Small cell lung carcinoma exhibits greater phospholipase C-beta1 expression and edelfosine resistance compared with non-small cell lung carcinoma.

Aberrant signal transduction pathways involved in the development of metastatic disease are poorly defined in both small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC). Neuropeptide-driven positive feedback loops stimulating cell proliferation are characteristic of SCLC. The activation of phospholipase C (PLC)-beta1 is an early and common response to stimulation of G protein-coupled receptors by these neuroendocrine growth factors. The importance of PLC-beta in neuropeptide signaling prompted us to compare PLC-beta isoform expression and activity in four independent SCLC cell lines and four independent NSCLC cell lines. We found that PLC-beta1 is more highly expressed in SCLC than in NSCLC, as indicated by Western blotting of cell lysates. All SCLC lines studied express PLC-beta1; only one of the NSCLC lines investigated showed detectable levels of the enzyme. NSCLC lines are significantly more sensitive to the antiproliferative effects of ET-18-OCH3 (edelfosine) compared with the SCLC lines, as indicated by [3H]thymidine uptake. The only SCLC cell line (NCI-H345) that is as sensitive as the NSCLC cell lines to ET-18-OCH3 also expresses uniquely low levels of PLC-beta1. The participation of PLC-beta1 in signaling by SCLC growth factor receptors is indicated by our finding that PLC-beta1 (but not PLC-beta3) coimnunoprecipitates with G(alpha)q/11 upon activation of neurotensin receptors; this association is inhibited by ET-18-OCH3. Ca2+ mobilization mediated by neurotensin receptors is also inhibited by ET-18-OCH3. The binding of GTPgammaS to G(alpha)q/11 upon treatment of SCLC cells with neurotensin is not inhibited by ET-18-OCH3. These findings indicate that ET-18-OCH3 does not interfere with G(alpha)q/11 activation but rather inhibits the association of G(alpha)q/11 with PLC-beta1. Our data suggest that PLC-beta is an important mediator of both SCLC and NSCLC proliferation. Differences in PLC-beta1 expression may be exploitable in the development of effective diagnostic and therapeutic tools.

Activation of transfected M1 or M3 muscarinic acetylcholine receptors induces cell-cell adhesion of Chinese hamster ovary cells expressing endogenous cadherins.

Expression of endogenous cadherins by Chinese hamster ovary (CHO) cells has not been previously reported. However, we observed that CHO cells adhere to one another upon activation of transfected muscarinic acetylcholine receptors (mAChR), suggesting that the cells express endogenous cadherins. A 160-base pair RT-PCR product with 100% homology to the cytoplasmic domain of human E-cadherin was amplified from CHO cells. A second RT-PCR product amplified from these cells has 92% homology to the cytoplasmic domain of human cadherin-9 and 86% homology to the cytoplasmic domain of human cadherin-6. Western blotting indicates that CHO cells express a 165-kDa protein recognized by E-cadherin antibodies and a 120-kDa protein recognized by an antibody to the cadherin C-terminus sequence. The ability of transfected mAChR subtypes to regulate cadherin-mediated adhesion of CHO cells was tested by measuring the permeation of horseradish peroxidase across confluent CHO cell monolayers, by microscopic examination of the cells, and by aggregation assays. Cell-cell adhesion is induced within 15 min of activating transfected M1 or M3 mAChR which functionally couple to protein kinase C (PKC). In contrast, CHO cell adhesion is not affected by activating transfected M2 mAChR which functionally couple to other effectors. Activation of PKC with phorbol esters also induces cell-cell adhesion of all CHO sublines tested. Immunofluorescence assays reveal that endogenous cadherins redistribute on the plasma membrane of CHO cells following mAChR or PKC activation. Inactivation of cadherins by removal of extracellular Ca2+ abrogates adhesion induced by mAChR or PKC activation. Our demonstration that activation of only odd-numbered mAChR subtypes induces cadherin-mediated adhesion suggests that the unique responses of cells to M1 or M3 mAChR stimulation may involve cadherin activation.

Reduced DNA synthesis and cell viability in small cell lung carcinoma by treatment with cyclic AMP phosphodiesterase inhibitors.

