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.

Effect of Nimesulide on proliferation and apoptosis of human hepatoma SMMC-7721 cells

AIM: Cyclooxygenase-2 (COX-2) has been suggested to be associated with carcinogenesis. We sought to investigate the effect of the selective COX-2 inhibitor, Nimesulide on proliferation and apoptosis of SMMC-7721 human hepatoma cells.METHODS: This study was carried out on the culture of hepatic carcinoma SMMC-7721 cell line. Various concentrations of Nimesulide (0, 200 micromol/L, 300 micromol/L, 400 micromol/L) were added and incubated. Cell proliferation was detected with MTT colorimetric assay, cell apoptosis by electron microscopy, flow cytometry and TUNEL.RESULTS: Nimesulide could significantly inhibit SMMC-7721 cells proliferation dose-dependent and in a dependent manner compared with that of the control group. The duration lowest inhibition rate produced by Nimesulide in SMMC-7721 cells was 19.06%, the highest inhibition rate was 58.49%. After incubation with Nimesulide for 72 h, the most highest apoptosis rate and apoptosis index of SMMC-7721 cells comparing with those of the control were 21.20%+/-1.62% vs 2.24%+/-0.26% and 21.23+/-1.78 vs 2.01+/-0.23 (P【0.05). CONCLUSION:The selective COX-2 inhibitor, Nimesulide can inhibit the proliferation of SMMC-7721 cells and increase apoptosis rate and apoptosis index of SMMC-7721 cells. The apoptosis rate and the apoptosis index are dose-dependent. Under electron microscope SMMC-7721 cells incubated with 300 micromol and 400 micromol Nimesulide show apoptotic characteristics. With the clarification of the mechanism of selective COX-2 inhibitors, These COX-2 selective inhibitors can become the choice of prevention and treatment of cancers.

The promoting molecular mechanism of alphafetoprotein on the growth of human hepatoma Bel7402 cell line

AIM: The goal of this study was to characterize the AFP receptor, its possible signal transduction pathway and its proliferative functions in human hepatoma cell line Bel 7402. METHODS: Cell proliferation enhanced by AFP was detected by MTT assay, 3H-thymidine incorporation and S-stage percentage of cell cycle analysis. With radioactive labeled 125I-AFP for receptor binding assay; cAMP accumulation, protein kinase A activity were detected by radioactive immunosorbent assay and the change of intracellular free calcium (Ca2+i) was monitored by scanning fluorescence intensity under TCS-NT confocal microscope. The expression of oncogenes N- ras, p 53, and p21( ras ) in the cultured cells in vitro were detected by Northern blotting and Western blotting respectively. RESULTS: It was demonstrated that AFP enhanced the proliferation of human hepatoma Bel 7402 cell in a dose dependent fashion as shown in MTT assay, (3)H-thymidine incorporation and S-phase percentage up to 2-fold. Two subtypes of AFP receptors were identified in the cells with Kds of 1.3 x 10(-9)mol.L(-1) and 9.9 x10(-8)mol. (-1)L respectively. Pretreatment of cells with AFP resulted in a significant increase (625%) in cAMP accumulation. The activity of protein kinase A activity were increased up to 37.5, 122.6, 73.7 and 61.2% at treatment time point 2, 6, 12 and 24 hours. The level of intracellular calcium were elevated after the treatment of alpha-fetoprotein and achieved to 204% at 4 min. The results also showed that AFP(20mg.L(-1)) could upregulate the expression of N- ras oncogenes and p 53 and p21( ras ) in Bel 7402 cells. In the later case,the alteration were 81.1%(12h) and 97.3%(12h) respectively compared with control. CONCLUSION: These results demonstrate that AFP is a potential growth factor to promote the proliferation of human hepatoma Bel 7402 cells. Its growth-regulatory effects are mediated by its specific plasma membrane receptors coupled with its transmembrane signaling transduction through the pathway of cAMP-PKA and intracellular calcium to regulate the expression of oncogenes.

