Apoptotic Effect of Cryptotanshinone on Human Melanoma A375 Cells

[Objectives] The aim was to investigate the effect of cryptotanshinone on apoptosis of human melanoma A375 cells and its related mechanism of mitochondrial pathway.[Methods]The cytotoxic effect of cryptotanshinone on apoptosis of human melanoma A375 cells was detected by MTT colorimetry.The apoptosis of melanoma A375 cells was detected with Annexin V-FITC/PI and observed by fluorescence inverted microscope.The expression of apoptosis-related proteins was detected by Western blotting.[Results]The viability of the A375 cells decreased with the increase of drug concentration.The fluorescence intensity of the cells increased with the treatment time.The expression of pro-apoptotic protein caspase-3 gradually increased,while the expression of apoptosis-inhibiting proteins p-AKT and Bcl-2 gradually reduced.[Conclusions]Cryptotanshinone induces apoptosis of human melanoma A375 cells via AKT signaling pathway,thus exerting a good cytotoxic effect on A375 cells.

Effects of histamine on growth and apoptosis of human melanoma cells A375

Objective: To investigate the effects of histamine on growth and apoptosis of human melanoma cells A375. Methods: The effect of histamine on growth of A375 cells in vitro was examined by MTT assay and Trypan blue exclusion assay. Cell cycle analysis, early apoptosis analysis by double staining with Annexin V-FITC and PI, and active caspase-3 analysis by staining FITC-conjugated monoclonal rabbit anti-active caspase-3 antibody were made by flow cytometer. StreptAvidin-Biotin Complex (SABC) immunocytochemical assays were adopted to detect Bax/Bcl-2 protein expressions.Results: Histamine inhibited proliferation of A375 cells in a dose- and time-dependent manner, and altered cell cycle distribution of A375 cells revealing an increase in G0/G1-phase population, a decrease in S-phase population and the inhibition of G1/S switching. Histamine induced apoptosis of A375 cells (P<0.05), elevated the cells population with detectable active caspase-3 (P<0.05), increased the number of cells forming Bax and decreased the number of cells forming Bcl-2 significantly (P<0.05). Conclusion: That histamine inhibits cell cycle progress of A375 cells is one of the possible mechanisms of proliferation arrest of A375 cells elicited by histamine. Histamine mediates apoptosis in A375 cells that may be caspase-dependent through mitochondria routine. Histamine with high concentration inhibits growth of A375 cells in vitro by interfering proliferation and inducing apoptosis of cells.

ETM study of electroporation influence on cell morphology in human malignant melanoma and human primary gingival fibroblast cells

Objective:To estimate electroporation(EP) influence on malignant and normal cells.Methods: Two cell lines including human malignant melanoma(Me-43) and normal human gingival fibroblast(HCFs) were used.EP parameters were the following:230,1000,1 730,2 300 V/cm;30 μ s by 3 impulses for every case.The viability of cells after EP was estimated by MTT assay. The ullrastructural analysis was observed by transmission electron microscope(Zeiss EM 900). Results:In the current study we observed the intracellular effect following EP on Me-43 and HGF cells.At the conditions applied,we did not observe any significant damage of mitochondrial activity in both cell lines treated by EP.Conversely,we showed that EP in some conditions can stimulate cells to proliferation.Some changes induced by EP were only visible in electron microscopy.In fibroblast cells we observed significant changes in lower parameters of EP(230 and 1 000 V/cm).After applying higher electric field intensities(2 300 V/cm) we detected many vacuoles,myelin-like bodies and swallowed endoplasmic reticulum.In melanoma cells such strong pathological modifications after EP were not observed,in comparison with control cells. The ultrastructure of both treated cell lines was changed according to the applied parameters of EP.Conclusions:We can claim that EP conditions are cell line dependent.In terms of the intracellular morphology,human fibroblasts are more sensitive to electric field as compared with melanoma cells.Optimal conditions should be determined for each cell line.Summarizing our study,we can conclude that EP is not an invasive method for human normal and malignant cells. This technique can be safely applied in chemotherapy for delivering drugs into tumor cells.

