Small Lung Carcinoma Cell Line Research Articles

Integrated classification of lung tumors and cell lines by expression profiling.

The utility of cancer cell lines depends largely on their accurate classification, commonly based on histopathological diagnosis of the cancers from which they were derived. However, because cancer is often heterogeneous, the cell line, which also has the opportunity to alter in vitro, may not be representative. Yet without the overall architecture used in histopathological diagnosis of fresh samples, reclassification of cell lines has been difficult. Gene-expression profiling accurately reproduces histopathological classification and is readily applicable to cell lines. Here, we compare the gene-expression profiles of 41 cell lines with 44 tumors from lung cancer. These profiles were generated after hybridization of samples to four replicate 7,685-element cDNA microarrays. After removal of genes that were uniformly up- or down-regulated in fresh compared with cell-line samples, cluster analysis produced four major branch groups. Within these major branches, fresh tumor samples essentially clustered according to pathological type, and further subclusters were seen for both adenocarcinoma (AC) and small cell lung carcinoma (SCLC). Four of eight squamous cell carcinoma (SCC) cell lines clustered with fresh SCC, and 11 of 13 SCLC cell lines grouped with fresh SCLC. In contrast, although none of the 11 AC cell lines clustered with AC tumors, three clustered with SCC tumors and six with SCLC tumors. Although it is possible that preexisting SCC or SCLC cells are being selected from AC tumors after establishment of cell lines, we propose that, even in situ, AC will ultimately progress toward one of two poorly differentiated phenotypes with expression profiles resembling SCC or SCLC.

MYO18B, a candidate tumor suppressor gene at chromosome 22q12.1, deleted, mutated, and methylated in human lung cancer.

Loss of heterozygosity on chromosome 22q has been detected in approximately 60% of advanced nonsmall cell lung carcinoma (NSCLC) as well as small cell lung carcinoma (SCLC), suggesting the presence of a tumor suppressor gene on 22q that is involved in lung cancer progression. Here, we isolated a myosin family gene, MYO18B, located at chromosome 22q12.1 and found that it is frequently deleted, mutated, and hypermethylated in lung cancers. Somatic MYO18B mutations were detected in 19% (14/75) of lung cancer cell lines and 13% (6/46) of primary lung cancers of both SCLC and NSCLC types. MYO18B expression was reduced in 88% (30/34) of NSCLC and 47% (8/17) of SCLC cell lines. Its expression was restored by treatment with 5-aza-2'-deoxycytidine in 11 of 14 cell lines with reduced MYO18B expression, and the promoter CpG island of the MYO18B gene was methylated in 17% (8/47) of lung cancer cell lines and 35% (14/40) of primary lung cancers. Furthermore, restoration of MYO18B expression in lung carcinoma cells suppressed anchorage-independent growth. These results indicate that the MYO18B gene is a strong candidate for a novel tumor suppressor gene whose inactivation is involved in lung cancer progression.

Cell Adhesion-Mediated Transformation of a Human SCLC Cell Line Is Associated with the Development of a Normal Phenotype

Small cell lung carcinoma (SCLC) is a highly metastatic disease with a poor prognosis due to its resistance to current modes of therapy. SCLC cells appear to arise by oncogenic transformation of self-renewing pulmonary neuroendocrine cells, which have the potential to differentiate into a variety of lung epithelial cell lineages. Epithelial–mesenchymal conversion involved in such cell type transitions leads to the acquisition of an invasive and metastatic phenotype and may be critical for neoplastic progression and its eventual resistance to therapy. In order to investigate mechanisms involved in such transitions, a SCLC cell line was exposed to 5-bromodeoxyuridine. This treatment induced a dramatic conversion from non-substrate-adherent aggregates to monolayers of cells exhibiting an epithelioid phenotype. The phenotypic transition was concomitant with downregulation of vimentin, upregulation of cytokeratins, and cell–cell and cell–matrix adhesion molecules as well as redistribution of the actin cytoskeleton. The changes in the levels and organization of cell–cell and cell–matrix adhesion molecules were correlated with an in vivo loss of tumorigenicity.

