Tomomichi Sone

Involvement of Carboxylesterase 1 and 2 in the Hydrolysis of Mycophenolate Mofetil

Mycophenolate mofetil (MMF) is the ester prodrug of the immunosuppressant agent mycophenolic acid (MPA) and is rapidly activated by esterases after oral administration. However, the role of isoenzymes in MMF hydrolysis remains unclear. Although human plasma, erythrocytes, and whole blood contain MMF hydrolytic activities, the mean half-lives of MMF in vitro were 15.1, 1.58, and 3.20 h, respectively. Thus, blood esterases seemed to contribute little to the rapid MMF disappearance in vivo. In vitro analyses showed that human intestinal microsomes exposed to 5 and 10 μM MMF exhibited hydrolytic activities of 2.38 and 4.62 nmol/(min · mg protein), respectively. Human liver microsomes exhibited hydrolytic activities of 14.0 and 26.1 nmol/(min · mg protein), respectively, approximately 6-fold higher than those observed for intestinal microsomes. MMF hydrolytic activities in human liver cytosols were 1.40 and 3.04 nmol/(min · mg protein), respectively. Because hepatic cytosols generally contain 5-fold more protein than microsomes, MMF hydrolysis in human liver cytosols corresponded to approximately 50% of that observed in microsomes. Fractions obtained by 9000g centrifugation of supernatants from COS-1 cells expressing human carboxylesterase (CES) 1 or 2 exhibited MMF hydrolytic activity, with CES1-containing fractions showing higher catalytic efficiency than CES2-containing fractions. The CES inhibitor bis-p-nitrophenylphosphate inhibited MMF hydrolysis in human liver microsomes and cytosols with IC50 values of 0.51 and 0.36 μM, respectively. In conclusion, both intestinal and hepatic CESs and in particular CES1 may be involved in MMF hydrolysis and play important roles in MMF bioactivation. Hepatic CES1 activity levels may help explain the between-subject variability observed for MMF usage.

Chromium(VI) inhibits mouse metallothionein-I gene transcription by preventing the zinc-dependent formation of an MTF-1-p300 complex.

Mouse MT-I (metallothionein-I) transcription is regulated by MTF-1 (metal-response-element-binding transcription factor-1) which is recruited to the promoter in response to zinc. Cr(VI) [chromium(VI)] pretreatment blocks zinc-activation of the endogenous MT-I gene and attenuates zinc-activation of MT-I-promoter-driven luciferase reporter genes in transient transfection assays. Chromatin immunoprecipitation assays revealed that Cr(VI) only modestly reduces recruitment of MTF-1 to the MT-I promoter in response to zinc, but drastically reduces the recruitment of RNA polymerase II. These results suggest that Cr(VI) inhibits the ability of MTF-1 to transactivate this gene in response to zinc. Zinc has recently been shown to induce the formation of a co-activator complex containing MTF-1 and the histone acetyltransferase p300 which plays an essential role in the activation of MT-I transcription. In the present study, co-immunoprecipitation assays demonstrated that Cr(VI) pretreatment blocks the zinc-induced formation of this co-activator complex. Thus Cr(VI) inhibits mouse MT-I gene expression in response to zinc by interfering with the ability of MTF-1 to form a co-activator complex containing p300 and recruiting RNA polymerase II to the promoter.

Ethanol-induced expression of glutamate–cysteine ligase catalytic subunit gene is mediated by NF-κB

Glutamate-cysteine ligase is a rate-limiting enzyme in the de novo synthesis of glutathione, a known scavenger of electrophiles and reactive oxygen species. Glutamate-cysteine ligase catalytic subunit (GCLC) is regulated transcriptionally by nuclear factor erythroid 2-related factor 2 (Nrf2). It has been reported that ethanol induces human GCLC production via Nrf2-mediated transactivation of the antioxidant-responsive element (ARE). Here, the luciferase reporter assay revealed the presence of an ethanol-responsive element in the human GCLC promoter; it spanned bases -1432 to -832 in hepatocytes and HepG2 cells transfected with cytochrome P450 2E1 (CYP2E1). The region lacked an ARE but had a putative nuclear factor-kappaB (NF-kappaB) element. NF-kappaB DNA-binding activity was activated in response to ethanol treatment. CYP2E1 expression was required for GCLC promoter-driven gene expression and the activation of NF-kappaB. Thus ethanol-induced GCLC transcription is mediated by not only Nrf2 but also NF-kappaB.

