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Activation of G(q) protein-coupled receptors can either stimulate or inhibit cell growth. Previously, these opposite effects were explained by differences in the cell models. Here we show that activation of m3 muscarinic acetylcholine receptors ectopically expressed in NIH3T3 cells can cause stimulation and inhibition of growth in the same cell. A clonal cell line was selected from cells that formed foci agonist dependently (3T3/m3 cells). In quiescent 3T3/m3 cells, carbachol stimulated DNA synthesis. In contrast, when 3T3/m3 cells were growing, either due to the presence of serum or after transformation with oncogenic v-src, carbachol inhibited growth. This inhibition was not due to reduction of extracellular signal-regulated kinase activity because carbachol induced extracellular signal-regulated kinase phosphorylation in both quiescent and growing 3T3/m3 cells. Investigating the cell cycle mechanisms involved in growth inhibition, we found that carbachol treatment decreased cyclin D1 levels, increased p21(cip1) expression, and led to hypophosphorylation of the retinoblastoma gene product (Rb). Proteasome inhibitors blocked the carbachol-induced degradation of cyclin D1. Effects on p21(cip1) were blocked by a protein kinase C inhibitor. Thus, m3 muscarinic acetylcholine receptors couple to both growth-stimulatory and -inhibitory signaling pathways in NIH3T3 cells, and the observed effects of receptor activation depend on the context of cellular growth.

In this study we used differential display reverse transcription-polymerase chain reaction to search for differentially expressed all-trans-retinoic acid (ATRA)-responsive genes in pancreatic carcinoma cells. We identified the kinesin-related protein HsEg5, which plays an essential role in spindle assembly and spindle function during mitosis, as a novel molecule involved in ATRA-mediated growth inhibition. Using Northern and Western blot analysis we demonstrated that ATRA significantly inhibits HsEg5 expression in various pancreatic carcinoma cell lines as well as in HaCat keratinocytes. Inhibition of HsEg5 expression by ATRA occurs at the posttranscriptional level. As a consequence, tumor cells synchronized in S-phase revealed a retarded progression through G2/M phase of the cell cycle indicating that HsEg5 inhibition results in a delayed progression through mitosis. Furthermore, a significant decrease of HsEg5 protein expression achieved by antisense transfection revealed a significant growth inhibition compared with control cells. Therefore, HsEg5 represents a novel molecule involved in ATRA-mediated growth inhibition, suggesting that vitamin A derivatives can interact with the bipolar spindle apparatus during mitosis.