Expression of Histidine Decarboxylase and Synthesis of Histamine by Human Small Cell Lung Carcinoma

We recently found that human small cell lung carcinomas (SCLCs) express, in addition to other neuroendocrine markers, vesicular monoamine transporters. Our present results indicate that SCLCs are histaminergic. We detected the biosynthetic enzyme histidine decarboxylase by immunohistochemistry in paraffin sections of 12 biopsies of SCLC tumors. This finding was supported by immunoblotting and reverse transcription-polymerase chain reaction experiments using established SCLC cell lines, frozen and paraffin-embedded SCLC tumors. Moreover, we found histamine to be synthesized, stored, and released by cultured SCLC cells. Our novel observations may be useful for developing new diagnostic tools for this frequent and highly malignant tumor.

Cyclooxygenase-2 expression influences the growth of human large and small cell lung carcinoma lines in athymic mice: Impact of an organoselenium compound on growth regulation

Increased expression of cyclooxygenase-2 (COX-2) significantly enhances carcinogenesis and inflammatory reactions. Regulation of COX-2 overexpression may be a reasonable target for cancer chemoprevention. We have tested the hypothesis that levels of COX-2 expression determine the growth of human lung cancer cells in nude mice. Two cell lines, NCI-H460 (non-small cell lung cancer) and NCI-H69 (small cell lung cancer) were selected because the former expresses high levels of COX-2 protein and the latter has no detectable levels. We also examined the effects of 1,4-phenylenebis(methylene)selenocyanate (p-XSC), a highly effective chemopreventive organoselenium compound and known inhibitor of COX-2 expression, in vivo, on cell growth and COX-2 expression in vitro in the NCI-H460 cancer cell line. Cells were exposed to p-XSC at levels between 10 and 100 microM for six days and showed toxicity at approximately 50 microM. Pre-exposure of NCI-H460 to non-toxic levels of p-XSC suppressed COX-2 protein expression in a dose-dependent manner. At 40 microM, p-XSC suppressed phorbol myristate acetate (PMA)-induced COX-2 expression in NCI-H460 cells by more than 66%. In vivo studies in athymic mice showed a significant difference in tumor volume between cell lines. Pre-treatment of NCI-H460 cells with a non-toxic dose of p-XSC, prior to their injection into nude mice, significantly suppressed tumor growth when compared to untreated cells. Collectively, the outcome of our in vitro and in vivo studies supports the hypothesis that levels of COX-2 expression determine the extent of human lung tumor growth in athymic mice. Therefore, inhibition of COX-2 expression by agents such as p-XSC provides a strong rationale for the development of future clinical prevention trials.

Failure of steroid regulation of the MMTV promoter in a small cell lung cancer cell line is caused by a DNA sequence flanking the glucocorticoid response element.

We have previously described a panel of human small cell lung carcinoma (SCLC) cell lines that have profound glucocorticoid resistance, resulting from various molecular defects in glucocorticoid signalling. However, in one SCLC cell line, CORL103, the cause of the resistance is unknown. These cells are refractory to dexamethasone stimulation of MMTV even when exogenous wild-type glucocorticoid receptor (GR) is co-transfected. This is in contrast to cell lines DMS79 and CORL24 where resistance is overcome by transfection of the wild-type receptor. Sequencing of the GR from CORL103 cells revealed two point mutations, but neither of these induced dominant negative activity. Steroid hormone resistance extended to mineralocorticoid and progesterone receptor (MR, PR) activation of MMTV-luc, whereas oestrogen and thyroid hormone receptor transactivation were normal. A simpler reporter, TAT3-luc, containing three copies of the tyrosine aminotransferase glucocorticoid response element (GRE), was responsive when transfected into CORL103 cells with GR, MR and PR expression vectors and activated with their respective ligands. Similarly, pHH-luc and pAH-luc (truncated MMTV variants containing the GRE region, both derived from a different strain of MMTV), were effectively transactivated with dexamethasone. This suggests that the minor changes in the flanking sequence of the MMTV promoter are critically important in determining steroid responsiveness in CORL103 cells. We propose that minor differences in MMTV may determine recruitment of co-factors, which destabilise GR binding to the MMTV GREs. These findings represent a new, selective, model of glucocorticoid resistance that may explain specific cell and target gene differences in glucocorticoid sensitivity.

