, only moderate bystander cell death depending on cell -to -cell contact was demonstrated in 9L / 9L -4B1 cocultures upon 2 -AA treatment and was even absent with 4 -IM, thereby contrasting with earlier reports. The absence of a strong bystander effect may limit, on one hand, the overall utility of the cyp4B1 systems for cancer gene therapy. On the other hand, the weak bystander effect together with the fast induction of cytotoxicity may provide marked advantages for the use of the cyp4B1 systems as biosafety enhancers for gene marking or replacement studies and donor lymphocyte infusions after allogeneic bone marrow transplantation.

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“…Examination of the bronchiolar epithelium of IPO-treated animals revealed the presence of squamous and irregularly shaped cuboidal cells in the distal end of the bronchiole (Figure 7C, D). Normal cuboidal epithelium was found more proximally ( Figure 7C (29,36). Immature Clara cells of neonatal rabbits have very low levels of these 2 isozymes at birth, and adult levels are reached only after 4 weeks of age (26).…”

Section: Long-term Repairmentioning

confidence: 99%

Acute Injury to Differentiating Clara Cells in Neonatal Rabbits Results in Age-Related Failure of Bronchiolar Epithelial Repair

et al. 2000

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4-Ipomeanol and 2-aminoanthracene cytotoxicity in C3H/10T12 cells expressing rabbit cytochrome P450 4B1

Cited by 29 publications

References 29 publications

Ligand characterization of CYP4B1 isoforms modified for high-level expression inEscherichia coliand HepG2 cells

Ligand characterization of CYP4B1 isoforms modified for high-level expression inEscherichia coliand HepG2 cells

Differential cytotoxicity and bystander effect of the rabbit cytochrome P450 4B1 enzyme gene by two different prodrugs: Implications for pharmacogene therapy

Acute Injury to Differentiating Clara Cells in Neonatal Rabbits Results in Age-Related Failure of Bronchiolar Epithelial Repair

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