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To study the apoptosis-inducing effect of Tanshinone and its molecular mechanism on human lung cancer cells.Human lung cancer cell line (SPC-A-1) was treated in vitro with 0.5 mg/L Tanshinone IIA for five days, and the cells treated with all trans retinoic acid (RA) or DDP as controls. Changes in cell morphology, apoptotic index and apoptosis related gene expression were detected by electron microscope, FCM and DNA-end-labeling.Many apoptotic cells were observed by light and electron microscopes. FCM examination showed that apoptotic index in Tanshinone group was much higher than that of DDP and control groups, but no difference was found statistically compared with RA group. The expression of p53, Fas and Bax genes in Tanshinone group was up-regulated markedly, but Bcl-2 was obviously down-regulated.Tanshinone IIA can induce apoptosis in human lung cancer cell line (SPC-A-1) . Up-regulating expression of p53, Bax, Fas and down-regulating Bcl-2 expression might be its molecular mechanisms.

To study the growth-inhibiting effect and its molecular mechanism of Tanshinone on human lung carcinoma cell line.Human lung adenocarcinoma cell line (SPC-A-1) was treated in vitro with 0.5μg/ml Tanshinone IIA for five days, and the cells treated with all trans retinoic acid (RA) and DDP as control. Changes in cell morphology, proliferation dynamics, cell cycle distribution and tumor-related gene expression were detected.The cell growth and rate of clone formation of SPC-A-1 cells were markedly inhibited in Tanshinone group than RA group. Flow cytometry demonstrated that S phase cells decreased and G₀/G₁ phase cells increased in Tanshinone group. Expression of p53, p21 was up-regulated obviously but CDKN₂ was down-regulated markedly by Tanshinone IIA.Tanshinone IIA can inhibit cell growth and clone formation in human lung cancer cell line (SPC-A-1) and its possible molecular mechanism may be inhibiting DNA synthesis by up-regulating p53, p21 and down-regulating CDKN₂.

To study the immuno-function of erythrocytes and the influence of surgical treatment on it in the patients with lung cancer.A total of 61 patients with lung cancer were studied, consisting of 35 non-operative patients and 26 post-operative patients. Their immuno-function of erythrocytes was analyzed and compared with 30 normal controls.In the post-operative group, the values of RBC C3BRRT and NTER were significantly higher than those of non-operative group (P0.01), but still lower than those of normal control group (P0.05); RFER, DTER, ETER and ATER higher than those of non-operative group (P0.01), and not significantly different from normal control group (P0.05); RBC ICR lower than that of non-operative group (P0.01), and not remarkably different from normal control group (P0.05); and RFIR lower than that of non-operative group (P0.01), however, still higher than that of normal control group (P0.01).The immuno-function of erythrocytes may be related to body tumor load of the patients with lung cancer, and it may gradually recover after the primary tumor is resected.

To explore the possibility of separating and establishing clonal cell subpopulations with different metastatic phenotype from a human lung large cell carcinoma cell line (WCQH-9801), to identify the difference of biological and molecular biology between NL9980 and L9981 cell lines.Two sub-cell lines (NL9980 and L9981) were isolated and established from a human lung large cell carcinoma cell line (WCQH-9801) by the single cell cloning techniques. The RELP, mRNA and protein transcript expression were detected in NL9980 and L9981 cell lines by Southern blot, RT-PCR and Western blot. The biological characteristics of vivo and vitro were determined in NL9980 and L9981 cell lines by MTT, plate, Boyden chamber methods and animal models.(1)Two sub-cell lines, NL9980 and L9981 which had different metastatic phenotype, were successfully isolated and established from a human lung large cell carcinoma cell line (WCQH-9801). (2)The L9981 cell line had LOH of nm23-H1 gene, deletion of mRNA and protein expression of nm23-H1, but the NL9980 cell line had neither LOH of nm23-H1 nor deletion of mRNA and protein expression of nm23-H1. (3)The proliferation, clone formation and vitro invastion of L9981 cell line were significantly higher than those of NL9980 cell line. (4)The tumorigenicity and lung metastatic rate in nude mouse of L9981 cell line were remarkably higher than those of NL9980. (5) No significant difference of the chromosome number was observed between NL9980 and L9981 cell lines.(1)NL9980 and L9981 cell lines established from a human lung large cell carcinoma cell line have different biological and molecular characteristics. (2)The high invsaion and metastasis ability of L9981 cell line might be related to the LOH of nm23-H1 gene.

To observe the growth-inhibiting effect of anticancer ketonon on A549 cell line and PLA-801D cell line and to explore its mechanism based on the antineoplastic effects of Tanshinon.A549 and PLA-801D cell lines were treated with anticancer ketonon by techniques of cell culture in vitro . The growth curves and dose-effect curves were drawn up. The ability of clone formation was determined. It was observed and analysed by light microscopy and flow cytometry.The growth of A549 and PLA 801D cell lines was evidently inhibited. Ability of clone formation was inhibited. The apoptosis index of cells was increased after treated with anticancer ketonon and the cell cycle was blocked at G0/G1 phase.Anticancer ketonon can significantly inhibit the growth of human lung cancer cells probably through inducing the apoptosis of cancer cells.

To construct recombinant adenoviral vector carrying Smad3D or Smad7 by a simplified means.Based on AdEasy System, adenoviral backbone plasmid vector and shuttle vector carrying the gene of interest were transferred into E.coli BJ5183 by chemical transformation methods in special order. The homologous recombination was performed.Recombinant adenoviral vector pAd-Smad3D and pAd-Smad7 were constructed successfully, which were confirmed by restriction enzyme digesting.Recombinant adenoviral vector may be constructed quickly and efficiently in E.coli by sequential chemical transformation methods.

To study chemosensitivity of anticancer drugs in the peripheral blood lymphocytes(PBL) of lung cancer patients and evaluate the correlation of chemosensitivity of tumor cells and peripheral blood lymphocytes in vitro.The sensitive rate of 15 kinds of anticancer drugs in the peripheral blood lymphocytes and the tumor cells of 74 cases of lung cancer in vitro were tested by the MTT method.There was no significant difference in the sensitivity of 12 anticancer drugs between PBL and tumor cells of patients with lung cancer (P0.05).The chemosensitive test of the peripheral blood lymphocytes is valuable for reference of selecting anti-cancer drugs in clinic for lung cancer.