Effect of luteolin on apoptosis and vascular endothelial growth factor in human choroidal melanoma cells

AIM:To investigate the effects of luteolin on apoptosis,the cell cycle,and the expression and secretion of vascular endothelial growth factor(VEGF)in human choroidal melanoma cells(C918 and OCM-1).METHODS:C918 and OCM-1 cells cultured in vitro were treated with various concentrations of luteolin(0,5,10,15μmol/L).Cell growth was observed with an inverted microscope,and cell cycle arrest was detected by propidium iodide(PI)staining using flow cytometry.Apoptosis was detected by Hoechst33342 staining,and apoptosis rate was determined by Annexin V-FITC/PI experiments using flow cytometry.The expression of apoptosis-related proteins Bcl-2,Bax and VEGF was analyzed using Western blots.The levels of VEGF secreted by the cells into the supernatant was analyzed using ELISA.RESULTS:After treating with 5 to 15μmol/L luteolin for 48 h,the fusion degree of C918 and OCM-1 cells decreased,and more floating apoptotic cells appeared.Luteolin treatment increased the G0-G1 phase ratio of the C918 and OCM-1 cells,blocked cell cycle progression,and increased the apoptosis rate of the C918 and OCM-1 cells.Western blot showed that luteolin decreased the expression of Bcl-2 and VEGF in the C918 and OCM-1 cells and increased the expression of Bax protein.The ELISA results showed that 10 to 15μmol/L luteolin decreased the cell secretion of VEGF.CONCLUSION:Luteolin may induce apoptosis by regulating the levels of apoptosis-related proteins in C918 and OCM-1 cells.Luteolin can induce cell cycle arrest,decrease the expression of VEGF.

REARRANGEMENT AND EXPRESSION OF T CELL RECEPTOR β GENE IN HUMAN HEMOPOIETIC CELL LINES AND PRIMARY CELLS FROM ACUTE LYMPHOCYTIC LEUKEMIAS

Using Southern blot, Northern blot and Quick blot methods, we examined the rearrangement and expression of TCR βgene in four early differentiation stage cell lines from human hemopoietic system, namely HL-60, Jurkat, Daudi and Raji cells as well as lymphocytes from 17 acute lymphocytic leukemia （ALL） patients. The results showed. Ⅰ) Rearrangement of TCR βgene was seen in Jurkat cells. A germline pattern was observed in HL-60, Daudi and Raji cells. 2) Eight of 9 patients with T-ALL had cells with rearranged TCR βgene. But two of 3 patients with B-ALL and three of 5 patients with nonT, nonB-ALL also had cells with rearranged TCR βgene. 3) A 1.3 kb full-length transcript and a 1.0 kb truncated transcript were detected in Jurkat cells by probing with 32P-TCR βcDNA. But some leukemic B cells also expressed an incompleted transcript. 4) TCR βmRNA was detected in six of 8 patients with T-ALL, four of 5 patients with nonT, nonB-ALL and one of 3 patients with B-ALL. But the level of expression was quite differ ent. The dual-rearrangement and the abnormal expression may give us a new clue for researching leukemogenesis.

Apoptotic effect and mechanisms of AHPN on human skin malignant melanoma cell A375

Objective： To study apoptotic effects of synthetic retinoic acid 6-[3-（1-adamantyl）-4-hydroxyphenyl]-2-naphthalene carboxylic acid（AHPN） on human skin malignant melanoma A375 cells in comparison with the natural ligand all-trans-retinoic acid（ATRA） in vitro and the mechanisms related to the actions of AHPN. Methods：MTT assay was used to determine the anti-proliferative effects of AHPN and ATRA on A375 cells. Flow cytometry was performed to investigate the influence of AHPN and ATRA on cell cycle and cell apoptosis. In addition, transfection and luciferase activity assays were employed to explore the mechanisms of how AHPN executes its proapoptotic function. Results：Firstly, AHPN promoted apoptosis and GI arrest in A375 cells compared with ATRA. Secondly, the activity of NF-K B in A375 cells treated with AHPN increased 2-3 times compared with solvent DMSO treatment. Conclusion：AHPN, in comparison with ATRA, is a more effective alternative for therapy of malignant melanoma. The potentially proapoptotic function of AHPN requires activation of NF-K B.

