[Preparation and Regeneration of Protoplasts from Monascus purpureus and Genetic Transformation System.].

Generation of fungal protoplast is an essential tool for genetic transformation system. To establish protoplast-mediated genetic transformation system of Monascus purpureus, conditions for the protoplast isolation and regeneration of the mycelia of various enzymes and osmotic stabilizers were examined. To investigate suitable cell age for the protoplast preparation of mycelia of M. purpureus, the mycelia were cultured in different ways at 30oC. Mycelia obtained through cellophane - mediated culture for 30~40h were adequate to protoplast preparation. When lysing enzyme, cellulase and snailase were added to the mycelia in combination or alone, combination of lysing enzyme, cellulase and snailase accordingly at the concentration of 0.3%, 0.1% and 1% was most benefit for protoplast yield. When we applied various osmotic stabilizers at different concentrations to protoplast preparation, 1 mol/L MgSO4 was most effective for the protoplast release. The suitable incubation time with enzyme for the maximum release of protoplasts was 2.5-hr. When we investigate various osmotic stabilizers for the regeneration of the protoplasts of mycelia of strain M34 and N18, the complete medium containing 0.6 mol/L sucrose induced highest hyphal growth with regeneration frequency of 8.5% and 36.4%, respectively. PEG and CaCl2- mediated protoplast co-transformation of strain M34 with pBC-Hygro and pNL1, hygromycin B as selective marker, was fulfilled and 100 stable transformants per microgram DNA were obtained.

Enzyme activity analysis of CYP2C18 with exon 5 skipped.

AIM: To study the enzyme activity of CYP2C18 variant with exon 5 skipped. METHODS: A full length CYP2C18 cDNA X1 and an exon 5 skipped variant CYP2C18 X2 were separately subcloned into mammalian expression vector pREP9 to transfect HepG2 cells. The expression of CYP2C18 mRNA in transgenic cells and human liver tissues were determined by RT-PCR. The enzyme activity of CYP2C18 to oxidate tolbutamide in postmitochondrial supernate (S9) fraction was determined by HPLC. The cytotoxicity of ifosfamide to transgenic cells was evaluated by MTT test. RESULTS: HepG2-CYP2C18 X1 cells showed strong expression of the full length CYP2C18 mRNA. On the other hand, HepG2-CYP2C18 X2 cells had only infinitesimal expression of the exon-skipped CYP2C18 as well as the full length CYP2C18, while non-transfected HepG2 cell only demonstrated an infinitesimal expression of the full length CYP2C18. The expression of CYP2C18 exons 2 to 7 was also analyzed by RT-PCR in 7 extratumoral liver tissues. Among them, 3 samples expressed only wild type mRNA, whereas 4 samples expressed both wild type and alternative splicing products. The tolbutamide hydroxylase activity of CYP2C18 was tested, and it was shown that HepG2-2C18 X1 cells had higher enzyme activity than those of HepG2-2C18 X2 and HepG2 cells. The relative survival of HepG2-CYP2C18 X1 cells was lower than that of HepG2 cells with 1, 2, and 4 mmol/L ifosfamide treatments. In contrast, the relative survival of HepG2-CYP2C18 X2 cell was the same as that of HepG2 cell in 0.5 and 1 mmol/L of ifosfamide, but lower than that of HepG2 cell in 2 and 4 mmol/L of ifosfamide. CONCLUSION: CYP2C18 X1 could metabolize tolbutamide and ifosfamide efficiently. The exon 5-skipped CYP2C18 X2 could not metabolize tolbutamide, and could not metabolize ifosfamide effectively at low concentrations.

[Establishment of a HepG2 cell line stably expressing human cytochrome P450 1A2 and its metabolic activity].