Differential Inactivation of Caspase-8 in Lung Cancers

Caspase-8 (CASP8) is an apoptosis inducing cysteine protease which is activated through the formation of a death-inducing signaling complex when death receptors are complexed to their specific ligands. Recent reports indicate that CASP8 expression is lost via a combination of promoter methylation and allelic loss in a subset of neuroblastomas. We investigated the state of the gene in lung tumors and cell lines. RT-PCR studies indicated that gene expression was lost in most (27 of 34, 79%) of small cell lung carcinoma (SCLC) cell lines, but expression was retained in all 22 non-SCLC (NSCLC) lines tested. Loss of gene expression at the RNA level was associated with absent protein expression by Western blotting and lack of CASP8 enzymatic activity. Methylation of the promoter region of the CASP8 gene was present in 16 of 27 (59%) of the SCLC lines lacking gene expression. All methylated cell lines lacked the presence of an unmethylated allele indicating biallelic methylation or loss of non-methylated allele. Promoter methylation was absent in all SCLC and NSCLC cell lines retaining gene expression, and all of these lines had the unmethylated form of the gene. One non-expressing SCLC cell line, NCI-H82, had a homozygous deletion at 2q33 encompassing the chromosomal location of the CASP8 gene. The mechanism of gene inactivation in the remaining 10 of 27 (37%) non-expressing SCLC cell lines is unknown. Using five polymorphic markers for 2q33 a high frequency of allelic loss was present in SCLC lines. Analyses of fresh tumors showed that 15 of 43 (35%) of the SCLC, seven of 40 (18%) of bronchial carcinoids and none of 44 NSCLC tumors had CASP8 promoter methylation. Because only approximately 60% of SCLC cell lines lacking CASP8 expression were methylated, extrapolating from the cell line data, we estimate that approximately 58% of SCLC and 30% of bronchial carcinoids lack CASP8 expression. Thus, CASP8 expression is absent in a subset of both high grade (SCLC) and low grade (carcinoid) neuroendocrine lung tumors but not in NSCLC, which usually lack neuroendocrine features. CASP8 may function as a tumor suppressor gene in neuroendocrine lung tumors.

Resistant mechanisms of anthracyclines--pirarubicin might partly break through the P-glycoprotein-mediated drug-resistance of human breast cancer tissues.

Juliano and Ling initially reported the expression of a 170 kDa glycoprotein in the membrane of Chinese hamster ovarian cells in 1976, and named this glycoprotein P-glycoprotein (P-gp) based on its predicted role of causing "permeability" of the cell membrane. After much research on anthracycline-resistance, this P-gp was finally characterized as a multidrug-resistant protein coded by the mdr1 gene. Multidrug resistance associated protein (MRP) was initially cloned from H69AR, a human small cell-lung carcinoma cell line which is resistant to doxorubicin (DXR) but does not express P-gp. MRP also excretes substrates through the cell membrane using energy from ATP catabolism. The substrate of MRP is conjugated with glutathione before active efflux from cell membrane. Recently, membrane transporter proteins were re-categorized as members of "ATP-Binding Cassette transporter"(ABC-transporter) superfamily, as shown at http://www.med.rug.nl/mdl/humanabc.htm and http://www.gene.ucl.ac.uk/nomenclature/genefamily/abc.html. A total of ABC transporters have been defined, and MDR1 and multidrug resistance associated protein 1 (MRP1) were reclassified as ABCB1 and ABCC1, respectively. Their associated superfamilies include 11 and 13 other protein, in addition to ABCB and ABCC, respectively. Lung resistance-related protein (LRP) is not a member of the superfamily of ABC transporter proteins, because it shows nuclear membrane expression and transports substrate between nucleus and cytoplasm. LRP was initially cloned from a non-small cell lung carcinoma cell line, SW1573/2R120 which is resistant to DXR, vincristine, etoposide and gramicidin D and does not express P-gp. The mechanisms of resistance remains unclear, and why some resistant cell lines express P-gp and others express MRP and/or LRP is likewise unclear.

