An inert control chemical for GM6001, designated NC GM6001, did not alter ACh-induced cell proliferation (not shown). and time-dependent increase in levels of mRNA and MMP7 protein. Similarly, ACh induced robust and gene transcription. ACh-induced gene transcription was attenuated by atropine, anti-EGFR antibody, and chemical inhibitors of EGFR and ERK activation. In contrast, inhibitors of phosphatidylinositol 3-kinase and NF-B activation did not alter gene transcription. Collectively, these findings indicate that MMP7-catalyzed release of HBEGF mediates ACh-induced transactivation of EGFR and consequent proliferation of colon cancer cells. ACh-induced activation of EGFR and downstream ERK signaling also regulates transcriptional activation of gene transcription that was also mediated by activation of M3R and EGFR and by post-EGFR ERK signaling. In a systematic analysis of gene transcription, we identified additional robust ACh-induced, EGFR/ERK-dependent transcriptional activation of and mRNA levels were measured by Q-PCR. Data are presented as values from cells treated with ACh relative to cells treated with water after normalization to a control. p-Synephrine MMP7 ELISA. H508 cells were seeded at 106 cells per 2 ml of medium per well in six-well plates. After serum starvation for 24 h, 100 M ACh was added. At desired times, supernatants were collected and centrifuged at 500 rpm for 5 min. MMP7 was measured using the Quantikine System (R & D Systems) according to the manufacturer’s instructions. Briefly, cell extracts and supernatants were diluted twofold in Calibrator Diluent RD6-28, and 50 l were added directly to coated ELISA plates in duplicate. After 2 h of incubation at room temperature, wells were washed three times, MMP7 antibody conjugates were added for 2 h, wells were washed again three times, MMP substrate was added, and color was developed. Optical density was measured at 450 nm, with wavelength correction set p-Synephrine at 540 nm. Statistical analysis. Values are means SE of at least three independent experiments. Statistical calculations were performed using Student’s unpaired 0.05 p-Synephrine was considered significant. RESULTS ACh induces dose-dependent proliferation of human colon cancer cells. Previously, we showed that ACh induces proliferation of H508 human cancer cells that coexpress M3R and EGFR (10). To select appropriate ACh concentrations for the experiments that follow, we examined the dose-response curve for ACh-induced H508 cell proliferation. As shown in Fig. 1and 0.05 vs. ACh-stimulated cells (Student’s 0.05). An inert control chemical for GM6001, designated NC ATM GM6001, did not alter ACh-induced cell proliferation (not shown). These data were consistent with the conclusion that MMP-catalyzed HBEGF release and binding to EGFR mediated ACh-induced transactivation of EGFR and downstream stimulation of H508 cell proliferation. To confirm the role of MMP7 in ACh-induced cell proliferation, we examined the actions of recombinant MMP7 (rMMP7) and neutralizing anti-MMP7 antibody (Fig. 1 0.05). As anticipated, control experiments showed that anti-MMP7 antibody did not alter proliferation induced by HBEGF (Fig. 1 0.05; Fig. 2 0.05 vs. ACh-stimulated cells. ** 0.05 vs. cells stimulated by H508 supernatant alone. *** 0.05 vs. cells stimulated by HBEGF alone in untreated H508 media. H508 cells express multiple EGFR ligands. To determine whether H508 cells express EGFR ligands other than HBEGF, we performed Q-PCR using primers shown in Table 1. Of seven known EGFR ligands, we detected abundant mRNA for transforming growth factor-, HBEGF, and AR (Fig. 3 0.05 vs. AR-stimulated cell proliferation. ACh enhances anchorage-independent growth of colon cancer cells. Anchorage-independent growth is a hallmark of malignant cell transformation (30). It requires fewer extracellular growth factors and is independent of cell-cell interaction (30). Hence, measurement of anchorage-independent growth is considered an accurate and stringent test for uncontrolled cell proliferation (18, 20, 39). To determine whether ACh promotes anchorage-independent growth of H508 colon cancer cells, we used a soft agar assay (see materials and methods). After 7 days of incubation, ACh (300 M) stimulated a 3.5-fold increase in the number of H508 cell colonies formed in agarose (Fig. 4, and 0.05 vs. ACh-stimulated colonies. Effect of ACh on MMP7 gene transcription and MMP7 protein expression. Having shown that M3R-induced transactivation of EGFR and cell.