To clone DNA sequence of human telomerase reverse transcriptase (hTERT) promoter, and study its transcriptional activities in various human lung cancer cells and normal lung cell.hTERT promoter of 1.1 kb was amplified with polymerase chain reaction (PCR) method, utilizing DNA of 293 cell as template. After DNA sequencing with correct result, the hTERT promoter was inserted into luci-ferase reporter vectors (pGL3-Basic) to reconstruct a recombinant plasmid named pGL3-hTERTp. Then the reconstructed plasmid was transiently transfected into lung cancer cell lines A549, SPC-A-1, LTEPa-2, NCI-H446, YTMLC, GLC-82, A2 and human embryonic pulmonary fibroblast cell line MRC-5. The transcriptional activities of hTERT promoter in various cells were determined by measuring the luciferase activities after 48 hours of transfection.Electrophoresis demonstrated that cloned hTERT promoter was about 1.1 kb, and DNA sequencing showed a same sequence as registered in GenBank. The cloned hTERT promoter was located between 1 126 bp and 43 bp in upstream of the transcription start site ATG and the length was 1 086 bp. The recombinant plasmid pGL3-hTERTp was confirmed by double digestion and PCR method with correct results. Luciferase assay showed there were different transcriptional activities of hTERTp in various lung cancer cell lines, but not in the MRC-5 cell line.The hTERT promoter cloned in this study has transcriptional activities in various lung cancer cell lines but not in normal cell. It may act as control element in tumor-targeting gene therapy with hTERT.

To explore the possibility of targeting blockage of Wnt signal transduction pathway of nm23-H1 gene transfection in human high-metastatic large cell lung cancer cell line L9981, and to provide evidence to elucidate the signal conductive mechanism of nm23-H1 mediated tumor metastasis suppression.The expression of GSK-3β and β-catenin of Wnt signal pathway was detected in cytoplasm and nucleus in L9981 cell line with nm23-H1 deletion, L9981-pLXSN cell line transfected with vector and L9981-nm23-H1 cell line transfected with nm23-H1 gene by Western blot.(1)GSK-3β expression in L9981-nm23-H1 cytoplasm (6 341±541) was significantly higher than those in L9981 (3 736±298) and L9981-pLXSN (3 613±383) cell lines ( P0.001); (2)GSK-3β expression in L9981-nm23-H1 nucleus (4 356±490) was significantly higher than those in L9981 (657±57) and L9981-pLXSN (705±75) cell lines ( P0.001); (3)β-catenin expression in L9981-nm23-H1 cytoplasm (3 649±118) was significantly higher than those in L9981 (1 401±31) and L9981-pLXSN (1 350±55) cell lines ( P0.001); (4)No statistical difference of the β-catenin expression in nucleus was observed among L9981-nm23-H1 (2 945±68), L9981 (2 604±23) and L9981-pLXSN (2 652±53)( P0.05); (5)No significant difference of GSK-3β or β-catenin expression in cytoplasm and nucleus was observed between L9981 and L9981-pLXSN ( P0.05).(1)nm23-H1 gene can remarkably upregulate the expression of GSK-3β in cytoplasm and nucleus, and β-catenin expression in cytoplasm in L9981-nm23-H1 cell, but can not induce the nucleus accumulation of β-catenin. (2)Regulation of GSK-3β and β-catenin expression, and targeting blockage of Wnt signaling pathway may be one of molecular mechanisms that nm23-H1 contributes to play a vital role in the "Lung Cancer Metastasis Suppressive Cascade".

To establish a human large cell lung cancer cell line L9981-nm23-H1 transfected with wild type nm23-H1 gene.pLXSN-nm23-H1-EGFP was constructed by gene clone technique, and L9981-nm23-H1 was established by infected virus of nm23-H1 gene. The DNA and protein expression of nm23-H1 were detected in the transgene large cell lung cancer cell line by PCR and Western blot.pLXSN-nm23-H1-EGFP was constructed successfully, and the nm23-H1 cDNA was inducted to L9981 cell. The protein of nm23-H1 could be detected in L9981-nm23-H1 cell.Protein of nm23-H1 is stably, continuously and high efficiently expressed in L9981-nm23-H1 cell.

To investigate gene diagnosis of micrometastasis in lymph nodes in patients with non-small cell lung cancer (NSCLC) and the feasibility of mucin 1 (MUC1) mRNA and cytokeratin 19 (CK19) mRNA as molecular marker to detect micrometastasis of lung cancer.Expression of MUC1 mRNA and CK19 mRNA was detected in 119 lymph nodes taken from 31 patients with NSCLC, 35 lymph nodes from 10 patients with pulmonary benign diseases as controls by nested reverse transcriptase-polymerase chain reaction (RT-PCR).In the 119 lymph nodes from lung cancer patients, CK19 mRNA expression was detected in 66 lymph nodes (55.5%) and MUC1 mRNA expression was detected in 65 lymph nodes (54.5%) by RT-PCR. Neither CK19 mRNA nor MUC1 mRNA expression was observed in all the 35 lymph nodes in the benign pulmonary lesion group.The results suggest that the detection of both MUC1 and CK19 mRNA might be helpful to diagnose NSCLC micrometastasis in lymph nodes. The establishment of this method may lead to an earlier diagnosis of metastasis for lung cancer.

To explore the influences of nm23-H1 gene transfection and protein kinase C (PKC) inhibitor Calphostin C on PKC signal transduction pathway in human high-metastasis large cell lung cancer cell line L9981, and to evaluate the effects of nm23-H1 gene on translocation and activation in subcellular region.The translocation of PKC in subcellular region was observed in L9981 before and after nm23-H1 gene transfection and Calphostin C treatment by Laser scanning confocal microscope (LSCM) method.PKC-α and PKC-βII were found to locate in different subcellular site in L9981 before and after nm23-H1 gene transfection. PKC-α and PKC-βII mainly located in nucleus and perinucleus in L9981 and L9981-pLXSN cell lines, which were in active status. PKC-α and PKC-βII mainly located in soluble cytosolic fraction in L9981-nm23-H1 cell line and were inactive status. PKC-α and PKC-βII mainly located in cytosolic fraction and were in inactive status in all the three cell lines after treatment with Calphostin C.The results suggest that nm23-H1 gene might make PKC to translocate from nucleus and perinucleus to soluble cytosolic fraction in L9981 cell line. PKC inhibitor, Calphostin C, can also make PKC to translocate from nucleus and perinucleus to soluble cytosolic fraction in L9981, L9981-pLXSN cell lines. Both transfection of nm23-H1 gene and treatment with Calphostin C can suppress the PKC signal transduction in L9981 cell line.

To construct a DC-Ad-Ki-ras(V12) vaccine and investigate the anti-tumor activities of lung cancer dendritic cell vaccine modified by mutant Ki-ras gene in vitro.Ki-ras(V12) cDNA was transfected into cultured bone marrow-derived DC with the recombinant adenovirus [(Ad-Ki-ras(V12)] containing human mutant Ki-ras gene. Anti-tumor activity of the vaccine was studied in vitro by flow cytometry, PCR, MLR and cytotoxicity assay.(1) The DC vaccine was confirmed not only to express Ki-ras(V12) gene, but also to remarkably stimulate lymphocyte proliferation and improve CTL activity. (2) The DC vaccine modified by mutant Ki-ras gene could induce specifical CTL activity of immunized mice against Lewis lung carcinoma that could express Ki-ras(V12) gene, but not to B16.The DC vaccine modified by mutant Ki-ras gene can induce obvious anti-tumor activities against Lewis lung carcinoma that can express Ki-ras(V12) gene.

