ABSTRACT
Most patients suffering from breast carcinoma do not die due to the primary tumor but from the development of metastases. Active migration of cancer cells is a prerequisite for development of these metastases. We used time-lapse videomicroscopy and computer-assisted cell tracking of MDA-MB-468 human breast carcinoma cells, which were incorporated into a three-dimensional collagen matrix, in order to analyze the migratory activity of these cells in response to different neurotransmitters. Our results show that met-enkephalin, substance P, bombesin, dopamine, and norepinephrine have a stimulatory effect on the migration of the breast cancer cells; moreover, these cells show positive chemotaxis towards norepinephrine as was analyzed by the directionality and persistence on a single-cell basis. Gamma-aminobutyric acid (GABA) however has an inhibitory effect. Endorphin and leu-enkephalin, as well as histamin and acetylcholine, had no influence on the migratory activity of the cells. In summary, we provide evidence for a strong regulatory involvement of neurotransmitters in the regulation of breast cancer cell migration, which might provide the basis for the use of the pharmacological agonists and antagonists for the chemopreventive inhibition of metastasis development.
Subject(s)
Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Chemotaxis/drug effects , Neurotransmitter Agents/metabolism , Signal Transduction/drug effects , Antineoplastic Agents/pharmacokinetics , Bombesin/metabolism , Breast Neoplasms/pathology , Breast Neoplasms/prevention & control , Cell Migration Inhibition , Dopamine/metabolism , Enkephalin, Methionine/metabolism , Female , Flow Cytometry , Humans , Microscopy, Video , Neurotransmitter Agents/pharmacology , Norepinephrine/metabolism , Substance P/metabolism , Tumor Cells, Cultured , gamma-Aminobutyric Acid/metabolismABSTRACT
rna1p is the Schizosaccharomyces pombe ortholog of the mammalian GTPase-activating protein (GAP) of Ran. Both proteins are essential for nuclear transport. Here, we report the crystal structure of rna1p at 2.66 A resolution. It contains 11 leucine-rich repeats that adopt the nonglobular shape of a crescent, bearing no resemblance to RhoGAP or RasGAP. The invariant residues of RanGAP form a contiguous surface, strongly indicating the Ran-binding interface. Alanine mutations identify Arg-74 as a critical residue for GTP hydrolysis. In contrast to RasGAP and RhoGAP, Arg-74 could be substituted by lysine and contributed significantly to the binding of Ran. Therefore, we suggest a GAP mechanism for rna1p, which constitutes a variation of the arginine finger mechanism found for Ras GAP and RhoGAP.
