JMJD5 cleaves monomethylated histone H3 N-tail under DNA damaging stress.

The histone H3 N-terminal protein domain (N-tail) is regulated by multiple posttranslational modifications, including methylation, acetylation, phosphorylation, and by proteolytic cleavage. However, the mechanism underlying H3 N-tail proteolytic cleavage is largely elusive. Here, we report that JMJD5, a Jumonji C (JmjC) domain-containing protein, is a Cathepsin L-type protease that mediates histone H3 N-tail proteolytic cleavage under stress conditions that cause a DNA damage response.

Proteomic analysis of cellular response to microcystin in human amnion FL cells.

Microcystins (MC), the potent inhibitor of protein phosphatase 1 and 2A, are hepatotoxins of increasing importance due to its high acute toxicity and potent tumor promoting activity. So far, the exact mechanisms of MC-induced hepatotoxicity and tumor promoting activity have not been fully elucidated. To better understand the mechanisms underlying microcystin-RR (MC-RR) induced toxicity as well as provide the possibility for the establishment of biomarkers for MC-RR exposure, differential proteome analysis on human amnion FL cells treated by MC-RR was carried out using two-dimensional gel electrophoresis (2-DE) followed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry.

N-methyl-N'-nitro-N-nitrosoguanidine sensitivity, mutator phenotype and sequence specificity of spontaneous mutagenesis in FEN-1-deficient cells.

Intact pZ189 DNA was allowed to replicate in FL-FEN-1(-) cell line that was established in this laboratory in which the expression of FEN-1 gene was blocked by dexamethasone-inducible expression of antisense RNA to FEN-1. E. coli MBM7070 was transfected with the replicated plasmid, and those with mutations in the supF gene were identified.

Three new alternative splicing variants of human cytochrome P450 2D6 mRNA in human extratumoral liver tissue.

Aim: To identify the new alternative splicing variants of human CYP2D6 in human extratumoral liver tissue with RT-PCR and sequencing.

Methods: Full length of human CYP2D6 cDNAs was amplificated by reverse transcription-polymerase chain reaction (RT-PCR) from a human extratumoral liver tissue and cloned into pGEM-T vector. The cDNA was sequenced.

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.

Stable expression of human cytochrome P450 2D6*10 in HepG2 cells.

Aim: Over 90% of drugs are metabolized by the cytochrome P-450 (CYP) family of liver isoenzymes. The most important enzymes are CYP1A2, 3A4, 2C9/19, 2D6 and 2E1. Although CYP2D6 accounts for <2% of the total CYP liver enzyme content, it mediates metabolism in almost 25% of drugs.

ATM and ATR: sensing DNA damage.

Cellular response to genotoxic stress is a very complex process, and it usually starts with the "sensing" or "detection" of the DNA damage, followed by a series of events that include signal transduction and activation of transcription factors. The activated transcription factors induce expressions of many genes which are involved in cellular functions such as DNA repair, cell cycle arrest, and cell death. There have been extensive studies from multiple disciplines exploring the mechanisms of cellular genotoxic responses, which have resulted in the identification of many cellular components involved in this process, including the mitogen-activated protein kinases (MAPKs) cascade.

Heterologous expression of human cytochrome P450 2E1 in HepG2 cell line.

Aim: Human cytochrome P-450 2E1 (CYP2E1) takes part in the biotransformation of ethanol, acetone, many small-molecule substrates and volatile anesthetics. CYP2E1 is involved in chemical activation of many carcinogens, procarcinogens, and toxicants. To assess the metabolic and toxicological characteristics of CYP2E1, we cloned CYP2E1 cDNA and established a HepG2 cell line stably expressing recombinant CYP 2E1.

[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.

[Establishment of a cell line with antisense-blocked POLH and the role of POLH in alkylating agent MNNG induced nontargeted mutagenesis].

Objective: To investigate the function of POLH(polymerase eta) through establishment of the POLH gene-blocked cell line FL-POLH(-).

Methods: A mammalian expression vector expressing antisense POLH gene fragment pMAMneo-amp-POLHA (-) was constructed by cloning the 1473 - 2131 fragment of POLH gene into the mammalian expression vector pMAMneo-amp(-) in antisense orientation. The FL cells were transfected with this antisense RNA expressing vector and selected by G418.

[Cloning and bioinformatics of human REV3 gene promoter region and its response to carcinogen N-methyl-N'-nitro-N-nitrosoguanidine].

Objective: To understand the up regulatory mechanism of human REV3 gene induced by the chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).

Methods: Bioinformatic analysis of human REV3 gene promoter region was based on BLAST alignment, promoter prediction software and recognition of transcriptional factor binding sites. Cloning of human REV3 gene promoter region was performed by nested PCR.

