Effects of bombesin on human small cell lung cancer cells: evidence for a subset of bombesin non-responsive cell lines

The effects of bombesin on three human small cell lung carcinoma cell (SCLC) lines (NCI-H69, NCI-H128, and NCI-H345) have been examined and compared to the effects of the peptide on the mouse fibroblast cell line Swiss 3T3, and the rat pituitary tumor cell line GH3W5. While all three SCLC lines expressed messenger RNA encoding pro-gastrin releasing peptide (GRP), only the NCI-H345 cells expressed detectable membrane receptors for GRP and responded to nanomolar concentrations of bombesin as shown by 125I-GRP binding, total inositol phosphate accumulation, and increased clonal growth in soft agarose. These data show that some SCLC lines are insensitive to bombesin and do not express detectable membrane receptors for GRP.     

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 beta3 chain, which is a unique component of laminin-5. Laminin-5 is a heterotrimer protein consisting of the alpha3, beta3, and gamma2 chains, and another unique component of laminin-5 is the gamma2 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 alpha6beta4 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.     

Expression of the neuron-specific protein, 14-3-2, and steroid sulfatase in neuroblastoma cell hybrids

Three series of neuroblastoma X fibroblast hybrid clones were isolated from crosses between mouse or human fibroblasts and mouse or human neuroblastoma cell lines by virus-mediated cell fusion. The expression of 14-3-2 protein (an acidic protein specific to neurons) and steroid sulfatase activity was studied in parental and hybrid cell lines. Steroid sulfatase was extinguished in hybrids when only one parent expressed the enzyme, but was expressed in one hybrid combination in which both parents expressed the enzyme. The neuron-specific 14-3-2 protein, on the other hand, continued to be expressed in all three series of neuroblastoma x fibroblast hybrids. In most cases where these pheno-types were expressed, they also exhibited temporal modulation; that is, specific activity is low during logarithmic growth and increases markedly during stationary phase. The glial-specific protein S-100 is absent from all parents and hybrids. The results are discussed in terms of mechanisms of regulation of differentiated phenotypes in mammalian cells.     

MicroRNA expression and clinical outcome of small cell lung cancer

The role of microRNAs in small-cell lung carcinoma (SCLC) is largely unknown. miR-34a is known as a p53 regulated tumor suppressor microRNA in many cancer types. However, its therapeutic implication has never been studied in SCLC, a cancer type with frequent dysfunction of p53. We investigated the expression of a panel of 7 microRNAs (miR-21, miR-29b, miR-34a/b/c, miR-155, and let-7a) in 31 SCLC tumors, 14 SCLC cell lines, and 26 NSCLC cell lines. We observed significantly lower miR-21, miR-29b, and miR-34a expression in SCLC cell lines than in NSCLC cell lines. The expression of the 7 microRNAs was unrelated to SCLC patients' clinical characteristics and was neither prognostic in term of overall survival or progression-free survival nor predictive of treatment response. Overexpression or downregulation of miR-34a did not influence SCLC cell viability. The expression of these 7 microRNAs also did not predict in vitro sensitivity to cisplatin or etoposide in SCLC cell lines. Overexpression or downregulation of miR-34a did not influence sensitivity to cisplatin or etoposide in SCLC cell lines. In contrast to downregulation of the miR-34a target genes cMET and Axl by overexpression of miR-34a in NSCLC cell lines, the intrinsic expression of cMET and Axl was low in SCLC cell lines and was not influenced by overexpression of miR-34a. Our results suggest that the expression of the 7 selected microRNAs are not prognostic in SCLC patients, and miR-34a is unrelated to the malignant behavior of SCLC cells and is unlikely to be a therapeutic target.     

Differences in expression of pro-caspases in small cell and non-small cell lung carcinoma.