Defective caspase-3 relocalization in non-small cell lung carcinoma.

Many anticancer drugs exert their cytotoxicity through DNA damage and induction of apoptosis. Small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC) have different sensitivity to treatment with radiation and chemotherapeutic agents with SCLC being more sensitive than NSCLC both in vitro and in vivo. This difference might be related to the different susceptibility of small and non-small cell lung carcinoma to undergo apoptosis. The aim of this study was to investigate if deficiencies in the apoptotic pathways can explain the intrinsic resistance of NSCLC to anti-cancer treatment. Three different triggers were used to induce apoptosis. Etoposide and gamma-radiation, which are important parts of clinical lung cancer treatment, induce DNA-damage, whereas Fas ligation induces receptor-mediated apoptotic pathways. NSCLC cells were cross-resistant to all treatments, whereas SCLC cells, which do not express pro-caspase-8, were resistant to alphaFas-, but not to DNA-damage-induced apoptosis. Cytochrome c release, activation of caspase-9 and the executioner caspase-3 were observed in both types of lung cancer cells. However, cleavage of known nuclear substrates for caspase-3, such as PARP and DFF45/ICAD, was documented only in the sensitive SCLC cells but not in the resistant NSCLC cells. Moreover, relocalization of active caspase-3 from the cytosol into the nucleus upon treatment was observed only in the SCLC cell line. These results indicate that the inhibition of apoptosis in NSCLC occurs downstream of mitochondrial changes and caspase activation, and upstream of nuclear events.

Collateral sensitivity to gemcitabine (2′,2′-difluorodeoxycytidine) and cytosine arabinoside of daunorubicin- and VM-26-resistant variants of human small cell lung cancer cell lines

Multidrug resistance (MDR), characterized by a cross-resistance to many natural toxin-related compounds, may be caused either by overexpression of a drug efflux pump such as P-glycoprotein, (P-gP), multidrug resistance proteins MRP1–3, or BCRP/MXR or, in the case of DNA topoisomerase II active drugs, by a decrease in the enzymatic activity of the target molecule termed altered topoisomerase MDR (at-MDR). However, human small cell lung carcinoma (SCLC) cell lines showed a collateral sensitivity to 2′,2′-difluorodeoxycytidine (gemcitabine, dFdC) and 1-β- d-arabinofuranosylcytosine (ara-C). H69/DAU, a daunorubicin (DAU)-resistant variant of H69 with a P-gP overexpression, and NYH/VM, a VM-26 (teniposide)-resistant variant of NYH with an at-MDR, were both 2-fold more sensitive to gemcitabine and 7- and 2-fold more sensitive to ara-C, respectively. MDR variants had a 4.3- and 2.0-fold increased activity of deoxycytidine kinase (dCK), respectively. dCK catalyzes the first rate-limiting activation step of both gemcitabine and ara-C. In addition, deoxycytidine deaminase, responsible for inactivation of dFdC and ara-C, was 9.0-fold lower in H69/DAU cells. The level of thymidine kinase 2, a mitochondrial enzyme that can also phosphorylate deoxycytidine and gemcitabine, was not significantly different between the variants. These differences most likely caused an increased accumulation of the active metabolites (dFdCTP, 2.1- and 1.6-fold in NYH/VM and H69/DAU cells, respectively) and of ara-CTP (1.3-fold in NYH/VM cells). Ara-CTP accumulation was not detectable in either H69 variant. The pools of all ribonucleoside and deoxyribonucleoside triphosphates were at least 3- to 4-fold higher in the NYH variants compared to the H69 variants; for dCTP and dGTP this difference was even larger. The higher ribonucleotide pools might explain the >10-fold higher accumulation of dFdCTP in NYH compared to H69 variants. Since dCTP is low, H69 cells might not need a high ara-CTP accumulation to inhibit DNA polymerase. This might be related to the lack of ara-CTP in H69 variants. In addition, the increased CTP, ATP, and UTP pools in the MDR variants might explain the increased ara-CTP and dFdCTP accumulation. In conclusion, the MDR variants of the human SCLC cell lines were collaterally sensitive due to an increased dCK activity, and consequently an increased ara-CTP and dFdCTP accumulation.