Inhibitory effect of M3 receptor antagonist 4-DAMP on melanoma proliferation of A375 cell line

Objective: he effect of M3 receptor antagonist 4-DAMP (4-diphenylacetoxy-N-methylpiperidine-methiodide) on the proliferation of melanoma were explored, and the development prospect and significance of M3 receptor antagonist in the field of anti-tumor were discussed. Methods: The viability of tumor cells was detected by MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) colorimetry. The optimal concentration of 4-DAMP was screened and the effect of the drug was confirmed by EdU (5-ethynyl-2'-deoxyuridine) fluorescence staining. Adult male BALB/c nude mice were divided into control group, 5-Fu (5-Fluorouracil, 5-Fluorouracil, 20 mg/kg/d) group and 4-DAMP (2 mg/kg/d) group to establishtumor-bearing model in vivo. The tumor volume curve was calculated and plotted. Mice were killed after 21 days of continuous administration. Strip the tumor, take pictures and meatured the weighs, and calculate the inhibition rate. [1]The spleen was weighed and the spleen index was culculated. H & E staining was used to observe the pathological changes of tumor sections. The gene expression of M3R、MIA (Melanoma inhibitory activity, MIA)、SP-1 (Transcription factor Sp1, SP-1) and Lnc-SPRY4-IT1 (SPRY4 Intronic Transcript 1,Lnc-SPRY4-IT1) in A375 cell line was detected by real-time quantitative PCR (quantitative reverse-transcription polymerase chain reaction, qRT-PCR). The protein level of ERK1/2 (extracellular signal-regulated kinase 1/2,ERK1/2) was detected by Western Blot. Results: 4-DAMP could significantly inhibit the activity of A375 cell line (P < 0.01), and EdU staining demonstrated that 4-DAMP inhibited the growth of A375 cell line. The results of animal experiments showed that compared with the control group, the volume and mass of tumor in 5-Fu group and 4-DAMP group were significantly reduced (P < 0.05) and H & E staining showed that the intercellular space became larger, connective tissue increased, and tumor growth was obstructed. Compared with the control group, the tumor inhibition rate of 4-DAMP group was 72.01% (P < 0.01). There was no significant difference in body mass and spleen index. Compared with the control group, the expression of MIA, SP-1 and LncRNA-SPRY4-IT1 was decreased by 4-DAMP or si-M3R transfection. Compared with the control group, the expression of ERK1/2 was decreased by 4-DAMP or si-M3R transfection. Conclusions: These results suggest that M3 receptor antagonist 4-DAMP can inhibit the proliferation of melanoma and reduce the expression of mRNA of MIA, SP-1 and LncRNA-SPRY4-IT1 and the protein level of ERK1/2 in A375 tumor cells.

Establishment of an untransfected human corneal epithelial cell line and its biocompatibility with denuded amniotic membrane

AIM: To establish an untransfected human corneal epithelial (HCEP) cell line and characterize its biocompatibility with denuded amniotic membrane (dAM). METHODS: The torn HCEP pieces were primarily cultured in DMEM/F12 media (pH 7.2) supplemented with 20% fetal bovine serum and other necessary factors, yielding an HCEP cell line which was its growth performance, chromosome morphology, tumorigenicity and expression of marker proteins analyzed. In addition, the biocompatibility of HCEP cells with dAM was evaluated through histological and immunocytochemistry analyses and with light, electron and slit-lamp microscopies. RESULTS: HCEP cells proliferated to confluence in 3 weeks, which have been subcultured to passage 160. A continuous untransfected HCEP cell line, designated as utHCEPC01, was established with a population doubling time of 45.42 hours as was determined at passage 100. The cells retained HCEP cell properties as were approved by chromosomal morphology and the expression of keratin 3. They, with no tumorigenicity, formed a multilayer epithelium-like structure on dAMs through proliferation and differentiation during air-liquid interface culture, maintained expression of marker proteins including keratin 3 and integrin p 1 and attached tightly to dAMs. The reconstructed HCEP was highly transparent and morphologically and structurally similar to the original. CONCLUSION: An untransfected and non-tumorigenic HCEP cell line was established in this study. The cells maintained expression of marker proteins. The cell line was biocompatible with dAM. It holds the potential of being used for in vitro reconstruction of tissue-engineered HCEP, promising for the treatment of diseases caused by corneal epithelial disorders.