Expression of several molecular determinants of apoptosis was analyzed in 10 untreated small cell (SCLC) and 6 untreated non-small cell (NSCLC) lung carcinoma cell lines. Although SCLC lines were more prone to spontaneous apoptosis compared with NSCLC lines, the former showed higher Bcl-2 expression and a higher Bcl-2/Bax ratio. In order to understand this apparent contradiction, the expression of pro-caspases as well as calpain was analyzed in these cell lines at the protein and mRNA levels. No differences in protein level of pro-caspases-2, -3, -7, and -9 and of calpain were detected between the SCLC and the NSCLC lines, but a striking difference in pro-caspase-8 expression was noted. All 6 NSCLC, but only 2 of the 10 SCLC lines, expressed pro-caspase-8 protein. Further experiments using the RNase protection assay indicated that the lack of pro-caspase-8 expression at the mRNA level was characteristic for SCLC. Using the same experimental approach, we found that SCLC cell lines in addition to pro-caspase-8 were deficient in mRNA expression of pro-caspases-1, -4, and -10, suggesting a different caspase-activating cascade in SCLC compared with NSCLC. This first systematic characterization of pro-caspase expression in lung cancer surprisingly showed that SCLC, which are more prone to undergo spontaneous apoptosis, are deficient in several pro-caspases and have a high Bcl-2/Bax ratio. Thus, the propensity of SCLC cells to undergo apoptosis cannot be explained only by the expression of factors involved in regulation or execution of apoptosis.     

Muscarinic signaling in carcinoma cells

We previously reported that activation of M(3) muscarinic acetylcholine receptors (mAChR) generates anti-proliferative signals and stimulates cadherin-mediated adhesion in the SCC-9 small cell lung carcinoma (SCLC) cell line. The current study was undertaken to determine the frequency of functional mAChR expression among different SCLC cell lines, and to test the ability of mAChR to generate anti-proliferative signals in different SCLC cell lines. The potential role of Rac1 in SCLC cell-cell adhesion was also investigated. Exposure to the mAChR agonist carbachol induces robust Ca(2+) mobilization (indicated by intracellular fluorescence of the Ca(2+)-binding dye Indo-1) in three SCLC cell lines (SCC-9, SCC-15, and NCI-H146), modest Ca(2+) mobilization in one SCLC cell line (NCI-H209), and no detectable Ca(2+) mobilization in two SCLC cell lines (SCC-18 and NCI-H82). The M(3) mAChR-selective antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide inhibits Ca(2+) mobilization in all SCLC cell lines responding to carbachol. Incubation with carbachol for four hours significantly inhibits [3H]thymidine uptake in three of the four SCLC cell lines expressing functional mAChR (SCC-9, SCC-15, and NCI-H146 cells), but does not significantly alter [3H]thymidine uptake in the other SCLC cell lines examined. These results indicate that SCLC cell lines often express functional mAChR which elicit anti-proliferative signals when activated. To investigate the role of Rac1 in SCLC adhesion, SCC-9 cells were transiently transfected with cDNA constructs coding for Rac1, constitutively active Rac1(Val-12), or dominant negative Rac1(Asn-17) tagged to green fluorescent protein (GFP). SCC-9 cells expressing GFP-tagged constitutively active Rac1(Val-12) exhibit increased cell-cell adhesion in comparison to cells expressing GFP-Rac1 or GFP-Rac1(Asn-17). Constitutively active GFP-Rac1(Val-12), but not GFP-Rac1 or GFP-Rac1(Asn-17), accumulates at cell-cell junctions in SCC-9 cells. These results indicate that activated Rac1 increases SCLC cell-cell adhesion, consistent with the possibility that Rac1 activation contributes to increased SCLC cell-cell adhesion induced by mAChR stimulation. These findings indicate that activation of mAChR may play a significant role in regulating the proliferation and adhesion of SCLC cells. The demonstration by other investigators that acetylcholine is expressed by a variety of cells in the airways supports the possibility that acetylcholine may activate mAChR expressed by SCLC cells in primary tumors.     