To study the regulation of nm23-H1 gene on the expression of integrins in human high-metastasis large cell lung cancer cell line L9981 by transfecting nm23-H1 into cell.Lipofect was used to transfect nm23-H1 into the L9981 cell line; semi-RT-PCR was used to detect the difference of integrin β1 and integrin β3 mRNA expressions between tranfected and non-transfected cell lines; flow cytometry was used to detect the difference of integrin β1 and integrin β3 protein expressions between transfected and non-transfected cell lines.(1) A transgenic cell line L9981-nm23-H1 was obtained, which could stably and effectively express nm23-H1. The mRNA expressions of integrin β1 and β3 in L9981-nm23-H1 cell line were remarkably lower than those in L9981 cell line. (2) The protein expressions of integrin β1 and β3 in L9981-nm23-H1 cell line were significantly lower than those in L9981 cell line too (P0.01).Transfection of nm23-H1 gene into the L9981 cell line can significantly down-regulate the mRNA and protein expressions of integrin β1 and β3 in this cell line, which indicates that nm23-H1 may reverse the metastasis potential of L9981 cell line through modulation of integrin expression.

To investigate the influence of tumor metastasis suppressor gene nm23-H1 on the activity of glycogen synthase kinase 3β (GSK-3β) in human high-metastasis large cell lung cancer cell line L9981.The levels of GSK-3β expression in cytoplasm and nucleus were determined with anti- GSK-3β antibody in human high-metastasis large cell lung cancer cell line L9981 (cell line with nm23-H1 gene deletion), L9981-nm23-H1 (cell line with nm23-H1 transfected) and L9981-pLXSN (cell line with vector transfected) by Western blot method. The activity of GSK-3β among those three cell lines was detected by immunoprecipitation and analysed by a radioactive isotope scintillation counter before and after treating with 20 mmol/L LiCl.(1) The expression indensity of GSK-3β of cytoplasm and nucleus was (6 341±541) and (4 356±490) IOD in L9981-nm23-H1, (3 613±383) and (705±75) IOD in L9981-pLXSN, and (3 736±298) and (675±57) IOD in L9981, respectively. A high significance in GSK-3β expressive indensity of both cytoplasm and nucleus existed among L9981-nm23-H1, L9981-pLXSN and L9981 (P0.01); Multiple comparison: A highly significant difference was observed when L9981-nm23-H1 was compared with L9981-pLXSN or L9981 (P0.01), but no significant difference was observed between L9981-pLXSN and L9981 (P0.05). (2) The GSK-3β activity of cytoplasm and nucleus was (28 955±2 509) and (9 247±924) CPM in L9981-nm23-H1, (11 241±1 495) and (1 492±176) CPM in L9981-pLXSN, and (12 505±1 469) and (1 763±125) CPM in L9981, respectively. A highly significant difference in GSK-3β activity of both cytoplasm and nucleus existed among L9981-nm23-H1, L9981-pLXSN and L9981 (P0.01); Multiple comparison: the GSK-3β activity in L9981-nm23-H1 was significantly higher than that in L9981-pLXSN and L9981 (P0.01), but no significant difference was observed between the L9981-pLXSN and L9981 (P0.05). (3) After treatment with 20 mmol/L LiCl, the expressive indensity of GSK-3β of cytoplasm and nucleus was (4 718±549) and (3 823±350) IOD in L9981-nm23-H1, (2 030±155) and (217±15) IOD in L9981-pLXSN, and (2 164±151) and (224±19) IOD in L9981, respectively. No significant difference in GSK-3β expressive indensity existed between before and after treatment with LiCl in L9981-nm23-H1 (P0.05). However, the GSK-3β expressive indensity in cytoplasm and nucleus before treatment was remarkably higher than those after treatment in both L9981-pLXSN and L9981 (P0.05). (4) After treatment with 20 mmol/L LiCl, the GSK-3β activity in cytoplasm and nucleus was (11 099±1 112) and (3 748±215) CPM in L9981-nm23-H1, (4 447±430) and (1067±159) CPM in L9981, and (4 435±427) and (909±156) CPM in L9981-pLXSN, respectively. The GSK-3β activity both in cytoplasm and nucleus after treatment with LiCl was remarkably lower than that before treatment in L9981-nm23-H1, L9981-pLXSN and L9981 (P0.01 or P0.05).(1) Transfection of nm23-H1 gene can significantly up-regulate the expression level and activity of GSK-3β in human high-metastasis large cell lung cancer cell line L9981; (2) LiCl can remarkably suppress the upregulation effects of nm23-H1 gene on GSK-3β activity in L9981 cell line; (3) The effects of nm23-H1 gene on suppressing the signal transduction of Wnt pathway might be carried out through upregulating GSK-3β expression and activity in human high-metastasis large cell lung cancer cell line L9981.

To investigate the expression features of the heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) in non-small cell lung cancer and its clinical significance.hnRNP A2/B1 expression of cancer tissues, paracancerous lung tissues, resected bronchial stump epithelium tissues was detected in 58 non-small cell lung cancer patients and pulmonary tissues in 30 patients with benigh pulmonary lesions as control by immunohistochemistry methods.The positive expressive rate of hnRNP A2/B1 in lung cancer tissue (63.79%) was significantly higher than those in paracancerous lung tissue (43.10%) and benign pulmonary lesion tissues (20.00%) (P=0.000), and the positive rate in paracancerous lung tissue was also significantly higher than that in benign pulmonary lesion tissues (P0.05). The positive rate of hnRNP A2/B1 in the hyperplastic and dysplastic epithelium (40.00%) of resected bronchial stump was remarkably higher than that in normal bronchial epithelium (15.15%) in lung cancer patients (P=0.032). The positive rate of hnRNP A2/B1 in poor differentiated lung cancer (78.13%) was remarkably higher than that in moderate and well differentiated lung cancer (46.15%) (P0.05). The positive rate of hnRNP A2/B1 in lung cancer with lymph node metastasis (75.00%) was significantly higher than that without lymph node metastasis (50.00%) (P0.05). The positive rate of hnRNP A2/B1 in stage III+IV disease (78.57%) was significantly higher than that in stage I+II disease (50.00%) (P0.05). The positive rate of hnRNP A2/B1 in T3+T4 cancer (77.42%) was significantly higher than that in T1+T2 cancer (48.15%) (P0.05). The positive rate of hnRNP A2/B1 expression in lung cancer tissues was not related to histological classification of the cancer (P0.05).The overexpression of hnRNP A2/B1 in cancer tissues may play an important role in the oncogenesis, development and metastasis of lung cancer. Detection of hnRNP A2/B1 expression may be helpful to diagnose lung cancer and to predict prognosis of the patients with lung cancer.

