Elsevier

Life Sciences

Volume 72, Issues 18–19, 28 March 2003, Pages 2173-2182
Life Sciences

Muscarinic signaling in carcinoma cells

https://doi.org/10.1016/S0024-3205(03)00080-8Get rights and content

Abstract

We previously reported that activation of M3 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 Ca2+ mobilization (indicated by intracellular fluorescence of the Ca2+-binding dye Indo-1) in three SCLC cell lines (SCC-9, SCC-15, and NCI-H146), modest Ca2+ mobilization in one SCLC cell line (NCI-H209), and no detectable Ca2+ mobilization in two SCLC cell lines (SCC-18 and NCI-H82). The M3 mAChR-selective antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide inhibits Ca2+ 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 Rac1Val-12, or dominant negative Rac1Asn-17 tagged to green fluorescent protein (GFP). SCC-9 cells expressing GFP-tagged constitutively active Rac1Val-12 exhibit increased cell-cell adhesion in comparison to cells expressing GFP-Rac1 or GFP-Rac1Asn-17. Constitutively active GFP-Rac1Val-12, but not GFP-Rac1 or GFP-Rac1Asn-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.

Introduction

Previous studies indicate that muscarinic acetylcholine receptors (mAChR) are expressed by carcinoma cells originating in the lung [1], [2], [3], [4], [5], prostate [6], [7], [8], colon [9], [10], [11], [12], ovary [13], pancreas [14], and digestive tract [15], [16]. These carcinomas predominantly express the M3 mAChR subtype [5], [7], [8], [9], [11], [12], [16] and less commonly express the M1 mAChR subtype [7]. In primary tumors, mAChR expressed by carcinoma cells may be activated by acetylcholine derived from a variety of non-neuronal cell types, including epithelial and immune cells (reviewed in Refs. [17], [18]). The possibility also exists that some carcinoma cells express acetylcholine, which may act as an autocrine agonist for the mAChR expressed by the carcinomas. This possibility is consistent with the expression of acetylcholine and mAChR by epithelial cells (reviewed in [Ref. 17]), from which carcinomas originate.

We previously reported that activation of M3 mAChR in small cell lung carcinoma (SCLC) cells generates inositol polyphosphates and mobilizes intracellular Ca2+ [1], [5]. We found that M3 mAChR activation induces several responses in the SCC-9 SCLC cell line, including decreased cell proliferation [19], increased E-cadherin-mediated cell-cell adhesion [1], and increased β1 integrin-mediated cell-substrate adhesion [20]. The ability of the M3 mAChR to regulate SCLC cell proliferation and adhesion is intriguing, because these responses may affect the metastatic potential of SCLC cells. Changes in carcinoma cell proliferation following mAChR activation may alter both the progression of primary tumors and the development of newly metastasized secondary tumors. Changes in carcinoma cell-cell or cell-substrate adhesion following mAChR activation may affect metastasis by altering the ability of tumor cells to dissociate from the primary tumor and to migrate through connective tissues. Thus, elucidating the mechanisms of M3 mAChR-mediated changes in carcinoma cell proliferation and adhesion may help identify novel pathways regulating tumor progression and metastasis.

The effects of M3 mAChR activation on the proliferation of SCLC cells other than SCC-9 cells have not been extensively characterized. This lack of information makes it difficult to determine whether the decreased proliferation of SCC-9 cells in response to M3 mAChR activation is unique to this cell line, or a general characteristic of most SCLC cells. The current study addresses this issue by examining the effects of M3 mAChR activation on cell proliferation in six independent SCLC cell lines.

In addition to examining SCLC cell proliferation, we also examined the potential involvement of the small GTPase Rac1 in SCLC cell-cell adhesion. These studies were conducted because the M3 mAChR-mediated activation of Rac1 stimulates cadherin-mediated adhesion in other cell types [21]. Activation of transfected M3 mAChR in Chinese hamster ovary cells (CHO-m3 cells) stimulates cadherin-mediated adhesion of the cells [21], [22], similar to the effects of activating endogenous M3 mAChR in SCLC cells [1]. This adhesive response of CHO-m3 cells requires the M3 mAChR-mediated activation of Rac1 [21]. Although Rac1 is implicated in regulating the cadherin-mediated adhesion of several cell types (reviewed in [Ref. 21]), the involvement of Rac1 in the cadherin-mediated adhesion of SCLC cells has not been previously characterized. In the current study, we investigated the participation of Rac1 in SCLC adhesion by examining the effects of transfected wild-type and mutant Rac1 on SCLC cell-cell adhesion.

Section snippets

Cell lines

The SCC-9, SCC-15, and SCC-18 SCLC cell lines were established as previously described [3] and generously provided by Dr. Vanda Lennon (Mayo Clinic, Rochester, MN). The NCI-H82, NCI-H146, and NCI-H209 SCLC cell lines were obtained from the American Type Culture Collection (Rockville, MD). All cell lines were cultured in complete SCLC medium as previously described [1], [5].

Intracellular Ca2+ mobilization

Intracellular Ca2+ concentrations in the SCLC cells were measured using the fluorescent Ca2+-binding dye Indo-1, as

Results

Exposure of six independent SCLC cell lines to the mAChR agonist carbachol (1 mM) results in intracellular Ca2+ mobilization that is robust in the SCC-9, SCC-15, and NCI-H146 cell lines, moderate in the NCI-H209 cell line, and undetectable in the SCC-18 and NCI-H82 cell lines (Fig. 1). Exposure of the cells to 1 μM 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), which is a selective antagonist for M3 mAChR, abolishes carbachol-induced Ca2+ mobilization in the SCC-9, SCC-15, NCI-H146,

Discussion

These studies provide evidence that a significant proportion of SCLC cell lines express functional M3 mAChR. Exposure of the cells to carbachol significantly diminishes [3H]thymidine uptake in the majority of the SCLC cell lines that express functional mAChR, indicating that diminished DNA synthesis is a frequent response to mAChR activation in SCLC cells. The effects of transfected wildtype and mutant Rac1 proteins on SCLC cell-cell adhesion indicate that Rac1 activation enhances SCLC

Conclusion

This study indicates that functional M3 mAChR are expressed by a significant proportion of SCLC cell lines. The frequent expression of M3 mAChR among SCLC cell lines is similar to the widespread expression of M3 mAChR among other types of carcinomas. Activation of functional mAChR generates anti-proliferative signals in the majority of SCLC cell lines. This response of SCLC cells to mAChR activation differs from the responses of other types of carcinomas, in which mAChR activation generally

Acknowledgements

I thank Cathy Cole Lanning for the generation of the cDNA constructs coding for the GFP-tagged Rac1 proteins and for excellent technical assistance. These studies were supported by Grant R29 CA52471 from the NCI and Grant R01 HL63921 from the NHLBI, National Institutes of Health.

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