Hepatitis C virus infection of human hepatoma cell line 7721 in vitro

AIM: To establish a cell culture system with long-term replication of hepatitis C virus in vitro. METHODS: Human hepatoma cell line 7721 was tested for its susceptibility to HCV by incubating with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various phases during the culturing periods. The presence of HCV RNA, the expression of HCV antigens in cells and/or supernatant were examined by RTPCR, in situ hybridization and immunohistochemistry respectively. RESULTS: The intracellular HCV RNA was first detected on d2 after infection and then could be intermittently detected in both cells and supernatant over a period of at least three months. The expression of HCV NS3,CP10 antigens could be observed in cells. The fresh cells could be infected by supernatant from cultured infected cells and the transmission of viral genome from HCV-infected 7721 cells to PBMCs was also observed. CONCLUSION: The hepatoma line 7721 is not only susceptible to HCV but also supports its long-term replication in vitro.

Changes of NF-kB,p53,Bcl-2 and caspase in apoptosis induced by JTE-522 in human gastric adenocarcinoma cell line AGS cells:role of reactive oxygen species

AIM: To identify whether JTE-522 can induce apoptosis in AGS cells and ROS also involved in the process, and to investigate the changes in NF-kB, p53, bcl-2 and caspase in the apoptosis process. METHODS: Cell culture, MTT, Electromicroscopy, agarose gel electrophoresis, lucigenin, Western blot and electrophoretic mobility shift assay (EMSA) analysis were employed to investigate the effect of JTE-522 on cell proliferation and apoptosis in AGS cells and related molecular mechanisms. RESULTS: JTE-522 inhibited the growth of AGS cells and induced the apoptosis. Lucigenin assay showed the generation of ROS in cells under incubation with JTE-522. The increased ROS generation might contribute to the induction of AGS cells to apoptosis. EMSA and Western blot revealed that NF-kB activity was almost completely inhibited by preventing the degradation of IkBalpha. Additionally, by using Western blot we confirmed that the level of bcl-2 was decreased, whereas p53 showed a great increase following JTE-522 treatment. Their changes were in a dose-dependent manner. CONCLUSION: These findings suggest that reactive oxygen species, NF-kB, p53, bcl-2 and caspase-3 may play an important role in the induction of apoptosis in AGS cells after treatment with JTE-522.

Effect of heparin on apoptosis in human nasopharyngeal carcinoma CNE2 cells

In order to study the mechanism of the effect of heparin on apoptosis in carcinoma cells, the nasopharyngeal carcinoma cell line CNE2 was used to identify the effect of heparin on apoptosis associated with the expression of c-myc, bax, bcl-2 proteins by use of Hoechst 33258 staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), agarose gel electrophoresis, and flow cytometry, as well as Western blot analysis. The results showed that heparin induced apoptosis of CNE2 cells including the morphologic changes such as reduction in the volume, and the nuclear chromatin condensation, as well as the 'ladder pattern' revealed by agarose gel electrophoresis of DNA in a concentration-dependent manner. The number of TUNEL-positive cells was dramatically increased to 33.6+/-1.2% from 2.8+/-0.3% by treatment with heparin in different concentrations (10 to approximately 40 kU/L). The apoptotic index was increased to 32.5% from 3.5% by detecting SubG1 peaks on flow cytometry. Western blot analysis showed that levels of bcl-2, bax and c-myc were significantly overexpressed by treatment with the increase of heparin concentrations. These results suggest that heparin induces apoptosis of CNE2 cells, which may be regulated by differential expression of apoptosis-related genes.

Transepithelial transport of putrescine across monolayers of the human intestinal epithelial cell line, Caco-2

AIM: To study the transepithelial transport characteristics of the polyamine putrescine in human intestinal Caco-2 cell monolayers to elucidate the mechanisms of the putrescine intestinal absorption. METHODS: The transepithelial transport and the cellular accumulation of putrescine was measured using Caco-2 cell monolayers grown on permeable filters. RESULTS: Transepithelial transport of putrescine in physiological concentrations (】 0.5 mM) from the apical to basolateral side was linear. Intracellular accumulation of putrescine was higher in confluent than in fully differentiated Caco-2 cells, but still negligible (less than 0.5%) of the overall transport across the monolayers in apical to basolateral direction.EGF enhanced putrescine accumulation in Caco-2 cells by four fold, as well as putrescine conversion to spermidine and spermine by enhancing the activity of S adenosylmethionine decarboxylase. However, EGF did not have any significant influence on putrescine flux across the Caco-2 cell monolayers. Excretion of putrescine from Caco-2 cells into the basolateral medium did not exceed 50 picomoles, while putrescine passive flux from the apical to the basolateral chamber, contributed hundreds of micromoles polyamines to the basolateral chamber. CONCLUSION :Transepithelial transport of putrescine across Caco2 cell monolayers occurs in passive diffusion, and is not influenced when epithelial cells are stimulated to proliferate by a potent mitogen such as EGF.