Epigenetic modification regulates both expression of tumor-associated genes and cell cycle progressing in human colon cancer cell lines: Colo-320 and SW1116

The aim of this study is to assess the effects of DNA methylation and historic acetylation, alone or in combination, on the expression of several tumor-associated genes and cell cycle progression in two established human colon cancer cell lines: Colo-320 and SW1116. Treatments with 5-aza-2'-deoxycytidine (5-aza-dC) and trichostatin A, alone or in combination, were applied respectively. The methylation status of the CDKN2A promoter was determined by methyla-tion-specific PCR, and the acetylated status of the histones associated with the p21WAF1 and CDKN2A genes was examined by chromatin immunoprecipitation. The expression of the CDKN2A, p21WAF1, p53, p73, APC, c-myc, c-Ki-ras and survivin genes was detected by real-time RT-PCR and RT-PCR. The cell cycle profile was established by flow cytometry. We found that along with the demethylation of the CDKN2A gene promoter in both cell lines induced by 5-aza-dC alone or in combination with TSA, the expression of both CDKN2A and APC genes increased. The treatment of TSA or sodium butyrate up-regulated the transcription of p21WAF1 significantly by inducing the acetylation of histones H4 and H3, but failed to alter the acetylation level of CDKN2A-associated histones. No changes in transcription of p53, p73, c-myc, c-Ki-ras and survivin genes were observed. In addition, TSA or sodium butyrate was shown to arrest cells at the G1 phase. However, 5-aza-dC was not able to affect the cell cycle progression. In conclusion, regulation by epigenetic modification of the transcription of tumor-associated genes and the cell cycle progression in both human colon cancer cell lines Colo-320 and SW1116 is gene-specific.

Melatonin and Doxorubicin synergistically induce cell apoptosis in human hepatoma cell lines

AIM:To investigate whether Melatonin has synergistic effects with Doxorubicin in the growth-inhibition and apoptosis-induction of human hepatoma cell lines HepG2 and Bel-7402.METHODS:The synergism of Melatonin and Doxorubicin inhibited the cell growth and induced cell apoptosis in human hepatoma cell lines HepG2 and Bel-7402.Cell viability was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide(MTT)assay.Cell apoptosis was evaluated using TUNEL method and flow cytometry.Apoptosis-related protein Bax,Bcl-2 and caspase-3 expressions were measured by immunohistochemical staining.RESULTS:Treatment with Melatonin(10 -8 -10 -5 mol/L) alone had a dose-related inhibitory effect on cell proliferation but no cytotoxic effect on hepatoma cell lines HepG2 and Bel-7402.Interestingly,when combined with Doxorubicin,Melatonin significantly increased the effects of cell growth inhibition and cell apoptosis.Furthermore,TUNEL staining and flow cytometry revealed that cooperative apoptosis induction was associated with decreased expression of Bcl-2 as well as increased expression of Bax and Caspase3.CONCLUSION:The synergism of Melatonin and Doxorubicin inhibits hepatoma cell growth and induces cell apoptosis.

Recombinant human zona pellucida proteins ZP1, ZP2 and ZP3 co-expressed in a human cell line

Aim: To produce biologically active recombinant human (rh) ZP proteins in a human cell for use in sperm function tests. Methods: The human embryonic kidney cell line 293T was employed to produce rhZP1, rhZP2 and rhZP3 proteins individually and together by co-expression. Presence of these proteins in the culture medium and cell lysate was assessed by Western blotting analysis. The effect of the recombinant proteins on the human AR was assessed. Results: RhZP2 and rhZP3 were secreted into the culture medium, whereas rhZPl was found only in the cell lysate. Interestingly, when all zona pellucida proteins were co-expressed in the same cells, rhZPl was also secreted into the culture medium. However, despite the presence of all three ZP proteins in sufficient concentration and evidence of heavy glycosylation on gel electrophoresis, biological activity to induce the AR was not observed. Conclusion: RhZP1, rhZP2 and rhZP3 were successfully expressed in the human embryonic kidney cell line 293T. It appears that an interaction amongst these proteins may be required for release of rhZPl from the cell. Although this approach is not satisfactory for producing active human ZP proteins, it makes a significant contribution to the understanding of the structural and functional characteristics of the ZP proteins.

