Human Large Cell Lung Cancer Research Articles

Establishment and characterization of a human large cell lung cancer cell line with neuroendocrine differentiation.

Characterization of a human lung cancer cell line is reported. This cell line was established from a patient referred to Nemazi Hospital of Shiraz University of Medical Sciences with a diagnosis of poorly differentiated carcinoma. Sterile sample from peritoneal effusion was taken and immediately cultured in RPMI-1640 medium containing 20% FBS, at 37 degrees C with 5% CO2. This cell line has been in continuous culture for more than one year and has been named as Mehr-80. Several features of the cell line were investigated, including growth characteristics, electron microscopic features, cloning efficiency in soft agar, expression of various antigenic markers, chromosomal and DNA analysis. On the basis of morphological and immunohistochemical analysis of Mehr-80, it is possible to conclude that this cell line is characterized by features similar to those reported for large cell carcinoma with neuroendocrine differentiation (LCCND). This cell line will be a valuable in vitro tool for further studies on lung cancers.

Targeting blockage of Wnt signal transduction pathway of nm23-H1 gene transfection in human high-metastatic large cell lung cancer cell line L9981

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 ( P < 0.001); (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 ( P < 0.001); (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 ( P < 0.001); (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)( P > 0.05); (5)No significant difference of GSK-3β or β-catenin expression in cytoplasm and nucleus was observed between L9981 and L9981-pLXSN ( P > 0.05). (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".

Establishment of a human large cell lung cancer cell line L9981-nm23-H1

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.

Experimental study of effects of nm23-H1 gene transfection on the change of PKA activity in human high-metastasis large cell lung cancer cell line L9981

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 (P < 0.01), but no significant difference in the PKA activity was observed between L9981 and L9981-pLXSN (P > 0.05). (2)The PKA activity was remarkably increased in all the three lung cancer cell lines after treatment with different concentration of forskolin (P < 0.01), 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.

Transfection of tumor metastasis suppressor gene nm23-H1 can up-regulate the activity of GSK-3β in human high-metastasis large cell lung cancer cell line L9981

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 (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). (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.

Regulation of nm23-H1 transfection on expression of integrins in human high-metastasis large cell lung cancer cell line L9981

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 (P < 0.01). 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.

Study on protein kinase C translocation before and after transfection of nm23-H1 gene in human lung cancer cells using Laser scanning confocal microscope

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.

Transfection of the tumor metastasis suppressor gene nm23-H1 can targetly suppress the activity of extracellular signal-regulated protein kinase (ERK) in human high-metastasis large cell lung cancer cell line L9981

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 ( P < 0.01), 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 ( P > 0.05). 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.

Cathepsin B and cysteine proteinase inhibitors in human lung cancer cell lines.

Cell lines derived from human squamous cell (EPCL), large cell (LCLC), and small cell lung cancer (SCLC) lines were investigated for the expression of cathepsin B (Cat B) and cysteine proteinase inhibitors (CPIs). The EPLC and LCLC lines expressed 5- to 50-fold more Cat B activity and contained more mature Cat B of M(r) 27-29 kDa (> 2.5 microg/mg total protein) than the SCLC lines (< 1.0 microg/mg total protein). The LPLC lines also secreted the highest amounts of Cat B precursor of M(r) about 46 kDa. Inhibitory activities against Cat B and papain were associated with high molecular mass (HMM) and low molecular mass (LMM) inhibitory proteins, both in cell extracts and in media. About 75% of the inhibitory activity was associated with HMM inhibitors, the majority of which were kininogens (M(r) > or = 67 kDa). The LMM inhibitors of M(r) 10-15 kDa were cystatin C and stefins A and B, which were quantitated by ELISA: stefins A and B were present in cell extracts and medium in similar concentrations (5-200 ng/10(6) cells), while 80-99% of the cystatin C was released in the medium (10-195 ng/10(6) cells). Phorbol ester (PMA), which induces protein-kinase C mediated signal transduction and enhances cellular differentiation in many non-small cell lung cancer (NSCLC) cell lines, increased intracellular Cat B activity and Cat B protein as well as its secretion in some cell lines but not in others, regardless of their histological type. PMA significantly (P < 0.049) decreased intracellular stefin A concentrations in two EPLC lines and non-significantly in two LCLC lines. PMA decreased secretion of stefin A in all EPLC lines, but not in LCLC lines, while IGF-I significantly increased stefin B secretion in both SCLC lines. These data showed that lung tumor cells produce both cysteine proteinases and cystatins. As the antagonistic molecules are regulated differently in histologically different types of lung tumor cells, it is possible that an imbalance between the proteinases and their specific inhibitors plays a role in progression of certain types of lung tumors in vivo.

Examination by laser scanning confocal fluorescence imaging microscopy of the subcellular localisation of anthracyclines in parent and multidrug resistant cell lines.

