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The microsomal epoxide hydrolase (mEH) catalyses the hydrolysis of reactive epoxides which are formed by the action of cytochromes P-450 from xenobiotics. In addition it has been suggested that mEH might mediate the transport of bile acids. For the mEH it has been shown that it is co-translationally inserted into the endoplasmic reticulum. Here we demonstrate that the N-terminal 20 amino acid residues of this protein serve as its single membrane anchor signal sequence and that the function of this sequence can also be supplied by a cytochrome P-450 (CYP2B1) anchor signal sequence. The evidence supporting this conclusion is as follows: (i) the rat mEH and a CYP2B1-mEH fusion protein, in which the CYP2B1 membrane anchor signal sequence replaced the N-terminal 20 amino acid residues of mEH, was co-translationally inserted into dog pancreas microsomes in a cell-free translation system, whereas a truncated epoxide hydrolase with a deletion of the 20 N-terminal amino acid residues was not co-translationally inserted. (ii) The mEH and the CYP2B1-mEH fusion protein, but not the truncated epoxide hydrolase, were anchored in microsomes in a cell-free translation system and in membrane fractions derived from fibroblasts which expressed these proteins heterologously. These fibroblasts were also used to evaluate the significance of the mEH membrane anchor for the catalytic activity of mEH. The mEH, the truncated mEH and the CYP-EH fusion protein were found to be enzymically active. This result shows that the membrane anchor signal sequence of mEH is dispensable for the catalytic activity of this protein. However, truncated mEH was only expressed at low levels, which might indicate that this protein is unstable.

Expression of mdm-2 mRNA was measured in 90 ovarian-cancer tissue specimens using the S1 nuclease assay, to investigate a possible association between MDM2 expression and prognosis. mdm-2 mRNA expression was an independent prognostic factor for patients with primary ovarian cancer, FIGO (International Federation of Gynecology and Obstetrics) stages III and IV (n 5 57), who all received chemother-apy with carboplatin or cisplatin and cyclophosphamide. Median survival time for patients (FIGO stages III and IV) with no detectable expression of mdm-2 mRNA (n 5 14) was 171 days, as compared with 839 days for patients (n 5 43) with detectable mdm-2 mRNA (p 5 0.0194; log-rank test). However, no association between mdm-2 mRNA expression and survival was observed for patients with FIGO stages I and II who did not receive chemotherapy. mdm-2 expression was not associated with FIGO stage, residual disease, histologic grade and type. Our results suggest that mdm-2, which is known to disrupt p53 function, sensitizes ovarian-cancer cells to cisplatin/cyclophosphamide, possibly by inhibition of p53-mediated G 1 cell-cycle arrest and p53-stimulated nucleotide-excision repair. Int.

An important feature of cytochrome P450 (CYP) 2B1 is its high ability to convert the prodrug cyclophosphamide (CPA) to therapeutically cytotoxic metabolites, resulting in interstrand DNA-cross-linking and cell death. We have examined whether and how the phosphorylation of CYP2B1 influences CPA metabolic activation in vitro and in vivo. We found first that only part of the total CYP2B1 pool undergoes phosphorylation. This part is fully inactivated. Second, phosphorylation of CYP2B1 in intact hepatocytes reduced by up to 75% toxification of CPA to mutagenic metabolites (totally dependent on the same preferentially CYP2B-catalyzed 4-hydroxylation of CPA as is the generation of highly cytotoxic species). Third, the phosphoacceptor-serine 128 of CYP2B1 in the consensus sequence for interaction with the protein kinase A represents an on/off switch for the activation of CPA depending on the phosphorylation conditions in the cell. Fourth, evidence is presented that the above-described events also occur in vivo. In conclusion, a successful therapy with CPA, helped by forced expression of CYP2B1 in tumor cells (as recently proposed) will, in addition, be profoundly modified by its phosphorylation status. © 2001 Wiley-Liss, Inc. Key words: cyclophosphamide; CYP2B1; phosphorylation; hormonal controlThe activity of many of the foreign compound metabolizing enzymes, e.g., CYP, is of obvious importance for both physiologic homeostasis as well as effects of drugs and environmental chemicals. 1 Much of the detoxification is performed by CYP enzymes. Many of them are inducible and have a broad and overlapping substrate specificity. 2 Interindividual differences in the expression level of CYP enzymes are known, 3 thus contributing to the variations in toxic symptoms and therapeutic response evoked by antineoplastic drugs. 4 CYP2B1 (the major phenobarbital-inducible form) has long been suggested as 1 of the most important enzymes involved in the activation of widely used antineoplastic prodrugs. It catalyzes oxidative desulfuration of thio-TEPA to TEPA as well as 1-electron reduction of Adriamycin to its semiquinone free radical [5][6][7] and, most importantly, is responsible for conversion of the oxazaphosphorines CPA and IFA to their active metabolites. 8,9 It has been shown that 3 specific rat liver CYP enzymes (CYP2B1, CYP2C6 [constitutively expressed, gender independent] and CYP2C11 [constitutively expressed, male specific]) are major catalysts of CPA activation in adult rat liver, 8,10 while a rat CYP3A enzyme additionally contributes to the activation of IFA. 9 The corresponding human enzymes CYP2B6 and CYP3A4 activate CPA and IFA in human liver, respectively. 11-13 It was demonstrated that among rat enzymes CYP2B1 (the ortholog of the human CYP2B6) is by far the most active catalyst. 8,11,12 Activation of CPA to the therapeutic active species has its initial step in the liver. 8 The CYP2B1-catalyzed 4-hydroxylation reaction forms a highly unstable metabolite, which undergoes spontaneous ␤-elimination and gives rise to 2 pro...