Establishment of an untransfected human corneal stromal cell line and its biocompatibility to acellular porcine corneal stroma

AIM: To establish an untransfected human corneal stromal (HCS) cell line and characterize its biocompatibility to acellular porcine corneal stoma (aPCS). METHODS: Primary culture was initiated with a pure population of HCS cells in DMEM/F12 media (pH 7.2) containing 20% fetal bovine serum and various necessary growth factors. The established cell line was characterized by growth property, chromosome analysis, tumorigenicity assay, expression of marker proteins and functional proteins. Furthermore, the biocompatibility of HCS cells with aPCS was examined through histological and immunocytochemistry analyses and with light, electron microscopies. RESULTS: HCS cells proliferated to confluence 2 weeks later in primary culture and have been subcultured to passage 140 so far. A continuous untransfected HCS cell line with a population doubling time of 41.44 hours at passage 80 has been determined. Results of chromosome analysis, morphology, combined with the results of expression of marker protein and functional proteins suggested that the cells retained HCS cell properties. Furthermore, HCS cells have no tumorigenicity, and with excellent biocompatibility to aPCS. CONCLUSION: An untransfected and non-tumorigenic HCS cell line has been established, and the cells maintained positive expression of marker proteins and functional proteins. The cell line, with excellent biocompatibility to aPCS, might be used for in vitroreconstruction of tissue-engineered HCS.

Protein profile of human hepatocarcinoma cell line SMMC-7721:Identification and functional analysis

AIM： TO investigate the protein profile of human hepatocarcinoma cell line SMMC-7721, to analyze the specific functions of abundant expressed proteins in the processes of hepatocarcinoma genesis, growth and metastasis, to identify the hepatocarcinoma-specific biomarkers for the early prediction in diagnosis, and to explore the new drug targets for liver cancer therapy. METHODS： Total proteins from human hepatocarcinoma cell line SMMC-7721 were separated by two-dimensional electrophoresis （2DE）. The silver-stained gel was analyzed by 2DE software Image Master 2D Elite. Interesting protein spots were identified by peptide mass fingerprinting based on matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry （MALDI-TOF-MS） and database searching. RESULTS： We obtained protein profile of human hepatocarcinoma cell line SMMC-7721. Among the twenty-one successfully identified proteins, mitofilin, endoplasmic reticulum protein ERp29, ubiquinol-cytochrome C reductase complex core protein I, peroxisomal enoyl CoA hydratase, peroxiredoxin-4 and probable 3-oxoacid CoA transferase 1 precursor were the six novel proteins identified in human hepatocarcinoma cells or tissues. Specific functions of the identified heat-shock proteins were analyzed in detail, and the results suggested that these proteins might promote tumorigenesis via inhibiting cell death induced by several cancer-related stresses or via inhibiting apoptosis at multiple points in the apoptotic signal pathway. Other identified chaperones and cancer-related proteins were also analyzed.CONCLUSION： Based on the protein profile of SMMC-7721 cells, functional analysis suggests that the identified chaperones and cancer-related proteins have their own pathways to contribute to the tumorigenesis, tumor growth and metastasis of liver cancer. Furthermore, proteomic analysis is indicated to be feasible in the cancer study.

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.