To explore the effects of nm23-H1 gene transfection and forskolin on PKA activity in human high-metastasis large cell lung cancer cell line L9981.Three cell lines, primary human large cell lung cancer cell line (L9981), vector transfection cell line (L9981-pLXSN) and nm23-H1 gene transfection cell line (L9981-nm23-H1-pLXSN), were treated with PKA activator forskolin. The PKA activity at different time points after treatment with forskolin was detected in the three lung cancer cell lines by radioimmunological method with SignaTECT cAMP-dependent PKA assay system.(1) Before forskolin treatment, the activity of PKA of L9981-nm23-H1-pLXSN was remarkably higher than those of L9981 and L9981-pLXSN (P0.01), but no significant difference in the PKA activity was observed between L9981 and L9981-pLXSN (P0.05). (2)The PKA activity was remarkably increased in all the three lung cancer cell lines after treatment with different concentration of forskolin (P0.01), and up to the highest level at the concentration of 100 μmol/L. It showed a dose-dependent relationship between the PKA activity and forskolin concentration; (3) The PKA activity in all the three cell lines was elevated to the highest level at 30 minutes after treatment with forskolin of 100 μmol/L, and it showed a time-dependent relationship between the PKA activity and action time of forskolin.(1)Transfection of nm23-H1 gene can up-regulate the PKA activity of human high-metastasis large cell lung cancer cell line L9981, and its function as a tumor metastasis suppressor gene may be related to its effects on regulation of PKA signal transduction pathways; (2)Forskolin can remarkably up-regulate the PKA activity of L9981 cell line, and the elevation of PKA activity has a time-dependent and dose-dependent relation to forskolin.

To investigate the influence of the tumor metastasis suppressor gene nm23 H1 on the activity of extracellular signal-regulated protein kinase (ERK) in human high metastasis large cell lung cancer cell line L9981.The levels of total ERK1/2 and phospho-pERK1/2 were determined with p44/42 MAP kinase antibody and dually phosphospecific phospho-44/42 MAP kinase antibody in human high-metastasis large cell lung cancer cell lines L9981 (cell line with nm23-H1 gene deletion), L9981-nm23-H1 (cell line with nm23-H1 transfected ) and L9981-PLXSN (cell line with vector transfected) by Western blot method, respectively. The activity of phospho-ERK1/2 was determined with an ERK1/2 assay kit by immunopreciptation and Western blot analysis.The expression levels of phospho-ERK1/2 kinase and the activity of phospho-ERK1/2 in the lung cancer cell line L9981-nm23-H1 were remarkably higher than those of the L9981 cell line and L9981-PLXSN cell line ( P0.01), but no significant difference in both the phospho-ERK1/2 expression and phospho-ERK1/2 activity was observed between the L9981 and L9981-PLXSN cell lines ( P0.05). There was no significant difference in the total ERK1/2 level among the three cell lines.nm23-H1 gene can obviously targetly suppress the activity of ERK1/2 in human high metastasis large cell lung cancer cell line L9981. This suggest that the mechanisms of nm23-H1 gene as a tumor metastasis suppressor gene may be related to its suppression to the MAPK/ERK signal transduction pathway.

To study the relationship between the serum levels of endostatin,VEGF and clinical and pathophysiological characteristics in the non-small cell lung cancer (NSCLC) patients.The serum levels of endostatin and VEGF were detected in 46 patients with NSCLC and 14 patients with benign pulmonary diseases as control by ELISA mothod.(1)The preoperative serum level of endostatin [( 20.85 ±4.56) μg/L] and VEGF [(1.75±0.37) μg/L] in lung cancer patients was significantly higher than those in patients with benign pulmonary diseases [(15.68±2.78) μg/L and (1.05±0.32) μg/L). (2)The preoperative serum level of endostatin and VEGF in lung cancer patients was closely related to P-TNM stages, distant metastasis, grade of cell differentiation and the size of the primary tumors ( P0.05), but not to the histological classification, type of the tumor, lymph node status, age, sex of the patients and smoking or not ( P0.05). (3)The serum level of endostatin in lung cancer patients on the 7th postoperative day [(23.41± 5.12 ) μg/L] was significantly higher than that before operation [(20.85±4.56) μg/L] ( P0.05) and on the 1st postoperative day [(18.89±4.67) μg/L] ( P0.001). The serum level of VEGF in lung cancer patients on the 7th postoperative day [(3.75±0.71) μg/L] was also significantly higher than that before operation [(1.72±0.46) μg/L] and on the 1st postoperative day [(2.22±0.58) μg/L] ( P0.001). (4)The preoperative serum level of endostatin was highly negatively correlated to serum VEGF level in lung cancer patients ( r=-0.380, P0.01).Elevation of serum endostatin and VEGF exists in patients with NSCLC. The serum levels of endostatin and VEGF in patients with NSCLC might be helpful to evaluate the biological behavior of lung cancer.

It has been known that oncogenesis and development of lung can- cer is a complex process regulated many genes and involved in multistages. Recent studies have demonstrated that signal transduction abnormality may play a very important role in these procedures. The aim of this study is to investigate the influence of exogenous MEK1/2 pathway inhibitor U0126 on the expression and activity of extracellular signal-regulated kinase (ERK1/2) of human high-metastatic large cell lung cancer cell line L9981 and its malignant biological behaviors.The expressive levels of total-ERK1/2, dually phosphorylated ERK1/2 and ERK1/2 relative activity of the human high-metastatic large cell lung cancer cell line L9981 (parent cell line with nm23-H1 gene hetero-deletion ) were detected by Western blot and immuno-precipitation technique after treating with different doses of U0126. The in vitro proliferative and invasive abilities of the lung cancer cell line were determined by MTT and modified Boyden chamber methods.The ERK1/2 relative activity of L9981 gradually decreased accompanied with increase of U0126 doses, and a highly significant difference of phosphorylated ERK1/2 expression level existed among the different concentration groups of U0126 (P0.01), but no significant difference of total ERK1/2 expression was found among the different concentration groups of U0126 (P=0.387). After treatment with same concentration of U0126 for different time, the ERK1/2 relative activity of L9981 gradually decreased as the treatment time of U0126 prolonging, and a highly significant difference of phosphorylated ERK1/2 expression level was observed among different treatment time groups (P0.01). But no significant difference of total ERK1/2 expression level was found among different time groups (P=0.689). The inhibition of ERK1/2 pathway by MEK1/2 specific inhibitor U0126 targeting the ERK1/2 pathway of L9981 was dose- and time-dependent. After treatment with different concentration of U0126, the proliferation of L9981 gradually decreased accompanied with increase of U0126 concentration and a highly significant difference existed among different groups of U0126 concentration (P0.001). The in vitro invasion of L9981 gradually decreased accompanied with increase of U0126 concentration. No significant difference of in vitro invasion of L9981 was found among 0, 10 and 20μmol/l of U0126 (P0.05). A highly significant difference was observed when U0126 concentration increased to 40 and 60μmol/l compared with 0, 10 and 20μmol/l of U0126 (P0.001).The inhibition of Ras-to-MAPK pathway by Ras-to-MAPK specific inhibitor U0126 targeting the Ras-to-MAPK pathway of the human high-metastatic large cell lung cancer cell line L9981 is dose-dependent and time-dependent. Suppressing or blocking of Ras-to-MAPK signal transduction pathway can reverse the invasive and metastatic phenotype of the human high-metastatic large cell lung cancer cell line L9981. These results suggest that the key kinase MEK1/2 of the ERK1/2 pathway may be a potent therapeutic target for human lung cancer.

