BACKGROUNDTo 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.

METHODSExpression 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).

RESULTSIn 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.

CONCLUSIONSThe 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.

BACKGROUNDTo establish a human large cell lung cancer cell line L9981-nm23-H1 transfected with wild type nm23-H1 gene.

METHODSpLXSN-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.

RESULTSpLXSN-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.

CONCLUSIONSProtein of nm23-H1 is stably, continuously and high efficiently expressed in L9981-nm23-H1 cell.

BACKGROUNDTo investigate the expression features of the heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) in non-small cell lung cancer and its clinical significance.

METHODShnRNP 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.

RESULTSThe 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 (P < 0.05). 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%) (P < 0.05). 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%) (P < 0.05). 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%) (P < 0.05). The positive rate of hnRNP A2/B1 in T3+T4 cancer (77.42%) was significantly higher than that in T1+T2 cancer (48.15%) (P < 0.05). The positive rate of hnRNP A2/B1 expression in lung cancer tissues was not related to histological classification of the cancer (P > 0.05).

CONCLUSIONSThe 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.

BACKGROUNDTo 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.

METHODSThe 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.

RESULTS(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 (P < 0.01); Multiple comparison: A highly significant difference was observed when L9981-nm23-H1 was compared with L9981-pLXSN or L9981 (P < 0.01), but no significant difference was observed between L9981-pLXSN and L9981 (P > 0.05). (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 (P < 0.01); Multiple comparison: the GSK-3β activity in L9981-nm23-H1 was significantly higher than that in L9981-pLXSN and L9981 (P < 0.01), but no significant difference was observed between the L9981-pLXSN and L9981 (P > 0.05). (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 (P > 0.05). 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 (P < 0.05). (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 (P < 0.01 or P < 0.05).

CONCLUSIONS(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.

BACKGROUNDTo 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.

METHODSTwo 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.

RESULTS(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.

CONCLUSIONS(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.

nm23-H1 is a proven tumor metastasis suppressive gene, tumor metastasis phenotype could be reversed by transfected nm23-H1 cDNA. This study was conducted to transfect nm23-H1 cDNA into L9981 cells and to explore the function of nm23-H1 in reversing the malignant phenotype of L9981 cells. The plasmid of pLXSN-nm23-H1-EGFP was constructed by gene clone technique, and the transfected nm23-H1 cDNA cell lines of L9981-nm23-H1 was established. The protein expression of nm23-H1 was detected by Western blot. The biologic features of L9981-nm23-H1 cells were studied in vitro and in vivo. The results showed that the fusion protein of nm23-H1-EGFP was stable, continuous and expressed with high efficiency in L9981-nm23-H1 cells. The cell proliferation, colon formation and invasive ability are significantly lowered in L9981 cells transfected nm23-H1 cDNA (P < 0.01); the tumorgenesis and the lung metastasis incidence was lower in tranfected nm23-H1 cells than in L9981 and L9981-Plxsn in nude mice (P < 0.01); the rate for inhibiting tumorgenesis of nm23 -H1 was 82.56%. These data suggest that the malignant phenotype could be reversed by wild nm23-H1 gene in L9981 cells.
Animals ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Lung Neoplasms ; pathology ; Mice ; Mice, Nude ; NM23 Nucleoside Diphosphate Kinases ; genetics ; Neoplasm Metastasis ; Neoplasm Transplantation ; Recombinant Fusion Proteins ; genetics ; Transfection ; Tumor Suppressor Proteins ; genetics

BACKGROUNDThe 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.

METHODSThe NIT1 cDNA was acquired by RT-PCR. EcoRI/NotI digested PCR product was directly cloned into procaryotic expression vector pET-32a.

RESULTSThe 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.

CONCLUSIONSThe 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.

BACKGROUNDIt 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.

METHODSThe 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.

RESULTSTwo 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 (P < 0.05). Metastasis rates of the lung in the three groups were not statistically different (P > 0.05). The apoptosis index of Ketonon group was significantly higher than that of control group (P < 0.05), but the cell cycle was not statistically changed compared with that of control group (P > 0.05).

CONCLUSIONSAnticancer Ketonon has antineoplastic effect on Lewis lung cancer in mice and the mechanism may be associated with inducing apoptosis of tumor cells.

BACKGROUNDLung 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.

METHODSPolymorphism 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.

RESULTSNo 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).

CONCLUSIONS(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.

BACKGROUNDMetastasis 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).

METHODSThe 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.

RESULTSA 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, P < 0.05). Fifteen protein spots were only detected in L9981, and 27 protein spots were only detected in NL9980.

CONCLUSIONSThe 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.