JTE-522-induced apoptosis in human gastric adenocarinoma cell line AGS cells by caspase activation accompanying cytochrome C release,membrane translocation of Bax and loss of mitochondrial membrane potential

AIM: To investigate the role of the mitochondrial pathway in JTE-522-induced apoptosis and to investigate the relationship between cytochrome C release, caspase activity and loss of mitochondrial membrane potential (Deltapsim). METHODS: Cell culture, cell counting, ELISA assay, TUNEL, flow cytometry, Western blot and fluorometric assay were employed to investigate the effect of JTE-522 on cell proliferation and apoptosis in AGS cells and related molecular mechanism. RESULTS: JTE-522 inhibited the growth of AGS cells and induced the apoptosis. Caspases 8 and 9 were activated during apoptosis as judged by the appearance of cleavage products from procaspase and the caspase activities to cleave specific fluorogenic substrates. To elucidate whether the activation of caspases 8 and 9 was required for the apoptosis induction, we examined the effect of caspase-specific inhibitors on apoptosis. The results showed that caspase inhibitors significantly inhibited the apoptosis induced by JTE-522. In addition, the membrane translocation of Bax and cytosolic release of cytochrome C accompanying with the decrease of the uptake of Rhodamin 123, were detected at an early stage of apoptosis. Furthermore, Bax translocation, cytochrome C release, and caspase 9 activation were blocked by Z-VAD.fmk and Z-IETD-CHO. CONCLUSION: The present data indicate a crucial association between activation of caspases 8, 9, cytochrome C release, membrane translocation of Bax, loss of Deltapsim and JTE-522-induced apoptosis in AGS cells.

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. Exons from 1 to 4 of human CYP2D6 cDNAs were also amplificated by RT-PCR from extratumoral liver tissues of 17 human hepatocellular carcinomas. Some RT-PCR products were sequenced. Exons 1 to 4 of CYP2D6 gene were amplified by PCR from extratumoral liver tissue DNA. Two PCR products from extratumoral liver tissues expressing skipped mRNA were partially sequenced. RESULTS: One of the CYP2D6 cDNAs had 470 nucleotides from 79 to 548 (3' portion of exons 1 to 5' portion of exon 4), and was skipped. Exons 1 to 4 of CYP2D6 cDNA were assayed with RT-PCR in 17 extratumoral liver tissues. Both wild type and skipped mRNAs were expressed in 4 samples, only wild type mRNA was expressed in 5 samples, and only skipped mRNA was expressed in 8 samples. Two more variants were identified by sequencing the RT-PCR products of exons 1 to 4 of CYP2D6 cDNA. The second variant skipped 411 nucleotides from 175 to 585. This variant was identified in 4 different liver tissues by sequencing the RT-PCR products. We sequenced partially 2 of the PCR products amplified of CYP2D6 exon 1 to exon 4 from extratumoral liver tissue genomic DNA that only expressed skipped mRNA by RT-PCR. No point mutations around exon 1, intron 1, and exon 4, and no deletion in CYP2D6 gene were detected. The third variant was the skipped exon 3, and 153 bp was lost. CONCLUSION: Three new alternative splicing variants of CYP2D6 mRNA have been identified. They may not be caused by gene mutation and may lose CYP2D6 activity and act as a down-regulator of CYP2D6.