It has been proved that tumor development and metastasis are dependent on angiogenesis. Suppression of tumor angiogenesis can inhibit tumor growth and metastasis. Collagen X VIII/endostatin is one of the most effective inhibitors of angiogenesis at present. The aim of this study is to study the relationship between transcription expression of endostatin mRNA and clinical and pathophysiological characteristics in non-small cell lung cancer (NSCLC).The transcription expression of endostatin mRNA was detected in 46 lung cancer tissues and paracancerous lung tissues, 14 benign pulmonary lesion tissues as control by RT-PCR method.(1)The transcription expression of endostatin mRNA in lung cancer tissues (0.872±0.071) was significantly higher than that in paracancerous tissues (0.717±0.073) and benign pulmonary lesion tissues (0.611±0.026) (P0.001).(2)The transcription expression of endostatin mRNA in lung cancer tissues was closely related to P-TNM stages, distant metastasis, grade of cell differentiation and size of the primary tumors (P0.05), but not to location of tumor, lymph node status, histological classification, age and sex of the patients and smoking or not (P0.05).The transcription expression of endostatin mRNA in NSCLC tissues is significantly higher than that in paracancerous tissues and benign pulmonary lesion tissues, and is closely related to P-TNM stages, distant metastasis, grade of cell differentiation and size of the primary tumors, hence it might be helpful to evaluate the biological behavior of lung cancer.

Some studies have supposed that glutathione S-transferases (GSTs) may be involved in detoxification of carcinogens, especially from tobacco smoke. Therefore, polymorphism of GSTs has been considered as potential protectors of individual cancer risk. The objective of this study is to investigate the relationship between genetic polymorphism of GSTT1 and inherent susceptibility to lung cancer in Han population in Sichuan, China.A case-control study was carried out to compare the distribution frequency of GSTT1 gene polymorphism between lung cancer (n=150) and control healthy individuals (n=152) with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method and to analyze the relationship between the GSTT1 gene polymorphism and smoking and the inherent susceptibility of lung cancer.(1) The distribution frequency of GSTT1(-) genotype was 54.7% (82/150) in lung cancer and 38.2% (58/152) in control group respectively (OR=1.681, 95%CI=1.009- 2.803 , P=0.046); (2) GSTT1(-) genotype remarkably increased the risk of squamous cell carcinoma (OR=2.969, 95%CI= 1.511 -5.834, P=0.002) and adenocarcinoma (OR=2.095, 95%CI=1.060-4.140, P= 0.033 ); (3) In smokers, GSTT1(-) genotype significantly increased the risk for lung cancer (OR=4.051, 95%CI=1.959-8.380 , P=0.000); (4) In people with GSTT1(-) genotype, smoking markedly increased the risk for lung cancer (OR=53.885, 95%CI=11.789-246.302, P=0.000); (5) In heavy smokers (≥20 packyears), GSTT1(-) genotype could remarkably increase the risk of lung cancer (OR=4.296, 95%CI=1.649-11.190, P=0.003).(1) People with GSTT1(-) genotype have significantly increased risk for lung cancer in Han population in Sichuan, China, especially for squamous cell carcinoma. (2) GSTT1(-) genotype interacts synergistically with smoking on lung cancer risk. The more the cigarettes smoke, the higher the risk of lung cancer increases in those people who are smokers with GSTT1(-) genotype.

The human FHIT gene at chromosome 3p14.2 is a tumor suppressor gene, and its abnormality in structure and function is related to carcinogenesis and progression of lung cancer. It is postulated that FHIT and NIT1 likewise collaborate in biochemical or cellular pathway in mammalian cells, but their molecular mechanisms in lung cancer cell are still unknown. The aim of this study is to construct procaryotic expression vector of human NIT1, providing a foundation to explore the expression of human NIT1 protein and to study the interaction of NIT1 and FHIT genes in lung cancer cell lines.The NIT1 cDNA was acquired by RT-PCR. EcoRI/NotI digested PCR product was directly cloned into procaryotic expression vector pET-32a.The sequence of NIT1 cDNA clone exactly corresponded with the sequence of NIT1 cDNA in GenBank. The expectant fragments of DNA were obtained after recombinant procaryotic expression vector was digested by EcoRI and NotI.The procaryotic expression vector of human NIT1 is successfully constructed by RT-PCR and direct clone. It provides an important basis to detect the expression of NIT1 protein and to further explore the relationship between NIT1 and FHIT genes in oncogenesis and development of human lung cancer.

Genetic polymorphism in metabolic enzymes, which are involved in metabolism of environmental carcinogens, have been thought to be related to susceptibility of lung cancer. The aim of this study is to investigate the cytochrome P450 2D6(CYP2D6) genetic polymorphism distribution in Han population in Sichuan, China, and to evaluate the relationship between CYP2D6 genetic polymorphism and lung cancer susceptibility.PCR-RFLP was used to identify CYP2D6ch genotypes among 150 patients with primary lung cancer and 152 healthy controls, in Han population in Sichuan, China, and case-control study was used to analyze the relationship between genetic polymorphism and lung cancer susceptibility.(1) The distribution frequency of CYP2D6ch C and T allele were 39.5% and 60.5% in control group and 46.3% and 53.7% in lung cancer group, respectively. There was no significant difference between the two groups (P=0.089). (2)The distribution frequency of C/C, C/T and T/T genotypes were 18.4%, 42.1% and 39.5% in control group, and 22.7%, 47.3% and 30.0% in lung cancer group, respectively. No significant difference was found between the two groups (P=0.215). (3) The individuals who carried with Non-T/T genotypes had a 2.084-fold increased risk with squamous cell carcinoma (95%CI 1.024-4.244, P=0.043) than those who carried with T/T genotype. (4) The lighter smokers (30 pack-years) who carried with Non-T/T genotypes had a 2.92-fold increased risk with lung cancer (95%CI 1.087-7.828, P=0.033) than those who carried with T/T genotype.CYP2D6ch Non-T/T genotypes are factors associated mail:zhouqh@mail.sc.cninfo.net) with increased risk of squamous cell carcinoma and also increase risk of lung cancer among lighter smokers.