This study highlights the usefulness of laser scanning confocal microscopy in the examination of subcellular disposition of anthracyclines in tumour cell lines. The distribution of anthracycline compounds has been studied in two pairs of parental and multidrug resistant (MDR) cell lines. For the parental EMT6 mouse mammary tumour cell line EMT6/P treated with doxorubicin (DOX) the anthracycline fluorescence was shown to be predominantly nuclear but with some particulate cytoplasmic fluorescence and very low levels of plasma membrane staining. In the same experiments much fainter fluorescence was seen for the EMT6/AR1.0 MDR subline which hyperexpresses P-glycoprotein. The loss of nuclear fluorescence was comparatively greater than loss of cytoplasmic fluorescence. For the human large cell lung cancer line COR-L23/P cellular DOX disposition was markedly nuclear with nuclear membrane staining and diffuse cytoplasmic fluorescence. For the MDR line COR-L23/R, which lacks P-glycoprotein expression, DOX fluorescence was reduced in the nucleus compared with the parental line, but an intense area of perinuclear staining was seen consistent with localisation to the Golgi apparatus. The morpholinyl-substituted analogue MR-DOX achieved very similar subcellular distribution in both parental and MDR lines, consistent with its retention of activity in the latter. The presence of verapamil during anthracycline exposure increased the intensity of fluorescence in the MDR lines, particularly in the nucleus. Relatively little effect was seen in the parental lines. Confocal microscopy provides high resolution images of the subcellular distribution of anthracyclines in parent and MDR cell lines. Differences in drug disposition in various cell lines may provide insights into the mechanism of multidrug resistance and suggest strategies for its therapeutic circumvention.

Chemosensitisation and drug accumulation effects of cyclosporin A, PSC-833 and verapamil in human MDR large cell lung cancer cells expressing a 190k membrane protein distinct from P-glycoprotein

The doxorubicin-selected multidrug resistant (MDR) human large cell lung cancer line COR-L23/R, lacks P-glycoprotein but shows a drug accumulation deficit. It does however overexpress a 190k membrane protein which shares an epitope with, but is otherwise distinct from, P-glycoprotein. The resistant cells show only a small sensitisation to vincristine and daunorubicin on treatment with cyclosporin A and its more potent analogue, PSC-833 despite an increase in drug accumulation. Verapamil, another effective resistance modifier in P-glycoprotein MDR cells, is slightly more effective. Fluorescent daunorubicin distributes in the cytoplasm and nucleus of sensitive parent COR-L23 cells but is confined to cytoplasmic perinuclear vesicles in resistant cells. Addition of cyclosporin A or PSC-833 slightly increases cytoplasmic fluorescence whereas verapamil also increases nuclear fluorescence. Resistance in this non-P-glycoprotein MDR line, COR-L23/R where these resistance modifiers have little effect may be associated with expression of the 190k protein.

Non-P-glycoprotein-mediated multidrug resistance with reduced EGF receptor expression in a human large cell lung cancer cell line

Non-P-glycoprotein-mediated multidrug resistance with reduced EGF receptor expression in a human large cell lung cancer cell line

Monoclonal antibodies that distinguish non-small cell from small cell lung cancer.

Two murine IgG2Ak monoclonal antibodies (703D4, 704A 1) were produced and characterized after immunization with a human large cell lung cancer line (NCI-H 157). These antibodies detect different epitopes on 31 kilodalton [35S]methionine incorporating protein(s). Radiobinding and immunohistochemical studies show these antibodies bind to most (11/13) human non-small cell lung cancer (adenocarcinoma, epidermoid, and large cell), but not to small cell lung cancer (0/11) tumors tested. The epitopes these antibodies recognized are also expressed on human melanomas (7/8), two other tumors (osteogenic sarcoma, renal cell carcinoma), but not on many other human tumors (breast, colon, neuroblastoma, lymphoid), and not on a panel of normal adult human tissues. Because the antigen(s) are preserved after fixation and because of their ability to distinguish lung cancer types from each other and normal tissues, they should be of clinical, as well as of biologic interest.

Activity of 9-10 anthracenedicarboxaldehyde bis[(4,5-dihydro-1 H-imidazol-2-yl)hydrazone]dihydrochloride (CL216,942) in a human tumor cloning system. Leads for phase II trials in man.

We have utilized a recently developed human tumor cloning system to screen for antitumor effects in vitro of a new anthracene derivative, CL216,942. The object was to determine whether the system is useful for pinpointing the types of tumors in patients which should be studied in early phase II clinical trials. Tumors from 684 patients were placed in culture (27 different histologic tumor types). Two hundred seventy-three tumors both grew and formed enough colonies for drug sensitivity assays. In vitro antitumor activity was noted for CL216,942 against human breast cancer, ovarian cancer, renal cancer, squamous cell, small cell and large cell lung cancer, lymphoma, acute myelogenous leukemia, melanoma, adenocarcinoma of unknown origin, adrenal cancer, gastric cancer, pancreatic cancer, and head and neck cancer. The drug definitely showed no in vitro activity against colon cancer. These data indicate that CL216,942 has a wide spectrum of in vitro antitumor activity. A comparison of these in vitro results with the results of phase II clinical trials with the drug should allow an evaluation of the utility of the human cloning system for predicting clinical activity of a new compound.