Identification of tumour-induced changes in endothelial cell surface protein expression: an in vitro model

The selective destruction of the supporting vasculature of tumours has been proposed as a means of therapy. Fundamental to this approach is the identification of suitable targets on tumour-endothelium. To detect proteins that may be up-regulated on the luminal (apical) surface of tumour-associated endothelium confluent endothelial cells were examined following incubation with tumour cell conditioned medium (TCM) from, or co-culture with, a range of breast carcinoma and small cell lung carcinoma (SCLC) cell lines. Exposed endothelial membrane proteins were labelled with sulpho-NHS-biotin and detected by enhanced chemiluminescence following two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and western blotting. TCM induced varying levels of proliferative activity in endothelial cells; generally breast TCM contained greater mitogenic activity than SCLC TCM. Exposure of human breast and lung microvascular, and umbilical vein endothelial cells to soluble tumour cell factors from several breast cancer and SCLC cells lines produced similar changes in luminal protein profiles: Breast cancer cells and in particular the MDA-MB-231 cell line induced the most pronounced changes. The expression of six proteins was altered consistently on endothelial cells stimulated with soluble tumour cell factors. However, similar changes were observed following incubation with ECGS suggesting that they were related to endothelial cell proliferation per se. As these proteins were altered in breast and lung microvascular, and umbilical vein endothelial cells stimulated by a variety of breast cancer and SCLC cell lines they support the potentially broad applicability of anti-vascular approaches targeted at the endothelium.

Differential sensitivity to etoposide (VP-16)-induced S phase delay in a panel of small-cell lung carcinoma cell lines with G1/S phase checkpoint dysfunction.

The highly schedule-dependent cytotoxic agent etoposide (VP-16) is initially effective in the treatment of small-cell lung cancer (SCLC), particularly in multidrug combination chemotherapy. Heterogeneity in cellular sensitivity to cell cycle arrest may underpin the inadequacy of low-dose extended-cycle single-agent regimes in tumours with partially dysfunctional apoptotic signalling pathways. We have studied the longevity and dose dependency of cell cycle and to a limited extent the apoptotic responses of a panel of seven unselected SCLC cell lines, screened for TP53 status. Cells were analysed using flow cytometry for the cell cycle responses and field inversion gel electrophoresis for apoptotic patterns. The mitotic inhibitor nocodazole was used to assess and correct cell line response data for differences in cell cycle traverse per se. An overall lack of G1/S arrest and muted DNA fragmentation were consistent with the presence of TP53 gene abnormalities. Maximal G2 arrest but with clear recovery potential occurred at an exposure dose (ED, concentration of drug x time) value of approximately 24 microM h. Higher doses (ED values >48 microM h) revealed a wide variation in S phase delay that was independent of population doubling time and could not be compensated for by drug concentration changes alone. The results suggest that heterogeneity in the in vitro sensitivity of unselected SCLC cell lines to S phase arrest is demonstrable at ED values projected to be critical for clinical activity. Such variation in S phase responsiveness may reflect differences in checkpoint activation and offer a functional target for the design of more-effective combination therapy.