Lung cancer is the leading cause of malignant tumor death among Chinese population. It has been known that the development of lung cancer may be associated with genetic po-lymorphism of some lung cancer related genes. The aim of this study is to evaluate the relationship between genetic polymorphism of metabolizing enzymes and susceptibility of lung cancer in Chinese population.Polymorphism of CYP2E1 RsaI/PstI and GSTM1 was detected in 99 patients with lung cancer and 66 patients with benign pulmonary disease by PCR-RFLP and PCR. The association between genetic polymorphism and susceptibility of lung cancer was analyzed.No significant difference in three RsaI/PstI genotype distribution of CYP2E1 was found between lung cancer group and control group (Chi-Square=1.374, P=0.241). (2) The frequency of GSTM1-null genotype in lung cancer group was significantly higher than that in control group (57.6% vs 40.9%, Chi-Square=4.401, P=0.036). (3) The individuals who carried with GSTM1-null genotype had a 1.96 fold increased risk of lung cancer (OR=1.96, 95%CI=1.042-3.689, P=0.037) than those who carried with GSTM1-present genotype. (4) When data were stratified by smoking status, the smokers who carried with c1/c1 genotype had a significantly higher risk of lung cancer (OR=3.525, 95%CI=1.168- 10.638, P=0.025) than those never-smokers who carried with at least one c2 allel. (5) When combination of polymorphism of CYP2E1 RsaI/PstI genotype and GSTM1 genotype was analyzed, compared with individuals who had concurrent present of GSTM1 and at least one c2 allel genotype, the risk of lung cancer for combination of GSTM1 null and c1/c1 genotype was increased significantly (OR=3.449, 95%CI=1.001- 11.886, P=0.050). Considering smoking status, compared with never-smokers who had concurrent present of GSTM1 and at least one c2 allel genotype, the risk of lung cancer for combination of GSTM1 null and c1/c1 genotype was remarkably increased (OR=11.553, 95%CI=1.068-124.944, P=0.044), as well as that for combination of GSTM1 null and at least one c2 allel genotype (OR=13.374, 95%CI=1.258-142.166, P= 0.032).(1)GSTM1 null genotype is an important factor associated with increased risk of lung cancer. (2) The combination of c1/c1 and GSTM1-null genotype can remarkably increase risk of lung cancer both in smokers and non-smokers.

It has been proved that Tanshinone has obvious anticancer effect, but its mechanisms of anticancer are still unknown. Anticancer Ketonon is complex antitumor drug which Tanshinone is combined with other anticancer elements. This study aims to explore the antineoplastic effects of Anticancer Ketonon on Lewis lung cancer and the mechanisms in mice.The mice were divided into three groups: Ketonon group, 5-fluorouracil (5-Fu) group and control group. The former two groups were treated with responsive drugs after subcutaneous inoculation of Lewis lung cancer. The last group was only treated with normal saline after inoculation. Apoptosis index and cell cycle were measured by flow cytometry.Two experiments were carried out in male and female mice respectively. The tumor inhibitory rates of Anticancer Ketonon were 38.9% and 32.2% respectively, those of 5-FU were 59.6% and 53.9%. Compared with those of control groups, the tumor weights in Ketonon group and 5-Fu group were statistically decreased (P0.05). Metastasis rates of the lung in the three groups were not statistically different (P0.05). The apoptosis index of Ketonon group was significantly higher than that of control group (P0.05), but the cell cycle was not statistically changed compared with that of control group (P0.05).Anticancer Ketonon has antineoplastic effect on Lewis lung cancer in mice and the mechanism may be associated with inducing apoptosis of tumor cells.

Metastasis is not only the malignant characteristics of lung can- cer, but also the chief cause of failure to cure and high mortality of lung cancer. To better explore and understand the mechanism of lung cancer metastasis and to search for potential markers for early diagnosing and reversing lung cancer metastasis, differential proteomic analysis is conducted in two human large cell lung cancer cell lines with high metastasis potentials (L9981) and low metastasis potentials (NL9980) by two-dimension gel electrophoresis (2-DE).The total proteins of the two cell lines were separated by immobilized pH gradient (IPG)-based 2-DE. The differentially expressed proteins of the two cell lines were analyzed using image analysis software.A high resolution and reproducible 2-DE image was successfully obtained. Average deviations for protein position in IEF direction were (0.858±0.076)mm and (1.514±0.127)mm in SDS-PAGE direction. The relative standard deviation for protein volume was (12.06±0.580)% in L9981 and (12.22±0.640)% in NL9980. The average total number of protein spots was 902±169 in L9981 cells and 941±173 in NL9980 cells in three repeated experiments. Image analysis of siliver-stained 2-DE image revealed that 4 protein spots had significant differential expressions in L9981 and NL9980 (student's t-test, P0.05). Fifteen protein spots were only detected in L9981, and 27 protein spots were only detected in NL9980.The results in this study suggest that an obviously differential proteomic expression exists between the human high- and low-metastatic large cell lung cancer cell lines. It will be helpful to further understand the molecular mechanisms of lung cancer invasion and metastasis, and provide new experimental evidence for searching metastatic-related molecule of lung cancer.

nm23-H1 gene is a well-known tumor metastasis suppression gene. Our previous study has found that transfection of wild type nm23-H1 gene can significantly downregulate the ERK1/2 activity of human high-metastatic large cell lung cancer cell line L9981. The aim of this study is to investigate the influence of nm23-H1 and exogenous ERK1/2 pathway inhibitor U0126 on the extracellular signal-regulated kinase (ERK1/2) of human high-metastatic large cell lung cancer cell line L9981 and its malignant biological behaviors.The expressive levels of total-ERK1/2, dually phosphorylated ERK1/2 and ERK1/2 relative activity of the human high-metastatic large cell lung cancer cell lines, L9981 (parent cell line with nm23-H1 gene hetero-deletion), L9981-nm23-H1 (transfected with nm23-H1 gene ) and L9981-PLXSN (transfected with vector) were detected by Western blot and immunoprecipitation technique after treating with U0126 (40μmol/L for 20 minutes). The in vitro proliferative and invasive abilities among the above three lung cancer cell lines were determined by MTT and improved Boyden chamber methods.The phosphorylated ERK1/2 expression level and relative activity in L9981-nm23-H1 lung cancer cell line were remarkably lower than those in L9981 and L9981-PLXSN lung cancer cell lines after being treated with U0126 (P0.01), but there was no significant difference between L9981 and L9981-PLXSN lung cancer cell lines. No significant difference of total ERK1/2 expression level was observed among the three lung cancer cell lines (P0.05) after being treated with U0126. The in vitro proliferation and invasion of L9981-nm-23H1 lung cancer cell line were remarkably lower than those of L9981 and L9981-PLXSN lung cancer cell lines (P0.01 ), but no significant difference was found between L9981 and L9981-PLXSN lung cancer cell lines (P0.05 ); U0126 could significantly down-regulate the in vitro proliferation and invasion of L9981 lung cancer cell line (P0.01).Blocking the activity of ERK1/2 in L9981 lung cancer cell line and transfecting the nm23-H1 gene into the L9981 lung cancer cell line may produce similar cell biological behavior changes, namely the significant reduction of in vitro proliferation and invasion of L9981 lung cancer cell line. These results indicate that the molecular mechanism which nm23-H1 gene reverses invasion and proliferation of the human high-metastatic large cell lung cancer cell line may be related to its effects of down-regulating the activity of the key kinase ERK1/2 of Ras-to-MAPK signal transduction pathway.