Growth inhibition and apoptosis induction Sulindac on Human gastric cancer cells

AIM: To evaluate the effects of sulindac in inducing growth inhibition and apoptosis of human gastric cancer cells in comparison with human hepatocellular carcinoma (HCC) cells. METHODS: The human gastric cancer cell lines MKN45 and MKN28 and human hepatocellular carcinoma cell lines HepG(2) and SMMC7721 were used for the study. Anti-proliferative effect was measured by MTT assay, and apoptosis was determined by Hoechst-33258 staining, electronography and DNA fragmentation. The protein of cyclooxygenase-2 (COX-2) and Bcl-2 were detected by Western dot blotting. RESULTS: Sulindac could initiate growth inhibition and apoptosis of MKN45, MKN28, HepG(2) and SMMC7721 cells in a dose-and time-dependent manner. Growth inhibitory activity and apoptosis were more sensitive in HepG(2) cells than in SMMC7721 cells, MKN45 and MKN28 cells. After 24 hours incubation with sulindac at 2mmol x L(-1) and 4mmol x L(-1), the level of COX-2 and Bcl-2 protein were lowered in MKN45, SMMC7721 and HepG(2) cells but not in MKN28 cells. CONCLUSION: Sulindac could inhibit the growth of gastric cancer cells and HCC cells effectively in vitro by apoptosis induction, which was associated with regression of COX-2 and Bcl-2 expression. The growth inhibition and apoptosis of HCC cells were greater than that of human gastric cancer cells. The different effects of apoptosis in gastric cancer cells may be related to the differentiation of the cells.

Antitumor activities of human autologous cytokineinduced killer(CIK)cells against hepatocellular carcinoma cells in vitro and in vivo

AIM: To characterize the anticancer function of cytokine-induced killer cells (CIK) and develop an adoptive immunotherapy for the patients with primary hepatocellular carcinoma (HCC), we evaluated the proliferation rate, phenotype and the antitumor activity of human CIK cells from healthy donors and HCC patients in vitro and in vivo. METHODS: Peripheral blood mononuclear cells (PBMC) from healthy donors and patients with primary HCC were incubated in vitro and induced into CIK cells in the presence of various cytokines such as interferon-gamma (IFN-gamma), interleukin-1 (IL-1), IL-2 and monoclonal antibody (mAb) against CD3. The phenotype and characterization of CIK cells were identified by flow cytometric analysis. The cytotoxicity of CIK cells was determined by (51)Cr release assay. RESULTS: The CIK cells were shown to be a heterogeneous population with different cellular phenotypes. The percentage of CD3+/CD56+ positive cells, the dominant effector cells, in total CIK cells from healthy donors and HCC patients, significantly increased from 0.1-0.13% at day 0 to 19.0-20.5% at day 21 incubation, which suggested that the CD3+ CD56+ positive cells proliferated faster than other cell populations of CIK cells in the protocol used in this study. After 28 day in vitro incubation, the CIK cells from patients with HCC and healthy donors increased by more than 300-fold and 500-fold in proliferation cell number, respectively. CIK cells originated from HCC patients possessed a higher in vitro antitumor cytotoxic activity on autologous HCC cells than the autologous lymphokine-activated killer (LAK) cells and PBMC cells. In in vivo animal experiment, CIK cells had stronger effects on the inhibition of tumor growth in Balb/c nude mice bearing BEL-7402-producing tumor than LAK cells (mean inhibitory rate, 84.7% vs 52.8%, P【0.05) or PBMC (mean inhibitory rate, 84.7% vs 37.1%, P【0.01). CONCLUSION: Autologous CIK cells are of highly efficient cytotoxic effector cells against primary hepatocellular carcinoma cells and might serve as an alternative adoptive therapeutic strategy for HCC patients.

Metastatic human hepatocellular carcinoma models in nude mice and cell line with metastatic potential

Metastatic human HCC model is needed for the studies on mechanism and intervention of metastatic recurrence. By using orthotopic implantation of histologically intact tissues of 30 surgical specimens, a patient-like metastatic model of human HCC in nude mice (LCI-D20) and a low metastatic model of human HCC in nude mice (LCI-D35) have been established. All mice with transplanted LCI-D20 tumors exhibited extremely high metastatic ability including spontaneous metastasis to liver, lungs, lymph nodes and peritoneal seeding. Remarkable difference was also found in expression of some of the invasiveness related genes and growth factors between the LCI-D20 and LCI-D35 tumors. PAI-1 increased gradually following tumor progression in LCI-D20 model, and correlated with tumor size and AFP level. Phasic expression of tissue intercellular adhesion molecule-1 in this model was also observed. Using corneal micropocket model, it was demonstrated that the vascular response induced by LCI-D20 tumor was stronger than that induced by LCI-D35 tumor. Similar report on metastatic human HCC model in nude mice and human HCC cell line with metastatic potential was rarely found in the literature. This LCI-D20 model has been widely used for the studies on intervention of metastasis, including anti-angiogenesis,antisense approach, metalloproteinase inhibitor, differentiation inducer, etc. It is concluded that the establishment of metastatic human HCC model in nude mice and human HCC cell line with metastatic potential will provide important models for the in vitro and in vitro study of HCC invasiveness, angiogenesis as well as intervention of HCC recurrence.