High affinity binding of VIP to human lung cancer cell lines

The binding of ¹²⁵I-VIP to human lung cancer cell lines was investigated. Radiolabeled VIP bound to adenocarcinoma, squamous cell carcinoma, large cell carcinoma and small cell lung cancer (SCLC) cell lines. As SCLC cell line NCI-N592 bound radiolabeled VIP well, its binding was further characterized. ¹²⁵I-VIP bound to membranes in a specific and time dependent manner. ¹²⁵I-VIP bound with high (Kd=0.8 nM) and moderate affinity (Kd=66 nM) to two classes of sites. Pharmacology studies indicated that the order of peptide potency was VIP PHI > secretin > VIP^10–28. Because VIP receptors are present on human lung cancer cells, VIP may function as a regulatory peptide in lung cancer.     

Adhesion of small cell lung cancer cells to E- and P-Selectin under physiological flow conditions: implications for metastasis formation

Haematogenous metastasis of small cell lung cancer (SCLC) is still a poorly understood process and represents the life threatening event in this malignancy. In particular, the rate-limiting step within the metastatic cascade is not yet clearly defined although, many findings indicate, that extravasation of circulating tumour cells is crucially important as most tumour cells within the circulation undergo apoptosis. If extravasation of SCLC tumour cells mimics leukocyte–endothelial interactions, SCLC cells should adhere to E- and P-selectins expressed on the luminal surface of activated endothelium. The adhesion to E- and P-selectin under physiological shear stress with regard to adhesive events, rolling behaviour and rolling velocity was determined in the human SCLC cell lines SW2, H69, H82, OH1 and OH3. OH1 SCLC cells adhered best to recombinant human (rh) E-selectin FC-chimeras and human lung endothelial cells (HPMEC), H82 SCLC cells adhered best to activated human umbilical vein endothelial cells (HUVEC) under physiological shear stress. As OH1 cells had also produced by far the highest number of spontaneous lung metastases when xenografted into pfp/rag2 mice in previous experiments our findings implicate that adhesion of SCLC cells to E-selectin is of paramount importance in SCLC metastasis formation.     

Human neuroblastoma cell lines having smooth muscle cell markers]

The neural crest gives rise to a variety of tissues, including peripheral neurons, Schwann cells, melanocytes and ectomesenchymal cells, which include the smooth muscle cells of large arteries. Cell lines derived from neuroblastoma (a neural crest tumor) have at least two distinct morphological cell types, a neuroblastic phenotype (N-type) and an epithelial-like phenotype (S-type) with characteristics of substrate-adhesiveness. We have analyzed 17 human neuroblastoma cell lines using a panel of monoclonal antibodies against cytoskeletal proteins. Three neuroblastoma cell lines (KP-N-SI, KP-N-YN and SMS-KCN) bound an alpha -smooth muscle actin antibody. In addition, one of these cell lines (KP-N-SI) bound anti-desmin monoclonal antibodies as determined by indirect immunofluorescence. A total of eight cloned cell lines were obtained from the above parent cell lines. These were composed of either N- or S-type cells and were confirmed to be the common neuroblastoma origin from each parent cell line by chromosomal analysis. Alpha-smooth muscle actin and desmin were demonstrated in the S-type cloned cells by indirect immunofluorescence, as well as by two-dimensional Western blot analysis. These results were confirmed by Northern blot analysis using a specific probe (pSH alpha SMA-3'UT) to human alpha-smooth muscle actin mRNA. This is the first report of the presence of alpha-smooth muscle actin and desmin in neuroblastoma cell lines. These data show that in addition to giving rise to cells with neural, Schwann cell and melanocyte markers, neuroblastoma can also give rise to the cells expressing smooth muscle cell markers.     

Harnessing the lysosome-dependent antitumor activity of phenothiazines in human small cell lung cancer