It has been proved that selenium has remarkable effects in the prevention of cancer and proliferation inhibition for breast cancer and prostate cancer. Up to now, little is known, however, if methylseleninic acid (MSA) has the anticancer effect on lung cancer or not. The objective of this study is to detect the effect of MSA on proliferation inhibition and apoptotic induction for human high-metastatic large cell lung cancer cell line L9981, and to explore the molecular mechanisms.The changes of proliferation, clone formation, apoptotic level and cell cycles were detected in L9981 by trypan blue staining, clone formation suppression test, and flow cytometry before and after treating with different concentration of MSA. The expression level of proliferative-related and apoptotic-related genes was also determined in L9981 by flow cytometry.(1)The proliferation ability of L9981 was remarkably inhibited at the concentration of 0.5μmol/L of MSA (P0.05), and the cells were arrested at G0/G1 phase after treating with the same concentration. (2)Apoptosis of L9981 was remarkably induced by MSA at the concentration of 2.5μmol/L (P0.05). (3)The clone formation ability of L9981 was significantly suppressed by MSA at the concentration of 5.0μmol/L (P0.05). (4)The expression levels of P53, P21, Fas, FasL and Bax were remarkably up-regulated after treatment with MSA.(1)MSA can significantly suppress the proliferation and clone formation ability of human high-metastatic large cell lung cancer cell line L9981, and also induce apoptosis of L9981. (2)The anticancer effects of MSA might be related to regulate the expression of cell cycle-related genes and apoptotic-related genes in the human high-metastatic large cell lung cancer line L9981.

The results of our previous studies have proven that nm23-H1 gene can suppress metastasis of lung cancer, which may be associated with suppression of the Wnt signal pathway through up-regulating the activity of glycogen synthase kinase 3β (GSK-3β), the key kinase of the Wnt signal pathway. The aim of this study is to investigate the effect of point mutation of nm23-H1 gene on GSK-3β activity in cytoplasm and nucleus in human high-metastatic large cell lung cancer cell line L9981.Using immunoprecipitation and a radioactive isotope scintillation counter, the activity of GSK-3β was detected in cytoplasm and nucleus of human low-metastatic large cell lung cancer cell line NL9980, human high-metastatic large cell lung cancer cell line L9981, L9981-pEGFP (transfected with vector), L9981-nm23-H1-pEGFP (transfected with wild type nm23-H1), L9981-nm23-H1 S44A -pEGFP mutant (transfected with serine 44 to alanineon of nm23-H1 gene), L9981-nm23-H1 P96S -pEGFP mutant (transfected with proline 96 to serine of nm23-H1 gene), L9981-nm23-H1 H118F -pEGFP mutant (transfected with histidine 118 to phenylalanine of nm23-H1 gene) and L9981-nm23-H1 S120G -pEGFP mutant (transfected with serine 120 to glycine of nm23-H1 gene).The GSK-3β activity in cytoplasm and nucleus was remarkably decreased in the transgene lung cancer cell lines transfected with mutant nm23-H1 cDNA (L9981-nm23-H1 S44A -pEGFP, L9981-nm23-H1 P96S -pEGFP, L9981-nm23-H1 H118F -pEGFP and L9981-nm23-H1 S120G -pEGFP). Significant differences of GSK-3β activity in cytoplasm and nucleus were observed (P0.05) when L9981-nm23-H1-pEGFP cell line was compared with L9981-nm23-H1 S44A -pEGFP, L9981-nm23-H1 P96S -pEGFP, L9981-nm23-H1 H118F -pEGFP and L9981-nm23-H1 S120G -pEGFP lung cancer cell lines. There was a highly significant difference in GSK-3β activity in the cytoplasm between L9981-nm23-H1-pEGFP cell line and L9981-nm23-H1 P96S -pEGFP lung cancer cell line (P0.01 ). A highly significant difference in GSK-3β activity in the nucleus was observed (P0.01) when L9981-nm23-H1-pEGFP lung cancer cell line was compared with L9981-nm23-H1 S44A -pEGFP, L9981-nm23-H1 P96S -pEGFP and L9981-nm23-H1 H118F -pEGFP lung cancer cell lines.(1) Point mutation of nm23-H1 gene can significantly influence the regulating effects on the GSK-3β activity in the cytoplasm and nucleus in the human high-metastatic large cell lung cancer cell line L9981. (2) The effects of nm23-H1 gene on metastatic phenotype may be related to the upregulation of GSK-3β activity in human high-metastatic large cell lung cancer cell line L9981.

Previous researches have proven that nm23-H1 gene is a tumor metastatic suppressor gene, however, it is still unknown about its exact molecular mechanisms. Site-directed mutagenesis can correctly change the base sequence and get mutant proteins. The aim of this study is to construct nm23-H1 mutant and EGFP fusion genes by site-directed mutagenesis, and to provide basement for studying the functional and biochemical mechanisms of nm23-H1 gene.Site-directed mutagenesis of nm23-H1 gene was performed by modified QuikChange™ Site-directed Mutagenesis Kit method. Pure plasmid containing fusion gene of nm23-H1 and EGFP (PLXSN-nm23-H1-EGFP) was mini-prepared. Four pairs of mutagenic primers were synthesized in vitro and the desired five mutations, S44A, P96S, H118F, S120G and P96S-S120G were introduced into nm23-H1-EGFP fusion gene by PCR.Five nm23-H1 mutant and EGFP fusion genes, nm23-H1(S44A)-EGFP, nm23-H1(P96S)-EGFP, nm23-H1 (H118F)-EGFP, nm23-H1(S120G)-EGFP and nm23-H1(P96S-S120G)-EGFP, were successfully constructed. The results of DNA sequencing confirmed that the base sequences of the mutant fusion genes were completely concordant with experiment design.Five nm23-H1 mutant and EGFP fusion genes are successfully constructed, which can be used in further studies. QuikChange™ site-directed mutagenesis is a simple, rapid and efficient method.