This study investigated the effects of the adenosine 3',5'-cyclic monophosphate (cAMP) phosphodiesterase inhibitors caffeine, theophylline, and 3-isobutyl-1-methyl-xanthine (IBMX) on the proliferation and viability of the small cell lung carcinoma (SCLC) cell lines NCI-H345, NCI-H128, and SCC-9. These effects were correlated with the ability of the drugs to induce intracellular Ca2+ mobilization. Treatment of NCI-H345 cells with caffeine resulted in rapid mobilization of Ca2+, as indicated by Fura-2 fluorescence. Incubation of NCI-H345 cells with 6.25 mM caffeine resulted in a 62% inhibition of [3H]thymidine uptake after 2 hr, indicating reduced DNA synthesis. Incubation with 25 mM caffeine resulted in almost total inhibition of [3H]thymidine uptake after 2 hr. Similar effects on [3H]thymidine uptake were seen upon treatment of NCI-H128 and SCC-9 cells with caffeine; however, these cells did not exhibit caffeine-induced Ca2+ mobilization. Inhibition of DNA synthesis (66-93%) also occurred upon incubation of all cell lines with theophylline and IBMX, which did not mobilize Ca2+. Treatment of NCI-H345, NCI-H128, and SCC-9 cells with caffeine, theophylline, or IBMX markedly reduced cell viability. Levels of cAMP increased in the cells following treatment with caffeine, theophylline, or IBMX, reflecting the ability of these drugs to inhibit cAMP phosphodiesterase. These results suggest that the decrease in DNA synthesis and the subsequent cell death induced by these drugs are due to reduced cAMP phosphodiesterase activity, rather than to changes in intracellular Ca2+. These findings indicate that drugs that alter cAMP signaling pathways are potentially valuable agents to inhibit SCLC survival.

Regulation of integrin-mediated adhesion by muscarinic acetylcholine receptors and protein kinase C in small cell lung carcinoma.

STUDY OBJECTIVES: Improved understanding of the phenotypic characteristics of small cell lung cancer (SCLC) cells may facilitate the development of new therapies for this bronchogenic malignancy with early metastases. Herein we investigate whether activation of the M3 subtype of muscarinic acetylcholine receptor (mAChR) expressed on SCLC cells affects beta1-integrin-mediated adhesion of these cells. DESIGN: Adhesion of the SCLC cell lines SCC-9 and NCI-H345 to extracellular matrix (ECM) proteins was investigated. Cell adhesion was quantified by labeling the cells with either toluidine blue dye and measuring optical density or 3H-thymidine and measuring beta-activity. Fluorescence-activated cell sorting was used to quantify the SCLC cell surface expression of beta1-integrins. SETTING: Experiments were conducted in the Molecular Pharmacology Laboratory, Guthrie Research Institute. MEASUREMENTS AND RESULTS: Activation of mAChR with the agonist carbachol (10 microM, 1.5 h) significantly increases adhesion of the SCC-9 SCLC cell line to the ECM proteins laminin and collagen types I and IV. In contrast, mAChR activation does not alter the adhesion of SCC-9 cells to vitronectin, fibronectin, poly-L-lysine, or bovine serum albumin. Carbachol also does not alter the adhesion of NCI-H345 SCLC cells that lack functional mAChR. Preincubation of SCC-9 cells with the AIIB2 blocking antibody to beta1-integrin inhibits mAChR-induced adhesion to ECM proteins. Immunofluorescence analysis indicates that mAChR activation does not alter the surface expression of beta1-integrins by SCC-9 cells. Direct stimulation of protein kinase C (PKC) by treatment with phorbol 12-myristate 13-acetate (PMA) (10 nM, 1.5 h) increases the adhesion of both the SCC-9 and NCI-H345 cell lines to ECM proteins. These results indicate that direct activation of PKC or stimulation of M3 mAChR (which results in increased PKC activity) increases the binding activity of beta1-integrins, resulting in increased adhesion of SCLC cells to ECM proteins. CONCLUSIONS: The ability of mAChR to regulate SCLC proliferation and adhesion suggests that activation of these receptors may be used to alter SCLC tumorigenesis and metastasis.

Absence of collagen stimulating factors for L929 cells in granulation tissue extracts.

We have attempted to find collagen synthesis stimulating activity in polyvinyl sponge granulation tissue extracts and inflammatory fluid. The cells studied in culture were mouse L929 cells grown in monolayers. A prior study by another group reported such factors to be present in healing wound extracts and in extracts of CHHl3 injured liver. We have been unable to find such stimulating activity for L929 cells in granuloma tissue fluid. In addition we have examined the procedures used by the prior group and have found major problems in their methodology which are discussed below.