Cloning and sequence analysis of a hamster liver cDNA encoding a novel putative carboxylesterase

A full-length cDNA encoding for a putative carboxylesterase was isolated from a hamster liver cDNA library. The cDNA consisting of 1911 base pairs contained an open reading frame of 1683 base pairs encoding for a polypeptide of 561 amino-acid residues, including 27 N-terminal amino-acid residues for signal peptide. The deduced amino-acid sequence of the cDNA is in 67% homology with the amino-acid sequence of rabbit form 2 carboxylesterase, which has not yet been cloned. It also had many structural features highly conserved among carboxylesterase isozymes.

Comparative study of the hydrolytic metabolism of methyl-, ethyl-, propyl-, butyl-, heptyl- and dodecylparaben by microsomes of various rat and human tissues

Abstract 1. Hydrolytic metabolism of methyl-, ethyl-, propyl-, butyl-, heptyl- and dodecylparaben by various tissue microsomes and plasma of rats, as well as human liver and small-intestinal microsomes, was investigated and the structure–metabolic activity relationship was examined. 2. Rat liver microsomes showed the highest activity toward parabens, followed by small-intestinal and lung microsomes. Butylparaben was most effectively hydrolyzed by the liver microsomes, which showed relatively low hydrolytic activity towards parabens with shorter and longer alkyl side chains. 3. In contrast, small-intestinal microsomes exhibited relatively higher activity toward longer-side-chain parabens, and showed the highest activity towards heptylparaben. 4. Rat lung and skin microsomes showed liver-type substrate specificity. Kidney and pancreas microsomes and plasma of rats showed small-intestinal-type substrate specificity. 5. Liver and small-intestinal microsomal hydrolase activity was completely inhibited by bis(4-nitrophenyl)phosphate, and could be extracted with Triton X-100. Ces1e and Ces1d isoforms were identified as carboxylesterase isozymes catalyzing paraben hydrolysis by anion exchange column chromatography of Triton X-100 extract from liver microsomes. 6. Ces1e and Ces1d expressed in COS cells exhibited significant hydrolase activities with the same substrate specificity pattern as that of liver microsomes. Small-intestinal carboxylesterase isozymes Ces2a and Ces2c expressed in COS cells showed the same substrate specificity as small-intestinal microsomes, being more active toward longer-alkyl-side-chain parabens. 7. Human liver microsomes showed the highest hydrolytic activity toward methylparaben, while human small-intestinal microsomes showed a broadly similar substrate specificity to rat small-intestinal microsomes. Human CES1 and CES2 isozymes showed the same substrate specificity patterns as human liver and small-intestinal microsomes, respectively.

Inhibition of prostate‐specific membrane antigen (PSMA)‐positive tumor growth by vaccination with either full‐length or the C‐terminal end of PSMA

For experimental immunotherapy of prostate cancer, we used a model system to target a defined region of the extracellular domain of prostate‐specific membrane antigen (PSMA). PSMA is a surface antigen expressed by prostate epithelium that is upregulated approximately 10‐fold in most prostate tumors. We vaccinated BALB/c mice with NIH3T3 cells cotransfected with pST/neo plus pEF‐BOS‐based vectors expressing either the full‐length 750–amino acid human PSMA or only the C‐terminal 180–amino acid region (PSMc). PSMc lies C‐terminal to the transferrin receptor‐like sequence in the extracellular domain of PSMA. BALB/c mice were injected i.p. 4 times at weekly intervals with vaccine cells. Vaccinated mice were then challenged s.c. with Renca/PSMA, a BALB/c renal cell carcinoma line transfected to express human PSMA. Growth of Renca/PSMA tumors was substantially retarded and host survival significantly prolonged in mice prevaccinated with either 3T3/PSMA or 3T3/PSMc. Furthermore, antiserum from vaccinated mice intensely immunocytochemically stained LNCaP, a PSMA‐positive human prostate cancer cell line. In contrast, control mice similarly prevaccinated i.p. with 3T3/neo (NIH3T3 cells transfected with pST/neo alone) developed Renca/PSMA tumors, which were palpable within 2 weeks and lethal by 5 weeks. Serum from 3T3/neo‐vaccinated mice did not immunocytochemically stain LNCaP cells. The antitumor activity induced by vaccination with 3T3/PSMc was also demonstrated via growth inhibition of established LNCaP tumors xenografted in athymic mice following passive transfer of immune serum from vaccinated mice. Our results suggest that vaccination with PSMc induces adaptive humoral activity, which is directed against the extracellular region of human PSMA and can significantly inhibit human prostate cancer growth in athymic mice, and that administration of antibodies to PSMA may provide a passive treatment modality for immunocompromised patients.

