CHANGES IN ACTIVITY OF INSULIN RECEPTOR TYROSINE KINASE AND CHARACTERISTICS OF ITS ENDOGENOUS SUBSTRATE IN TRANSFORMED HUMAN LYMPHOCYTES

The cascade reaction of tyrosine phosphorylation provides an attractive mechanism to regulate the metabolism and growth of normal and transformed cells. Thus, considerable effort has been made to identify the cellular substrates of the tyrosine kinases. In this work, the activity of the Insulin receptor tyrosine kinase and endogenous substrate in transformed lymphocytes were studied. Purified human T lymphocytes incubated with phytohemagglutinin (PHA) for 72 hours served as transformed cells, when labeled with [32P] - orthophosphate it appeared that the insulin-dependent protein kinase activity in the transformed cells increased 9- fold. In search for the physiologically significant substrates by using polyclonal antiphosphotyrosine antibody to immunoprecipitate phosphotyrosine-containing proteins that produced in the intact cell during insulin stimulation, a protein with molecular weight of 45kDa as identified and designated as PP45, which occurred during the 5 minutes response of lymphocytes to insulin.

Detection of CYP2E1,a Genetic Biomarker of Susceptibility to Benzene Metabolism Toxicity in Immortal Human Lymphocytes Derived from the Han Chinese Population

Objective Cytochrome P450 2E1 （CYP2E1） is an important metabolizing enzyme involved in oxidative stress responses to benzene, a chemical associated with bone marrow toxicity and leukemia, We aimed to identify the CYP2E1 genetic biomarkers of susceptibility to benzene toxicity in support of environmental and occupational exposure prevention, and to test whether a model using immortal human lymphocytes might be an efficient tool for detecting genetic biomarkers. Methods Immortalized human lymphocyte cell lines with independent genotypes on four CYP2E1 SNP sites were induced with 0.01% phenol, a metabolite of benzene. CYP2E1 gene function was evaluated by mRNA expression and enzyme activity. DNA damage was measured by Single-Cell Gel Electrophoresis （SCGE）. Results Among the four SNPs, cells with rs2070673TT and rs2030920CC showed higher levels of ~YP2E1 transcription and enzymatic activity than the other genotypes in the same SNP site. Cells with higher gene expression genotypes also showed higher comet rates compared with lower gene expression genotypes. Conclusion These results suggest that CYP2E1 rs2070673 and rs2030920 might be the genetic biomarkers of susceptibility to benzene toxicity and that the immortalized human lymphocytes model might be an efficient tool for the detection of genetic biomarkers of susceptibility to chemicals.

Genotoxicity evaluation of drinking water sources in human peripheral blood lymphocytes using the comet assay

The potential harm of organic pollutants in drinking water to human health is widely focused on in the wodd; more and more pollutants with genotoxic substances are released into the aquatic environment. Water source samples were collected from 7 different localities of Nanjing City. The potential genotoxicity of organic extracts from drinking water sources were investigated by means of the comet assay in human peripheral lymphocytes. The results showed that all the organic extracts from all the water source samples could induce DNA damages of human peripheral blood lymphocytes at different levels. A significant difference （P 〈 0.01） was observed when compared with the solvent control, The DNA damage increased with the increase of the dosage of the original water source. Significant differences of DNA damage were observed in different drinking water sources, as shown by the multiple comparisons analysis at the dosage of 100x; the degree of DNA damage treated by Hushu waterworks （at town level） was the most serious, the arbitrary units （AU） was 141.62±6.96, however, that of Shangyuanmen waterworks （at city level） was only 109.64±2.97. The analysis also revealed that the genotoxicity of town＇s water sources was higher than that of the city. The results demonstrated that the comet assay can be successfully applied to the genotoxicity monitoring programs of drinking water sources.

Metallothionein 1 Isoform Gene Expression Induced by Cadmium in Human Peripheral Blood Lymphocytes

Objective To study the gene expression of metallothionein 1 （MT-1） isoforms in human peripheral blood lymphocytes （HPBLs）. Methods The expression of mRNA representing the seven active MT-I genes was determined in HPBLs by quantitative RT-PCR before and after exposure to cadmium. Results Basal expressions of MT-IX, and MT-1A in HPBLs were similar to expression of housekeeping gene. In contrast, the basal gene expressions of MT- 1 H, IF, 1E, and 1G were a little transcripts in human HPBLs. No signal was detected for MT-1B. There was a sex difference （P〈0.05）. in basal gene expression of MT-1E. The levels of gene expression of MT-1A, 1E, IF, 1G, 1H, and 1X increased, but the level of MT-1B did not increase after exposure to cadmium. Conclusions Gene expressions of MT-1 G, MT-1 H, MT-1 F, and MT-1X in HPBLs can be used as a potential biomarker of cadmium exposure.