Lung cancer is one of the most malignant cancers which is hazarding the people's health and life in the world. At present, it is a highlight to exploit antitumor drug from plant at home and abroad. The aim of this study is to observe the effects of polysaccharid (PS-T) on expression of angiogenic-related gene mRNA in human high-metastatic large cell lung cancer cell line L9981, and to explore its possible molecular mechanism.L9981 in vitro was cultured, and the growth data were obtained by trypan blue staining. The mRNA transcript expression of β-catenin, E-cadherin, TIMP-1, CD44V6, MMP-2, endostatin, VEGF was detected in L9981 by RT-PCR before and after treating with PS-T. The ability of invasion of L9981 was determined by Boyden chamber method.(1)PS-T had remarkably inhibitive effects on the growth of L9981 in vitro. The inhibitive rate of PS-T on L9981 was concentration-dependent. No significant difference of inhibitive rate was found among the PS-T (1g/L), cisplatin (3mg/L) and PS-T (0.05g/L) + cisplatin (1.5mg/L)(P0.05). (2)The mRNA expression level of β-catenin, E-cadherin, TIMP-1, endostatin and MMP-2 was upregulated, while that of VEGF and CD44V6 was downregulated. Out of them the mRNA expression level of TIMP-1 and endostatin was remarkably upregulated, the expression level of CD44V6 was significanyly downregulated. (3)The in vitro invasive abilities of L9981 was significantly decreased in the PS-T, DDP and PS-T+DDP groups compared with that in blank control group.(1)PT-S could inhibit the growth of human high-metastatic large cell lung cancer cell line L9981 in vitro, the effect is dose-dependent. (2)PS-T can down- or up-regulate the mRNA transcript expression of some angiogenic-related gene mRNA. (3)PS-T has remarkably coordinating effects with cisplatin in the L9981 lung cancer cell line.

The present experimental data have showed that the function of kinase suppressor of Ras (KSR) is mainly as a scaffold protein that coordinates the assembly of a multiprotein complex containing MAPK and its upstream regulators. But whether KSR has kinase activity is still the point of argument until now. The aim of this study is to construct eukaryotic expression vectors of carboxyl terminus and amino terminus of KSR and to detect their expression in 293T cell line.N-KSR and C-KSR were amplified by PCR. The eukaryotic expression vectors of pCMV-Tag2b-N-KSR and pCMV-Tag2b-C-KSR were constructed by gene recombination technique and the recombinant plasmids were verified by restriction enzyme analysis and sequencing. Then positive clones were transfected into 293T cell line. Expression of target proteins was analyzed by Western blot.The sequences and open read frames of the two vectors were both completely concordant with experiment design. The target proteins could be observed in transfected 293T cells by Western blot.Eukaryotic expression vectors of pCMV-Tag2b-N-KSR and pCMV-Tag2b-C-KSR are successfully constructed, and they can be expressed in 293T cells. It provides an experimental base for further research work.

It has been proved that lysosome-associated protein transmembrane 4 beta (LAPTM4B) plays an important role in tumorigenesis, the *2/*2 genotype of LAPTM4B was closely related to liver cancer susceptibity. The aim of this study is to investigate the possible association between the allelic variation of LAPTM4B and the genetic susceptibility of lung cancer.The genotype of LAPTM4B was detected in 131 patients with lung cancer and 104 unrelated healthy adult individuals as control by polymerase chain reaction based on special primers. The genotypic distribution of LAPTM4B was analyzed by Chi-square test.The allelic frequencies of the *1 and *2 were 74.8% and 25.2% in the lung cancer group and 72.1% and 27.9% in the healthy control group respectively, but no significant difference was observed between the two groups (Chi-square=0.433, P=0.510). The genotypic distribution of the *1/*1,*1/*2 and *2/*2 were 53.4%, 42.7% and 3.8% in the lung cancer group, 54.8%, 34.6% and 10.6% in the healthy control group respectively, there was no significant difference between the two groups (Chi-square=4.89, P=0.087). No remarkable association was observed between the genotypic distribution of LAPTM4B and the clinical information in the patients with lung cancer, including pathological type and TNM staging.The results suggest that the allele of LAPTM4B is not closely associated with genetic susceptibility of lung cancer.

Ras to MAPK pathway plays a critical role in the transmission of many growth and developmental signals. A new component of this pathway which is termed kinase suppressor of Ras (KSR) was found in 1995. KSR is as a scaffolding protein that coordinates the assembly of a multiprotein complex containing mitogen-activated protein kinase (MAPK) and its upstream regulators. It has been proven that KSR has many phosphorylation sites, and phosphorylation state changes response to signaling events. Site-directed mutagenesis can precisely change the base sequence and get mutant proteins. The aim of this study is to construct two mutant proteins of KSR by using site-directed mutagenesis, and to express and purify them, therefore to provide basement for studying the functional and biochemical mechanisms of KSR.Site-directed mutagenesis of pCMV-Tag2b-KSR gene was performed by modified QuikChangtm site-directed mutagenesis kit method. Two pairs of mutagenic primers were synthesized in vitro and two mutations desired, the recombinant plasmids were verified by restriction enzyme analysis and DNA sequencing. Then positive clones were transfected into 293T cell line. The purified mutant proteins were analyzed by Western blot.Two mutants were successfully constructed. The results of DNA sequencing confirmed that the base sequences of the mutant genes were completely concordant with experiment design, which could be used to be transfected into 293T cell line. The purified mutants were identified by Western blot.Two mutant KSR genes are successfully constructed. It provides experimental basement for further functional research of KSR.

It has been confirmed that nm23-H1 gene is one of the tumor metastasis suppressor genes. Up to now, the exact mechanism of nm23-H1 gebe is uncertain. The aim of this study the mechanism of metastasis suppressor gene nm23-H1 involving in the Ras signaling of lung cancer.The wild and mutant type of pEGFP-nm23-H1 plasmids [WT (wild type), H118F, S120G, P96S, S44A] were transfected into the L9981 lung cancer cell lines through liposome method, and the complex of KSR and nm23-H1 was detected through co-immunoprecipitation and Western blot assay.The human KSR could be detected in the nm23-H1 immunoprecipitations in all the trasfected L9981 lung cancer cell lines. But no significant difference of KSR expression was found in the wild and mutant nm23-H1 trasfected cell lines (F =0.190, P =0.938).There was a close interaction between nm23-H1 and KSR, which was independent of the nm23-H1 mutation. Nm23-H1 involving in the Ras signaling of lung cancer may be through the KSR gene.