Nicotinic receptor-mediated activation by the tobacco-specific nitrosamine NNK of a Raf-1/MAP kinase pathway, resulting in phosphorylation of c-myc in human small cell lung carcinoma cells and pulmonary neuroendocrine cells.

Small cell lung carcinoma (SCLC) expresses phenotypic features of pulmonary neuroendocrine cells and demonstrates a strong etiologic association with smoking. SCLC cell lines express a Raf-1-dependent mitogenic signal transduction pathway, which is thought to transduce the mitogenic signals initiated by neuropeptide autocrine growth factors. Recent studies have identified the tobacco-specific carcinogenic nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) as a site-selective high-affinity agonist for the alpha7 nicotinic acetylcholine receptor (alpha7 nAChR), which regulates the growth of a significant subset of SCLC in vitro by stimulating the release of the autocrine growth factor serotonin. The purpose of this study was to identify signaling events initiated by binding of NNK to the alpha7 nAChR. We have used a human SCLC cell line and fetal hamster pulmonary neuroendocrine cells with in vitro kinase activation assays and western blots to assess the levels of expression and activation of Raf-1, MAPK and c-myc to address this issue. Our data show that NNK activates the Raf-1, MAP kinase pathway, resulting in phosphorylation of c-myc. The activation of this signal transduction pathway by NNK was inhibited by the site-selective antagonist for the alpha7 nAChR alpha-bungarotoxin (alpha-BTX) or by the serotonin reuptake inhibitor imipramine, suggesting that the responses to NNK were mediated by nicotinic receptor-initiated release of serotonin. Accordingly, NNK-induced 5-HT release was blocked by alpha-BTX while NNK-induced DNA synthesis was inhibited by alpha-BTX, imipramine, the PKC inhibitor sphingosine or the MEK inhibitor PD98059. SCLC cells demonstrated high basal levels of 5-HT release, DNA synthesis, and over-expressed Raf-1 and MAPK protein suggesting the constitutive activation of an upstream regulator such as the alpha7 nAChR. Our findings link, for the first time, the stimulation of a nicotinic acetylcholine receptor by a cancer-causing agent with the activation of a Raf-1/MAPK/c-myc signaling pathway. Furthermore, our data suggest that serotonin uptake inhibitors may protect against the development or be useful in the clinical management of SCLC.

Expression and fine mapping of murine vasoactive intestinal peptide receptor 1.

Vasoactive intestinal peptide (VIP) plays multiple roles in the nervous, endocrine, and immune systems as a neurotransmitter, a hormone, and a cytokine. VIP is widely distributed in neurons of the central and peripheral nervous systems (CNS/PNS), and recently has been found to be an important neuroprotective agent. VIP actions are mediated through specific G protein-coupled receptors. We have cloned the cDNA of VIP receptor subtype 1 (VIPR1 or VPAC1) and have demonstrated the quantitative expression profile in mice. Fluorometric real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrated that VPAC1 is expressed in all tissues examined. Expression was highest in the small intestine and colon followed by the liver and brain. The high level of VPAC1 expression in forebrain and cerebellum suggests that VPAC1 may mediate the neuroprotective effect of VIP. We have refined the chromosomal localization of the mouse, rat, and human VPAC1 genes. This fine mapping of the VPAC1 gene extends the respective regions of synteny between the distal region of mouse chromosome 9, rat chromosome 8q32, and human chromosome 3p21.33-p21.31. Thus, VPAC, constitutes a functional-positional candidate for the tumor-suppressor function mapped to human 3p22-p21 where loss-of-heterozygosity is observed in small-cell lung carcinoma (SCLC) cell lines and primary tumors. Availability of the cDNA sequences for mouse VPAC1 will facilitate the generation of VPAC1 null mutant animals. Such studies will ultimately enhance our understanding of the role of VIP in the nervous system.