OBJECTIVE: To establish a HepG2 cell line stably expressing the human cytochrome P450 1A2 and to study its metabolic activity. METHODS: The human wild-type CYP1A2 cDNA was subcloned into a mammalian expression vector pREP9. A transgenic cell line was established by transfecting the recombinant plasmid of pREP9-CYP1A2 to HepG2 cells. The expression of CYP1A2 mRNA was validated by RT PCR. The metabolic activation of HepG2 CYP1A2 cells on aflatoxin B1 (AFB1) was assayed by cytotoxicity test. RESULT: The HepG2-CYP1A2 cells expressed CYP1A2 mRNA and could increase the cytotoxicity to AFB1 in comparison with that of wild type HepG2 cells. CONCLUSION: The established HepG2-CYP1A2 can express the mRNA and has the metabolic activity to AFB1. The cell line may be useful for testing the toxicity and metabolism of xenobiotics, which might possibly be activated or metabolized by CYP1A2.

Establishment of a transgenic cell line stably expressing human cytochrome P450 2C18 and identification of a CYP2C18 clone with exon 5 missing.

AIM: The human cytochrome P-450 2C18(CYP2C18) has been characterized. However, the protein has not been purified from liver and very little is known regarding the specific substrate of CYP2C18. In order to study its enzymatic activity for drug metabolism, the CYP2C18 cDNA was cloned and a stable CHL cell line expressing recombinant CYP2C18 was established. METHODS: The human CYP2C18 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR) from total RNAs extracted from human liver and cloned into pGEM-T vector. The cDNA segment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9. A transgenic cell line was established by transfecting the recombinant plasmid of pREP9-CYP2C18 to Chinese hamster lung (CHL) cell. The enzyme activity of CYP2C18 catalyzing oxidation of tolbutamide to hydroxytolbutamide in postmitochondrial supernant(S9) fraction of the cell was determined by high performance liquid chromatography(HPLC). RESULTS: The amino acid sequence predicted from the cloned cDNA segment was identical to that of reported by Romkes et al (GenBank accession number: M61856, J05326). The S9 fraction of the established cell line metabolizes tolbutamide to hydroxytolbutamide. Tolbutamide hydroxylase activity was found to be 0.509+/-0.052 micromol x min(-1) x g(-1) S9 protein or 8.82+/-0.90 mol x min(-1) x mol(-1) CYP, but was undetectable in parental CHL cell. In addition, we have identified a CYP2C18 cDNA clone with exon 5 missing. CONCLUSION: The cDNA of human CYP2C18 was successfully cloned and a cell line, CHL-CYP2C18, efficiently expressing the protein of CYP2C18, was established. A spliced variant of CYP2C18 with exon 5 missing was identified in the cloning process.

Cloning of cytochrome P-450 2C9 cDNA from human liver and its expression in CHL cells.

AIM: Using bacterial, yeast, or mammalian cell expressing a human drug metabolism enzyme would seem good way to study drug metabolism-related problems. Human cytochrome P-450 2C9(CYP2C9) is a polymorphic enzyme responsible for the metabolism of a large number of clinically important drugs. It ranks among the most important drug metabolizing enzymes in humans. In order to provide a sufficient amount of the enzyme for drug metabolic research, the CYP2C9 cDNA was cloned and expressed stably in CHL cells. METHODS: After extraction of total RNA from human liver tissue, the human CYP2C9 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR), and cloned into cloning vector pGEM-T. The cDNA fragment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9. A transgenic cell line was established by transfecting the recombinant vector of pREP9-CYP2C9 into CHL cells. The enzyme activity of CYP2C9 catalyzing oxidation of tolbutamide to hydroxy tolbutamide in S9 fraction of the cell was determined by high performance liquid chromatography(HPLC). RESULTS: The amino acid sequence predicted from the cDNA segment was identical to that of CYP2C9*1, the wild type CYP2C9. However, there were two base differences, i.e. 21T>C, 1146C>T, but the encoding amino acid sequence was the same, L7, P382. The S9 fraction of the established cell line metabolizes tolbutamide to hydroxy tolbutamide; tolbutamide hydroxylase activity was found to be 0.465 +/- 0.109 micromol.min(-1).g(-1) S9 protein or 8.62 +/- 2.02mol.min(-1).mol(-1) CYP, but was undetectable in parental CHL cell. CONCLUSION: The cDNA of human CYP2C9 was successfully cloned and a cell line of CHL- CYP2C9, efficiently expressing the protein of CYP2C9, was established.