Effect of 5-Aza-2'-deoxycytidine on the P16 tumor suppressor gene in hepatocellular carcinoma cell line HepG2

INTRODUCTIONHepatocellular carcinoma (HCC) is one of the mostcommon human malignancies worldwide[1,2], and isclosely associated with infection of HBV and HCVand contamination of aflatoxin B1[3-6]. Althoughthe molecular mechanisms of hepatocarcinogenesisremain poorly understood, an increasing number ofgenetic abnormalities have been recognized[7-10],for example, the p16 gene[11,12] the p53gene[13-18], the E-cadherin gene[19], and the c-mycgene[20].

Cloning of UGT1A9 cDNA from liver tissues and its expression in CHL cells

AIM: To clone the cDNA of UGT1A9 from a Chinese human liver and establish the Chinese hamster lung (CHL) cell line expressing human UGT1A9. METHODS: cDNA of UGT1 A9 was transcripted from mRNA by reverse transcriptase-ploymerase chain reaction, and was cloned into the pGEM-T vector which was amplified in the host bacteric E.Coli DH5(alpha). The inserted fragment, verified by DNA sequencing, was subcloned into the Hind III /Not I site of a mammalian expression vector pREP9 to construct the plasmid termed pREP9-UGT1A9. CHL cells were transfected with the resultant recombinants, pREP9-UGT1A9, and selected by G418 (400 mg x L(-1)) for one month. The surviving clone (CHL-UGT1A9) was harvested as a pool and sub-cultured in medium containing G418 to obtain samples forUGT1A9 assays. The enzyme activity of CHL-UGT1A9 towards propranolol in S9 protein of the cell was determined by HPLC. RESULTS: The sequence of the cDNA segment cloned, which was 1666 bp in length, was identical to that released by Gene Bank (GenBank accession number: AF056188) in coding region. The recombinant constructed, pREP9-UGT1A9, contains the entire coding region, along with 18 bp of the 5' and 55 bp of the 3' untranslated region of theUGT1A9 cDNA, respectively. The cell lines established expressed the protein of UGT1A9, and the enzyme activity towards propranolol in S9 protein was found to be 101+/- 24 pmol x min(-1) x mg(-1) protein (n=3), but was not detectable in parental CHL cells. CONCLUSION: The cDNA of UGT1A9 was successfully cloned from a Chinese human liver and transfected into CHL cells. The CHL-UGT1 A9 cell lines established efficiently expressed the protein ofUGT1A9 for the further enzyme study of drug glucuronidation.

Reduction of tumorigenicity of SMMC-7721 hepatoma cells by vascular endothelial growth factor antisense gene therapy

AIM: To test the hypothesis to block VEGF expression of SMMC-7721 hepatoma cells may inhibit tumor growth using the rat hepatoma model. METHODS: Amplify the 200 VEGF cDNA fragment and insert it into human U6 gene cassette in the reverse orientation transcribing small antisense RNA which could specifically interact with VEGF165, and VEGF121 mRNA. Construct the retroviral vector containing this antisense VEGF U6 cassette and package the replication-deficient recombinant retrovirus. SMMC-7721 cells were transduced with these virus and positive clones were selected with G418. PCR and Southern blot analysis were performed to determine if U6 cassette integrated into the genomic DNA of positive clone. Transfected tumor cells were evaluated for RNA expression by ribonuclease protection assays. The VEGF protein in the supernatant of parental tumor cells and genetically modified tumor cells was determined with ELISA. In vitro and in vivo growth properties of antisense VEGF cell clone in nude mice were analyzed. RESULTS: Restriction enzyme digestion and PCR sequencing verified that the antisense VEGF RNA retroviral vector was successfully constructed.After G418 selection, resistant SMMC-7721 cell clone was picked up. PCR and Southern blot analysis suggested that U6 cassette was integrated into the cell genomic DNA. Stable SMMC-7721 cell clone transduced with U6 antisense RNA cassette could express 200 bp small antisense VEGF RNA and secrete reduced levels of VEGF in culture condition. Production of VEGF by antisense transgene-expressing cells was 65+/-10 ng/L per 10(6) cells, 42045 ng/L per 10(6) cells in sense group and 485+/-30 ng/L per 10(6) cells in the negative control group, (P【 0.05). The antisense-VEGF cell clone appeared phenotypically indistinguishable from SMMC-7721 cells and SMMC-7721 cells transfected sense VEGF. The growth rate of the antisense-VEGF cell clone was the same as the control cells. When S.C. was implanted into nude mice, growth of antisense-VEGF cell lines was greatly inhibited compared with control cells. CONCLUSION: Expression of antisense VEGF RNA in SMMC-7721 cells could decrease the tumorigenicity, and antisense-VEGF gene therapy may be an adjuvant treatment for hepatoma.