Induction of Antibodies against Prostate-Specific Membrane Antigen (PSMA) by Vaccination with a PSMA DNA Vector

Abstract Introduction and Objectives: Prostate-specific membrane antigen (PSMA) is a 750 amino acid surface protein expressed primarily in prostate epithelium, and is upregulated 10-fold in prostate cancer. It is therefore an attractive target for immunotherapy. However, most reported antibodies to PSMA apparently recognize epitopes in the residue 43–570 region of the extracellular domain, and upon binding are rapidly removed from the cell surface by internalization. This would potentially limit their ability to mediate Fc-dependent cytoxicity. In this study, we constructed a DNA expression vector, pV/TM–PSMc, in which this region was deleted from full-length PSMA cDNA. Mice were vaccinated with pV/TM–PSMc DNA to determine whether humoral responses directed against PSMA-positive human prostate cancer cells could be induced by this C-terminal region. Methods: Polymerase chain reaction (PCR)-based techniques were used to delete codons 50–570 from the coding region of human PSMA cDNA, thereby joining the C-terminal end (PSMc) to the N-terminal cytoplasmic/transmembrane domain (TM). This truncated product, TM–PSMc, was cloned into the vector pNGVL3 (pV). The resulting vector, pV/TM–PSMc, was confirmed by DNA sequencing, and by expression studies using reverse transcriptase (RT)–PCR for transcripts and immunohistochemical (IHC) staining with the PSMA monoclonal antibody (mAb) 7E11.C5. BALB/c mice were injected in the tibialis anterior muscle four times, at biweekly intervals, with 100μg vector DNA per injection. One week after the last injection, blood was drawn for serum preparation. The serum was assayed for antibodies against PSMA by IHC staining of LNCaP, a PSMA-positive human prostate cancer line. Expression in vaccinated muscle cells was determined by RT–PCR assay for TM–PSMc transcripts. Results: NIH3T3 cells transfected with pV/TM–PSMc stained positively by IHC reaction with mAb 7E11.C5. 48h after one intramuscular (i.m.) injection of mice with 100μg pV/TM–PSMc vector DNA, TM–PSMc transcripts were detectable in muscle RNA by RT–PCR analysis. Anti-serum from pV/TM–PSMc-DNA vaccinated mice, at a dilution of 1:20, intensely IHC-stained both live and fixed LNCaP cells. Conclusions: These results demonstrate that anti-PSMA humoral responses were induced by i.m. injection of mice with pV/TM–PSMc DNA. Antibodies in the anti-serum were directed against extracellular epitopes of native PSMA expressed by human prostate cancer cells. Vaccination with DNA expression vectors such as pV/TM–PSMc may provide an immunotherapeutic approach for the treatment of prostate cancer.

C-terminal deletion mutant of MRE-binding transcription factor-1 inhibits MRE-driven gene expression.