IN VITRO ANTITUMOR ACTIVITY OF TUMOR-INFILTRATING LYMPHOCYTES FROM HUMAN GASTRIC CARCINOMA

Tumor-infiltrating lymphocytes (TIL) isolated from 11 gastric carcinoma were studied. TIL could grow for a long-term in medium containing recombi-nant interleukin-2(rlL-2). The mean expansion fold achieved in 6 long-term cultures of 11 specimens was 15.1. RIL-2 expanded gastric TIL exhibited significant cytotoxicity against K562, BGC823, MCF-7 and more effective antitumor cytotoxicity against fresh autologous tumor targets and human gastric cancer cell line. Peak cytotoxicity was shown in the third or fourth week after cultures. Cryopreservation of gastric TIL didn't influence their expansion capacity and antitumor activity. Phenotypic analysis was demonstrated in this study. The results of present study indicate that TIL from human gastric carcinoma could be expanded and reach high levels of antitumor effector function in long-term cultures with rIL-2. Their function may be of clinical importance.

Nicotine protects against ulcerative colitis through regulating microRNA-124 and STAT3

OBJECTIVE Although it is generally believed that nicotine accounts for the beneficial effect of smoking on ulcerative colitis,the underlying mechanisms remain not well-understood.Our previous finding that nicotine inhibits inflammatory responses through inducing miRNA-124 prompted us to ask whether the miRNA is involved in the protective action of nicotine on UC.METHODS Mi R-124 expression in colon tissues and cells was determined by q-PCR and in situ hybridization.The effect of miR-124 on protective role of nicotine in ulcerative colitis was evaluated in DSS-treated mice and IL-6-treated Caco-2 colon epithelial cells.Expression of p-STAT3/STAT3 was detected by immunohistochemistry and Western blot analysis.RESULTS miR-124 expression is upregulated in colon tissues from patients and DSS-induced colitis.Nicotine treatment further elevated miR-124 level in colon tissues of the mice,in infiltrated lymphocytes and epithelial cells,and augmented miR-124 expression in lymphocytes isolated from human ulcerative colon tissues.Administration of nicotine also reduced weight loss,improved DAI and decreased HE score in DSS-induced colitis.Moreover,knockdown of miR-124 in vivo significantly diminished the beneficial effect of nicotine,and in vitro on IL-6-treated Caco-2 colon epithelial cells.Further analysis indicated that nicotine inhibited STAT3 activation in vivo and in IL-6-treated Caco-2 colon epithelial cells and Jurkat human T lymphocytes,in whichmiR-124 knockdown led to increased activation of STAT3.CONCLUSION These data indicated that nicotine exerts its protective action in UC through inducing miR-124 and its effect on STAT3,suggesting that the miR-124/STAT3 system is a potential target for the therapeutic intervention of UC.

Investigation of the cytotoxicity,antioxidative and immune-modulatory effects of Ligusticum porteri(Osha) root extract on human peripheral blood lymphocytes

OBJECTIVE： Ligusticum ported is a traditional Native American herb. The roots of L. ported are traditionally used in the treatment of many diseases, however, its cytotoxicity, antioxidative and immune- modulatory effects need to be investigated. In this study, we evaluated the effects of the root extract at different doses on human peripheral blood lymphocytes （PBLs）. METHODS： The lymphocytes were incubated with different concentrations of the root extracts （0, 50, 100, 200, and 400 μg/mL） and harvested every 6 h for 2 d （P〈0.05）. The protective effect of the herb against oxidative damage was determined by inducing oxidative stress with the administration of 50 μmol/L of hydrogen peroxide （H202）. RESULTS： Treatments with L. ported at 200 and 400 pg/mL increased the viability of PBLs. The deleterious effect of H2O2 was ameliorated by 400μg/mL L. ported treatment. Addition of 400 μg/mL L. ported reduced lipid peroxidation in stressed PBLs by 94% （P〈0.05）. Treatment with 400 μg/mL of L. ported resulted in a 26.4% increase of reduced glutathione levels. Activities of superoxide dismutase and catalase increased by 17.5% and 55.2% respectively, when stressed PBLs were treated with 400 μg/mL L. ported for 2 d （P〈0.05）. Treatment with 400 μg/mL L. ported increased interferon-γand interleukin-2 expressions in H2O2-challenged PBLs （P〈0.05）, however, the root extract did not cause a significant difference in interleukin-10 levels compared to the control （P〉0.05）. CONCLUSION： The findings suggest that L involving protective effects against oxidative ported might be a potential immune-modulating agent damage.