Bcl-2 over-expression and activation of protein kinase C suppress the Trail-induced apoptosis in Jurkat T cells

Trail, a tumor necrosis factor-related apoptosis-inducing ligand, is a novel potent endogenous activator of the cell death pathway through the activation of cell surface death receptors Trail-R1 and Trail-R2. Its role, like FasL in activation-induced cell death (AICD), has been demonstrated in immune system. However the mechanism of Trail induced apoptosis remains unclear. In this report, the recombinant Trail protein was expressed and purified. The apoptosis-inducing activity and the regulation mechanism of recombinant Trail on Jurkat T cells were explored in vitro. Trypan blue exclusion assay demonstrated that the recombinant Trail protein actively killed Jurkat T cells in a dose-dependent manner. Trail-induced apoptosis in Jurkat T cells were remarkably reduced by Bcl-2 over expression in Bcl-2 gene transfected cells. Treatment with PMA (phorbol 12-myristate 13-acetate), a PKC activator, suppressed Trail-induced apoptosis in Jurkat T cells. The inhibition of apoptosis by PMA was abolished by pretreatment with Bis, a PKC inhibitor. Taken together, it was suggested that Bcl-2 over-expression and PMA activated PKC actively down-regulated the Trail-mediated apoptosis in Jurkat T cell.

Studies on mechanism of Sialy Lewis-X antigen in liver metastases of human colorectal carcinoma

INTRODUCTIONSialyl Lewis-X antigen ,correlated with carcinoma, is a group of carbohydrate antigen containing oligosaccharide expressed of embryonic tisue and glycoproteins on cell surface of embryonic tissue[1].The SLeX antigen located on cell surface is synthesized principally by two enzymes ,al ,3fucosyltransfrease and a2, 3sialyctransferase.In adults ,SLeX antigen is expressed principally on the surfaces of granulocytic cells and some tumor cells .

Mast cells and human hepatocellular carcinoma

AIM: To investigate the density of mast cells (MCs) in human hepatocellular carcinoma (HCC), and to determine whether the MCs density has any correlations with histopathological grading, staging or some baseline patient characteristics.METHODS: Tissue sections of 22 primary HCCs were histochemically stained with toluidine blue, in order to be able to quantify the MCs in and around the neoplasm using a computer-assisted image analysis system. HCC was staged and graded by two independent pathologists. To identify the sinusoidal capillarisation of each specimen 3μm thick sections were histochemically stained with sirius red, and semi-quantitatively evaluated by two independent observers. The data were statistically analysed using Spearman′s correlation and Student′s t-test when appropriate.RESULTS: MCs density did not correlate with the age or sex of the patients, the serum alanine aminotransferase (ALT) or aspartate aminotransferase (AST) levels, or the stage or grade of the HCC. No significant differences were found between the MCs density of the patients with and without hepatitis C virus infection, but they were significantly higher in the specimens showing marked sinusoidal capillarisation.CONCLUSION: The lack of any significant correlation between MCs density and the stage or grade of the neoplastic lesions suggests that there is no causal relationship between MCs recruitment and HCC. However, as capillarisation proceeds concurrently with arterial blood supply during hepatocarcinogenesis, MCs may be considered of primary importance in the transition from sinusoidal to capillary-type endothelial cells and the HCC growth.