Heavy metal‐induced transcriptional activation of the genes coding for metallothionein (MT) is mediated by a cis‐acting DNA element, the metal‐responsive element (MRE). MRE‐binding transcription factor‐1 (MTF‐1) is a highly conserved heavy metal‐induced transcriptional activator. MTF‐1 also activates transcription in response to oxidative stress and regulates the expression of several cytoprotective factor genes, including MT, γ‐glutamylcysteine synthetase, and Cu/Zn‐superoxide dismutase. It is thus thought that MTF‐1 plays a role in cellular stress response. The physiological role of MTF‐1 remains unclear because of the lack of MTF‐1‐specific activators and/or inhibitors. To obtain an MTF‐1‐specific inhibitor, we constructed an MTFΔC (amino acids 1–317), a C‐terminal deletion mutant of MTF‐1. MTFΔC could bind MRE and competed with MTF‐1 for MTF–MRE complex formation. Transient expression of MTFΔC in HepG2 cells reduced MRE‐driven gene expression, demonstrating that MTFΔC is dominant to MTF‐1. HepG2 cells stably expressing MTFΔC showed increased susceptibility to the cytotoxic effects of tert‐butyl hydroperoxide (tBH). Furthermore, we constructed Ad5MTFΔC, a recombinant adenovirus that expresses MTFΔC. Infection with the virus induced MTFΔC expression and increased susceptibility to the cytotoxic effects of tBH. These results indicate that MTF‐1 participates in controlling the cellular redox state.

Role of metal-responsive transcription factor-1 (MTF-1) in EGF-dependent DNA synthesis in primary hepatocytes.

Metal‐responsive transcription factor‐1 (MTF‐1), which is involved in sensing heavy metal load, induces the transcription of several protective genes. The mouse Mtf‐1 gene is essential, and Mtf‐1−/− embryos die from liver degeneration. We showed that DNA synthesis induced in hepatocytes by epidermal growth factor (EGF) was delayed by inhibition of MTF‐1. To inhibit MTF‐1 activity, MTFΔC, a C‐terminal deletion mutant of MTF‐1, was expressed by infection with the virus Ad5MTFΔC. Lactate dehydrogenase (LDH) release and/or caspase‐3/7 activation was not observed under our experimental conditions. The inhibitory effect of MTFΔC on EGF‐dependent DNA synthesis in hepatocytes was not eliminated by zinc addition. EGF‐dependent extracellular signal‐related kinase (ERK) phosphorylation, an essential reaction for EGF‐dependent DNA synthesis, was decreased in MTF‐1‐inhibited hepatocytes. Moreover, decrease of ERK phosphorylation was observed by using siRNA in MTF‐1‐downregulated hepatocytes. These results indicate that MTF‐1 is particularly important for proper hepatocyte proliferation. This is the first report to suggest the function of MTF‐1 in the ERK pathway. J. Cell. Biochem. 99: 485–494, 2006.

Comparative study of hydrolytic metabolism of dimethyl phthalate, dibutyl phthalate and di(2-ethylhexyl) phthalate by microsomes of various rat tissues

Phthalates are used in food packaging, and are transferred to foods as contaminants. In this study, we examined the hydrolytic metabolism of dimethyl phthalate (DMP), dibutyl phthalate (DBP) and di(2-ethylhexyl) phthalate (DEHP) by rat tissue microsomes. We found that carboxylesterase and lipase contribute differently to these activities. When DMP, DBP and DEHP were incubated with rat liver microsomes, DBP was most effectively hydrolyzed to the phthalate monoester, followed by DMP, and the activity toward DEHP was marginal. In contrast, small-intestinal microsomes exhibited relatively higher activity toward long-side-chain phthalates. Pancreatic microsomes showed high activity toward DEHP and DBP. Liver microsomal hydrolase activity toward DMP was markedly inhibited by bis(4-nitrophenyl)phosphate, and could be extracted with Triton X-100. The activity toward DBP and DEHP was partly inhibited by carboxylesterase inhibitor, and was partly solubilized with Triton X-100. Ces1e, Ces1d and Ces1f expressed in COS cells exhibited the highest hydrolase activity toward DBP, showing a similar pattern to that of liver microsomes. Ces1e showed activity towards DMP and DEHP. Pancreatic lipase also hydrolyzed DBP and DEHP. Thus, carboxylesterase and lipase contribute differently to phthalate hydrolysis: short-side-chain phthalates are mainly hydrolyzed by carboxylesterase and long-side-chain phthalates are mainly hydrolyzed by lipase.