CHARACTERIZATION OF A HUMAN HERPES VIRUS－6（HHV－6） AND EPSTEIN－BARR VIRUS（EBV） ASSOCIATED LEUKEMIC CELL LINE，J6－1

This report characterizes the J6-1 cell line derived from a Chinese acute myelomonocytic leukemia patient and previously reported to be associated with EBV. These studies showed that J6-1 cells were also infected with HHV-6 as demonstrate at the DNA level by PCR and Southern blot hybridization and by expression of HHV-6 early membrane antigen on the J6-1 cell surface. Further characterization showed J6-1 was co-infected with EBV type 2. Generally, cells infected with EBV type 2 do not grow well in vitro. However, J6-1 , although difficult to maintain in vitro, has been grown for 15 years. Possibly, co-infection with HHV6 confers this property. In this regard, J6-1 cells exhibited density dependent growth which could be inhibited with an anti-HHV-6-MA monoclonal antibody(MAb). In contrast, anti-HHV-6-VCA MAb stimulated the J6-1 cell proliferation. Electron microscopic analysis showed that, morphologically, there were two types of J6-1 cell, one with lymphoblastoid features and one with a monocytoid appearance. Accordingly, the flow profile of the J6-1 cell line showed heterogeneity. with two populations comprised of CD15-, CD19+ cells with low light scatter(small cells) and a population with greater light scatter(larger cells) which was CD15+ , CD19+. The population was negative for progenitor cell markers(CD33, 34 ), and T cell markers. Southern analysis showed no T cell receptor rearrangement, however there was a clonal JH and kappa light chain expressing population. Glycocytochemical analysis showed several endogenous lectin receptors on the J6-1 cell surface: BSA-Xylose, BSA-Rhamnose, BSAGal. BSA-Lac. This cell line shares many characteristics with other monocytic/ lymphoblastoid cell lines isolated elsewhere and provides circumstantial evidence linking Herpes viruses, as least as co- factors,to leukemia cell growth.

Apoptosis mechanisms of human gastric cancer cell line MKN-45 infected with human mutant p27

AIM: To explore the inducing effect of human mutant p27 gene on the apoptosis of the human gastric cancer cell line MKN-45 and its associated mechanisms. METHODS: The recombinant adenovirus Ad-p27mt was constructed to infect the human gastric cancer cell line MKN-45. Using flow cytometry, TUNEL assay and DNA fragment analysis, we measured the apoptotic effect of Ad-p27mt on the human gastric cancer cells. RESULTS: Ad-p27mt was successfully constructed and the infection efficiency reached 100%. After 18 h of infection, we observed an apoptotic hypodiploid peak on the flow cytometer before G1-S and apoptotic characteristic bands in the DNA electrophoresis. The apoptotic rate detected by TUNEL method was significantly higher in the Ad-p27mt group (89.4±3.12%)compared to the control group (3.12±0.13%, P ＜ 0.01).CONCLUSION: Human mutant p27 can induce apoptosis of the human gastric cancer cells in vitro.

Human embryonic stem cell lines derived from the Chinese population

Six human embryonic stem cell lines were established from surplus blastocysts. The cell lines expressed alkaline phosphatase and molecules typical of primate embryonic stem cells, including Oct-4, Nanog, TDGF1, Sox2, EBAF, Thy-1, FGF4, Rex-1, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. Five of the six lines formed embryoid bodies that expressed markers of a variety of cell types; four of them formed teratomas with tissue types representative of all three embryonic germ layers. These human embryonic stem cells are capable of producing clones of undifferentiated morphology, and one of them was propagated to become a subline. Human embryonic stem cell lines from the Chinese population should facilitate stem cell research and may be valuable in studies of population genetics and ecology.

Effect of a nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester on invasion of human colorectal cancer cell line SL-174T