补骨脂素调控SKP2/PTHrP/SIRT1轴缓解炎症环境下髓核细胞退变的作用

目的探讨补骨脂素对白细胞介素-1β(IL-1β)和肿瘤坏死因子α(TNF-α)诱导的人髓核细胞(HNPCs)退变的影响及机制。方法体外培养HNPCs,用10 ng/ml IL-1β和50 ng/ml TNF-α诱导HNPCs 24 h使其发生退变,设置对照组、IL-1β+TNF-α组、补骨脂素(10 μmol/L、25 μmol/L和50 μmol/L)组、补骨脂素(25 μmol/L)+S期激酶相关蛋白2(SKP2)干扰或过表达组、补骨脂素+甲状旁腺激素相关蛋白(PTHrP)重组蛋白(100 nmol/L)组、补骨脂素+PTHrP重组蛋白(100 nmol/L)+沉默信号调节子1(SIRT1)激活剂SRT1720(1 μmol/L)组。采用流式细胞术检测髓核细胞凋亡情况;ELISA法检测髓核细胞中IL-6、基质金属蛋白酶3(MMP-3)和MMP-13的表达水平;DCFDA探针法检测髓核细胞中活性氧(ROS)的水平;Western blotting检测髓核细胞中SKP2、PTHrP、SIRT1、细胞外基质(ECM)相关蛋白[二型胶原蛋白(COL Ⅱ)和蛋白聚糖蛋白]的表达水平;蛋白质免疫共沉淀(Co-IP)实验检测SKP2与PTHrP蛋白的相互作用;泛素化实验检测补骨脂素对SKP2介导的PTHrP泛素化水平的影响。结果补骨脂素以剂量依赖的方式抑制炎症环境下髓核细胞凋亡以及IL-6、MMP-3、MMP-13和ROS的生成(P<0.05或P<0.01),促进SKP2和ECM相关蛋白的表达(P<0.05)。与补骨脂素+杂乱干扰组比较,补骨脂素+SKP2干扰组SKP2和ECM相关蛋白的表达水平明显降低(P<0.05),髓核细胞凋亡率、IL-6、MMP-3、MMP-13和ROS水平明显增高(P<0.05或P<0.01)。Co-IP实验结果显示,SKP2与PTHrP蛋白存在相互作用。泛素化实验结果显示,与补骨脂素+空载体组比较,补骨脂素+SKP2过表达组PTHrP蛋白表达水平明显降低,SKP2蛋白表达水平明显升高(P<0.05)。与补骨脂素组比较,补骨脂素+PTHrP重组蛋白组SIRT1和ECM相关蛋白表达水平明显降低(P<0.05),髓核细胞凋亡率、IL-6、MMP-3、MMP-13和ROS水平明显升高(P<0.05);与补骨脂素+PTHrP重组蛋白组比较,补骨脂素+PTHrP重组蛋白+SRT1720组PTHrP蛋白表达水平无明显变化(P>0.05),SIRT1和ECM相关蛋白表达水平明显升高(P<0.05),髓核细胞凋亡率、IL-6、MMP-3、MMP-13和ROS水平明显降低(P<0.05)。结论补骨脂素可通过促进SKP2介导的PTHrP泛素化降解而活化SIRT1,抑制炎症环境下髓核细胞的凋亡。

红细胞终末分化期出现与珠蛋白基因表达增强子HS2序列特异结合的蛋白质因子

利用Ｓｏｕｔｈｗｅｓｔｅｒｎ印迹和电泳迁移率改变分析法，研究了处于终末分化阶段的人胚肝中、晚幼红细胞和经ｈｅｍｉｎ诱导分化前后的Ｋ５６２细胞核抽提物中与地高辛标记的ＨＳ２探针特异结合的蛋白质。发现经ｈｅｍｉｎ诱导后的Ｋ５６２细胞样品中出现一些为诱导前缺如的，分子量为１２，１４，１８，４５和５０ｋＤ的ＨＳ２结合蛋白。这些结合蛋白在印迹图谱中的位置（分子量大小）与存在于正常终末分化期红细胞中ＨＳ２结合蛋白的相近。同样，诱导后Ｋ５６２细胞核蛋白质与ＨＳ２形成复合物的电泳迁移图谱与正常分化的人胚肝红细胞核蛋白质－ＨＳ２复合物的相似，而与未经诱导的Ｋ５６２细胞核蛋白质－ＨＳ２复合物的迁移图谱存在差异。提示诱导分化和正常分化的红细胞表达一些新的ＨＳ２结合蛋白，这些仅在终末分化期红细胞中出现的ＨＳ２结合蛋白，可能是参与红细胞分化和珠蛋白基因表达调控的重要因素。