Presynaptic neuronal antigens expressed by a small cell lung carcinoma cell line

Small cell lung carcinoma (SCLC) is a tumour of neuroendocrine origin often found in association with autoimmune paraneoplastic neurological disorders. We established a SCLC cell line from a woman with Lambert–Eaton myasthenic syndrome (LEMS) who developed antibodies to both the P/Q-type voltage-gated calcium channels (VGCC) and the muscle acetycholine receptor (AChR). We used a range of techniques to establish which neuronal antigens were expressed in her tumour cell line. The results show that many proteins involved in exocytosis are present in the SCLC cells, and that depolarisation-dependent release of [ 3H]-serotonin is linked to calcium influx through P/Q-type VGCCs. In addition, some of the subunits encoding the AChR and both agrin and ARIA, molecules released from the motor nerve during development, were expressed. These results suggest that many potential antigenic targets are present in SCLC, and indicate a surprising ‘motor nerve terminal’-like characteristic of this line.

Identification of a 428-kb homozygously deleted region disrupting the SEZ6L gene at 22q12.1 in a lung cancer cell line.

Frequent allelic losses on chromosome 22q in small cell lung carcinomas (SCLCs) and advanced non-small cell lung carcinomas indicate the presence of tumor suppressor gene(s) on this chromosome arm. We detected a homozygous deletion at 22q12.1 in a SCLC cell line, Lu24. Cloning of the breakpoints of the Lu24 deletion revealed that the deletion was interstitial and 428, 131 bp in size. The deleted region contained the SEZ6L (Seizure 6-like) gene, whose structure had been partially determined by the chromosome 22 sequencing project. We determined the full length cDNA sequence for the SEZ6L gene based on the genomic sequence for the SEZ6L locus using the GENSCAN program and the RT - PCR method. The deduced SEZ6L protein was a transmembrane protein of 1024 amino acids with multiple domains involved in protein - protein interaction and signal transduction. SEZ6L expression was detected in a variety of human tissues, including lung, while its expression was detected in 14 (30%) of 46 lung cancer cell lines examined. Missense mutations were detected in three (7%) of the 46 cell lines, and a 1 bp deletion in the polypyrimidine tract preceding exon 4 was detected in one (2%) of 46 primary lung cancers. Therefore, it is possible that genetic and/or epigenetic SEZ6L alterations are involved in the development and/or progression in a subset of lung cancer, although functional analysis of the SEZ6L gene as well as molecular analysis of other genes in the homozygously deleted region is necessary to understand the pathogenetic significance of 22q deletions in human lung carcinogenesis.

Differential Expression of the LAMB3 and LAMC2 Genes between Small Cell and Non-Small Cell Lung Carcinomas

To identify genes differentially expressed between small cell lung carcinoma (SCLC) cells and non-SCLC cells, mRNA differential display was applied to 3 SCLC cell lines and 6 non-SCLC cell lines. The LAMB3 gene was identified as being expressed only in non-SCLC cells and not in SCLC cells. The LAMB3 gene encodes the laminin β3 chain, which is a unique component of laminin-5. Laminin-5 is a heterotrimer protein consisting of the α3, β3, and γ2 chains, and another unique component of laminin-5 is the γ2 chain encoded by the LAMC2 gene. RT-PCR analysis of the LAMB3 and LAMC2 genes in 45 lung cancer cell lines revealed that both the LAMB3 and LAMC2 genes were co-expressed in 21 of 32 non-SCLC cell lines (66%) but only in one of 13 SCLC cell lines (8%). Coexpression of the LAMB3 and LAMC2 genes was also observed in all 4 cases of primary non-SCLC cells examined but not in the corresponding non-cancerous lung cells. Since α6β4 integrin, the specific laminin-5 binding receptor, is known to be expressed only in non-SCLC cells and not in SCLC cells, it was indicated that laminin-5 is a critical microenvironmental factor for the growth of non-SCLC cells but not of SCLC cells. The differences in the expression of integrins and laminins would be critical factors to distinguish SCLC and non-SCLC cells, and such differences might be associated with the unique biological properties of SCLC cells, including metastatic potential and drug sensitivity.

Prohormone convertase and autocrine growth factor mRNAs are coexpressed in small cell lung carcinoma.