[Activation of transcription factors induced by low concentration of N-methyl-N'-nitro-N-nitrosoguanidine].

Objective: To investigate the effect of MNNG on some of the transcription factors such as NF- kappaB, CREB, AP-1 and c-Myc.

Methods: The activities of these transcription factors were measured by transient transfection assay of SEAP vectors.

Result: The expressions of AP-1, CREB and NF- kappaB driven reporter genes were elevated for about 1.

[Activation of nucleus-independent signals triggered by N-methyl-N'-nitro-N- nitrosoguanidine].

Objective: To study the effect of MNNG on inducement of non-targeted mutation and activation of several cellular signal transduction pathways, and to determine whether the activation of these signaling pathways was dependent on the DNA-damage.

Methods: Vero cells were enucleated by discontinuous density centrifugation. The PKA activities were measured by enzyme-linked immunosorbent assay.

[Altered of zinc finger proteins expression in FL cells following benzo [a] pyrene treatment].

Objective: To understand benzo[a]pyrene (B[a]P) mediated cellular responses, and to provide clues to explore molecular mechanism of mutagenesis and carcinogenesis induced by B[a]P.

Methods: Two-dimensional electrophoresis (2-DE) was used to investigate the protein expression levels of FL cells after B[a]P exposure, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) combined with database search was applied to identify the differentially expressed proteins.

Result: Statistical analysis showed that the volumes of 47 protein spots were altered after B[a]P treatment (P<0.

[Proteomic analysis of cellular responses to low concentration of N-methyl-N'-nitro-N-nitrosoguanidine in human amnion FL cells].

Objective: To investigate the protein profile after treatment of low concentration of N- methyl-N'-nitro-N-nitrosoguanidine (MNNG) in human FL cells.

Methods: After MNNG treatment, whole cellular proteins were separated using two-dimensional gel electrophoresis and visualized by silver staining; the digitized images were analyzed with 2D analysis software. The differentially expressed protein spots were identified by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS).

[Stereoselective determination of propranolol enantiomer in transgenic cell lines expressing human cytochrome P450].

Objective: To establish a chiro chromatography for studying the stereoselective metabolism of propranolol (PL) in S(9) incubates prepared from transgenic cell lines expressing human cytochrome P450.

Methods: The concentration of each enantiomer in S(9) incubates was determined through precolumn derivatization with GITC, followed by RP-HPLC assay using S-(+)-propafenone as internal standard.

Results: Baseline separations among the diastereomers of S(-)-P, internal standard and R(+)-PL were achieved on Shimpack CLC C(18)ODS column, with UV detection and methanol:water:glacial acetic acid (67/33/0.

Response of human REV3 gene to gastric cancer inducing carcinogen N-methyl-N'-nitro-N-nitrosoguanidine and its role in mutagenesis.

Aim: To understand the response of human REV3 gene to gastric cancer inducing carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and its role in human mutagenesis.

Methods: The response of the human REV3 gene to MNNG was measured in human 293 cells and FL cells by RT-PCR. By using antisense technology, mutation analysis at HPRT locus (on which lesion-targeted mutation usually occurs) was conducted in human transgenic cell line FL-REV3(-) by 8-azaguanine screening, and mutation occurred on undamaged DNA template was detected by using a shuttle plasmid pZ189 as the probe in human transgenic cell lines 293-REV3(-) and FL-REV3(-).

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.

Establishment of Cell Line with the FEN-1 Gene Blocked by Its Antisense.

FEN-1 is a structure-specific endo/exonuclease, which is involved in the process of both DNA duplication and DNA repair. In this work a mammalian expression vector expressing antisense FEN-1 gene fragment pMAMneoAmp(-)FNB(-) was constructed, after cloning the NcoI-BamHI fragment of FEN-1 gene into the mammalian expression vector pMAMneoAmp(-) in antisense orientation. After FL cell was transfected with pMAMneoAmp(-)FNB(-) and selected by G418, the FL-FEN-1(-) cell line, in which the FEN-1 gene expression was blocked, was established.

The Influence of FEN-1 Gene on Cell Cycle and Genetic Stability.

FEN-1 is essential in the cell replication, repair and in the maintenance of cellular genetic stability. In this report, it was verfied that FEN-1 antisense mRNA fragment was expressed in the cell line FL-FEN-1(-),constructed in our lab, blocking FEN-1 gene expression. It was found by the flow cytometer analysis that the cell cycle of FL-FEN-1(-) cells was delayed in the S-phase DNA synthesis process and arrested in G(1) phase.

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.