AIML To investigate the effect and mechanism of action of the nitric oxide synthase （NOS） inhibitor NG-nitro-L-arginine methyl ester （L-NAME） on invasion and metastasis of human colorectal cancer cell line SL-174T. METHODS： Human colorectal cancer cel4 line SL-174T was cultured and treated separately with four different dosages of L-NAME for 72 h, Nitric oxide （NO） production was measured with Griess reagent, The effect of L-NAME on invasion and migration of SL-174T cells were evaluated by using Transwell chambers attached with polycarbonate filters and reconstituted basement membrane （Matrigel）, RT-PCR was performed to determine the mRNA levels of matrix metalloproteinase-2 （MMP-2） and tissue inhibitor metalloproteinase-2 （TIMP-2）,RESULTS： L-NAME could significantly inhibit NO production of SL-174T in a dose-dependent manner. After being treated for 72 h with 0.2, 0.4, 0.8, and 1.0 mmol/L L- NAME, respectively, the ability of the L-NAME treated SL- 174T cells to invade the reconstituted basement membrane decreased significantly （t = 8.056, P〈0.05; t= 14.467, P〈0.01; t= 27.785, P〈0.01; and t= 29.405, P〈0.01, respectively） and the inhibition rates were 10.29%, 19.62%, 34.08%, and 42.23%, respectively. Moreover, L-NAME could inhibit migration of SL-174T cells, and the inhibition rates were 20.76%, 24.95%, 39.43%, and 46. 85% for L-NAME at 0.2, 0.4, 0.8, and 1.0 mmol/L, respectively （t = 15.116, P〈0.01）. In addition, after treatment with L-NAME, expression of MMP-2 mRNA was significantly decreased （t = 71.238, P〈0.01） and that of TIMP-2 mRNA was markedly increased （t = -13.020, P〈0.01）. CONCLUSION： L-NAME exerts anti-invasive and anti- metastatic effects on SL-174T cell line via downregulating MNP-2 mRNA expression and upregulating TIMP-2 mRNA expression.

Alterations of FHIT Gene and P16 Gene in Nickel Transformed Human Bronchial Epithelial Cells

Objective To study the alterations of FHIT gene and P16 gene in malignant transformed human bronchial epithelial cells induced by crystalline nickel sulfide using an immortal human bronchial epithelial cell line, and to explore the molecular mechanism of nickel carcinogenesis. Methods 16HBE cells were treated 6 times with different concentrations of NiS in vitro, and the degree of malignant transformation was determined by assaying the anchorage-independent growth and tumorigenicity. Malignant transformed cells and tumorigenic cells were examined for alterations of FHIT gene and P16 gene using RT-PCR, DNA sequencing, silver staining PCR-SSCP and Western blotting. Results NiS-treated cells exhibited overlapping growth. Compared wkh that of negative control cells, soft agar colony formation efficiency of NiS-treated cells showed significant increases （P〈0.01） and dose-dependent effects. NiS-treated cells could form tumors in nude mice, and a squamous cell carcinoma was confirmed by histopathological examination. No mutation of exon 2 and exons 2-3, no abnormal expression in pl6 gene and mutation of FHIT exons 5-8 and exons 1-4 or exons 5-9 were observed in transformed cells and tumorigenic cells. However, aberrant transcripts or loss of expression of the FHIT gene and Fhit protein was observed in transformed cells and tumorigenic cells. One of the aberrant transcripts in the FHIT gene was confirmed to have a deletion of exon 6, exon 7, exon 8, and an insertion of a 36 bp sequence replacing exon 6-8. Conclusions The FHIT gene rather than the P16 gene, plays a definite role in nickel carcinogenesis. Alterations of the FHIT gene induced by crystalline NiS may be a molecular event associated with carcinogen, chromosome fragile site instability and cell malignant transformation. FHIT may be an important target gene activated by nickel and other exotic carcinogens.

DAPT suppresses the proliferation of human glioma cell line SHG-44

Objective:To explore the suppressing effect ofγ-secretase inhibitor DAPT on proliferation of human glioma cell line SHG-44 in vitro and its mechanism.Methods:The SHG-44 cell was treated by DAPT with different concentration.The proliferation of cells was detected by MTT assay;cell cycle and TSC of CD133^+were determined by flow cytometry analysis technique;the key factor in Notch signaling pathway(Notch-1,Delta-1,Hes-1)was measured by reverse transcrip tase-polymerase chain reaction and western blotting.Results:DAPT inhibited the growth and proliferation of SHG-44 cells significantly(P<0.05).And the inhibiting effect on SHG-44 cells produced by DAPT showed a dose-dependent manner.DAPT increased the rate of cells in G_0/G_1 phase of SHG-44 cells,while it decreased the rate of cells in S phase.TSC of CD133^+was significantly reduced after DAPT treated SHC-44 cells.The expression of protein and mRNA of Notch-1,Delta-1 and Hes-1 were gradually downregulated with the increase of DAPT doses.Conclusions:DAPT can downregulate these key factor in Notch signaling pathway,reduce the TSC of CD133+and inhibit the proliferation of SHC-44 cells.