A hallmark of small cell lung carcinoma (SCLC) is the expression of autocrine growth factors such as neurotensin and gastrin-releasing peptide, which bind to cellular receptors and stimulate cell division. The biological activity of autocrine growth factors requires the concurrent expression of prohormone convertases that cleave the growth factors to their active form, suggesting the expression of these genes is linked in SCLCs. RNase protection assays were used to detect the expression of autocrine growth factor and prohormone convertase mRNAs in a panel of lung cancer cell lines. These mRNAs are coexpressed in SCLC and lung carcinoid cell lines, but not in normal lung epithelium or in non-small cell lung cancers. These findings, together with earlier results from our laboratory, suggest the expression of prohormone convertases has an important role in the development and maintenance of the SCLC phenotype and that autocrine growth factor and prohormone convertase genes respond to a common transcriptional activator in SCLC.

Functional deficiencies of components of the MHC class I antigen pathway in human tumors of epithelial origin.

An association between oncogenic transformation and repression of different components of the MHC class I antigen processing machinery (APM) have been described in murine model systems. In order to discover whether a similar correlation exists, human tumor cell lines of distinct histology with altered ras protein were analyzed for the expression of APM components utilizing RT-PCR and Western blot analyses. A heterogeneous expression pattern of MHC class I antigens, TAP peptide transporter, proteasome subunits, proteasome activator PA28 and the chaperones calnexin, calreticulin as well as tapasin was displayed by these tumor cell lines. Single or combined deficiencies in the expression and/or function of TAP, LMP2, LMP10 and tapasin were demonstrated in 11 of 12 cell lines studied, whereas the expression of calnexin, calreticulin, beta2-microglobulin, LMP7 and PA28alpha was unaltered or only weakly decreased. The impaired expression of TAP, LMP subunits and tapasin was not associated with altered ras, but resulted in reduced MHC class I surface expression. In particular, a significant allele- and locus-specific downregulation of the HLA-A and HLA-B haplotypes was found. IFN-gamma treatment corrected the TAP, LMP and tapasin deficiencies and enhanced the constitutive PA28alpha, LMP7, calnexin and calreticulin expression which was accompanied with increased levels of MHC class I antigens. Thus, dysregulation rather than structural alterations of different APM components might be one mechanism of colon carcinoma, small cell lung carcinoma and pancreatic carcinoma cell lines to evade immune recognition.

O2 Sensing by Airway Chemoreceptor-derived Cells: PROTEIN KINASE C ACTIVATION REVEALS FUNCTIONAL EVIDENCE FOR INVOLVEMENT OF NADPH OXIDASE

Accumulating evidence suggests that neuroepithelial bodies are airway O(2) sensors. Recently, we have established the H-146 small cell lung carcinoma line as a suitable model to study the biochemical basis of neuroepithelial body cell chemotransduction. Here we explore the possibility that hypoxic modulation of K(+) channels is intimately linked to activity of NADPH oxidase. Graded hypoxia caused graded inhibition of whole cell K(+) currents, which correlated well with membrane depolarization. Pretreatment with the phorbol ester, 12-O-tetradecanoyl (TPA), inhibited K(+) currents at all potentials. Although 4alpha-phorbol 12,13-didecanoate and TPA in the presence of bisindolylmaleimide were also able to depress K(+) currents, only TPA could significantly ameliorate hypoxic depression of these currents. Thus, protein kinase C (PKC) activation modulates the sensitivity of these cells to changes in pO(2). Furthermore, because the addition of H(2)O(2), a downstream product of NADPH oxidase, could only activate K(+) currents during hypoxia (when endogenous H(2)O(2) production is suppressed), it appears likely that PKC modulates the affinity of NADPH oxidase for O(2) potentially via phosphorylation of the p47(phox) subunit, which is present in these cells. These data show that PKC is an important regulator of the O(2)-transduction pathway and suggests that NADPH oxidase represents a significant component of the airway O(2) sensor.