Phenothiazines are a family of heterocyclic compounds whose clinical utility includes treatment of psychiatric disorders as well as chemotherapy-induced emesis. Various studies have demonstrated that these compounds possess cytotoxic activities in tumor cell lines of different origin. However, there is considerable confusion regarding the molecular basis of phenothiazine-induced cell death. Lung cancer (LC) remains one of the most prevalent and deadly malignancies worldwide despite considerable efforts in the development of treatment strategies, especially new targeted therapies. In this work, we evaluated the potential utility of phenothiazines in human LC. We show that phenothiazines as single treatment decreased cell viability and induced cell death preferentially in small cell lung carcinoma (SCLC) over non small cell lung carcinoma (NSCLC) cell lines. Sensitivity to phenothiazines was not correlated with induction of apoptosis but due to phenothiazine-induced lysosomal dysfunction. Interestingly, the higher susceptibility of SCLC cells to phenothiazine-induced cell death correlated with an intrinsically lower buffer capacity in response to disruption of lysosomal homeostasis. Importantly, this effect in SCLC occurred despite mutation in p53 and was not influenced by intrinsic sensitivity/resistance toward conventional chemotherapeutic agents. Our data thus uncovered a novel context-dependent activity of phenothiazines in SCLC and suggest that phenothiazines could be considered as a treatment regimen of this disease, however, extended cell line analyses as well as in vivo studies are needed to make such conclusion.     

An antibody to de-N-acetyl sialic acid containing-polysialic acid identifies an intracellular antigen and induces apoptosis in human cancer cell lines

Polysialic acid (PSA), an α2,8-linked homopolymer of N-acetylneuraminic acid (Neu5Ac), is developmentally regulated and its expression is thought to be restricted to a few tissues in adults. Recently, we showed that two human pathogens expressed a derivative of PSA containing de-N-acetyl sialic acid residues (NeuPSA). Here we show that an epitope identified by the anti-NeuPSA monoclonal antibody, SEAM 3 (SEAM 3-reactive antigen or S3RA), is expressed in human melanomas, and also intracellularly in a human melanoma cell line (SK-MEL-28), a human T cell leukemia cell line (Jurkat), and two neuroblastoma cell lines (CHP-134 and SH-SY5Y). SEAM 3 binding induced apoptosis in the four cell lines tested. The unusual intracellular distribution of S3RA was similar to that described for the PSA polysialyltransferases, STX and PST, which are also expressed in the four cell lines used here. Interestingly, suppression of PST mRNA expression by transfection of SK-MEL-28 cells with PST-specific short interfering RNA (siRNA) resulted in decreased SEAM 3 binding. The results suggest further studies of the utility of antibodies such as SEAM 3 as therapeutic agents for certain malignancies.     

Neurotensin may function as a regulatory peptide in small cell lung cancer.

Neurotensin (NT) has been postulated to act as a modulatory agent in the central nervous system. Besides its presence in mammalian brain, NT is produced by small cell carcinoma of the lung (SCLC) and cell lines derived from these tumors. Receptors have also been characterized in some SCLC cell lines leading to the suggestion that NT could regulate the growth of SCLC in an autocrine fashion similar to bombesin/GRP. Previously, we had reported that a 10 nM dose of NT and NT(8-13), but not NT(1-8), elevated cytosolic Ca2+, indicating that SCLC NT receptors may use Ca2+ as a second messenger. Using intact SCLC cells we report that time-course incubations with NT lead to the formation of the amino-terminal fragment NT(1-8) and small amounts of the C-terminal fragment NT(9-13). These fragments are formed by metalloendopeptidase 3.4.24.15 cleaving enzyme at the Arg8-Arg9 bond of NT. Significant levels of soluble 3.4.24.15 (10-17 nmoles/mg Pr-/min) are present in SCLC cell lines. Using the in vitro clonogenic assay we tested the effect of 0.5, 5.0 and 10.0 nM doses of NT, NT(1-8) and NT(8-13) on SCLC clonal growth. NT and the C-terminal fragment NT(8-13) stimulated colony formation whereas the N-terminal fragment did not. In summary, NT may function as a regulatory peptide in SCLC through the formation of peptide fragments.     

Neurotransmitter-synthesizing enzymes in 14 human neuroblastoma cell lines

Fourteen human neuroblastoma cell lines were studied for expression and regulation of neurotransmitter-synthesizing enzymes. All cell lines contained enzyme activities of adrenergic and/or cholinergic neurons and 13 expressed activities for both. None contained enzymes for serotonergic or G AB Aergic neurons. Enzyme activity was characteristic for a given cell line. Enzyme activity in cell lines was sensitive to growth phase, culture medium, and concentration of fetal bovine serum.