DNAs coding for seven murine connexins (Cx) (Cx26, Cx31, Cx32, Cx37, Cx40, Cx43, and Cx45) are functionally expressed in human HeLa cells that were deficient in gap junctional communication. We compare the permeabilities of gap junctions comprised of different connexins to iontophoretically injected tracer molecules. Our results show that Lucifer yellow can pass through all connexin channels analyzed. On the other hand, propidium iodide and ethidium bromide penetrate very poorly or not at all through Cx31 and Cx32 channels, respectively, but pass through channels of other connexins. 4,6 Diamidino-2-phenylindole (DAPI) dihydrochloride shows less transfer among Cx31 or Cx43 transfectants. Neurobiotin is weakly transferred among Cx31 transfectants. Total junctional conductance in Cx31 or Cx45 transfected cells is only about half as high as in other connexin transfectants analyzed and does not correlate exactly with any of the tracer permeabilities. Permeability through different connexin channels appears to be dependent on the molecular structure of each tracer, i.e. size, charge and possibly rigidity. This supports the hypothesis that different connexin channels show different permeabilities to second messenger molecules as well as metabolites and may fulfill in this way their specific role in growth control and differentiation of cell types. In addition, we have investigated the function of heterotypic gap junctions after co-cultivation of two different connexin transfectants, one of which had been prelabeled with fluorescent dextran beads. Analysis of Lucifer yellow transfer reveals that HeLa cells expressing Cx31 (beta-type connexin) do not communicate with any other connexin transfectant tested but only with themselves. Two other beta-type connexin transfectants, HeLa-Cx26 and -Cx32, do not transmit Lucifer yellow to any of the alpha-type connexins analyzed. Among alpha- type connexins, Cx40 does not communicate with Cx43. Thus, connexins differ in their ability to form functional heterotypic gap junctions among mammalian cells.

Introduction:

A hallmark of astrocytic tumors is their infiltrative nature. Although their aggressive and typically widespread dispersal in the adult brain differs fundamentally from that of other brain tumors, little is known about their cellular basis. Astrocytic tumors express the gap junction protein connexin 43 (Cx43), and we show here that Cx43 expression induced the morphological transformation of glioma cells into an epithelial phenotype. In a short-term aggregation assay, Cx43 expression was associated with a several-fold increase in the competence of glioma cells to aggregate. Antibodies directed against the extracellular domain of Cx43 restored the connexin-deficient phenotype, as manifested by a dose-dependent reduction in aggregation. Apart from their role in gap junction formation, connexins may therefore be considered a distinct class of membrane proteins with adhesive properties. Moreover, implanted Cx43-expressing glioma cells established functional gap junction channels with host astrocytes and dispersed through a substantially greater volume of brain parenchyma than mock- and mutant Cx43-transfected sister cells. Cx43 expression therefore may modulate not only the adhesion of astrocytes to one another, but the spread of glial tumor cells throughout astrocytic syncytia. These observations widen our concept of the potential interactions between tumor cells and their surroundings and suggest that both connexin proteins and their derived gap junctions are critical determinants of the invasiveness of central gliomas.

No abstract

The grofit package was developed to fit many growth curves obtained under different conditions in order to derive a conclusive dose-response curve, for instance for a compound that potentially affects growth. grofit fits data to different parametric models and in addition provides a model free spline method to circumvent systematic errors that might occur within application of parametric methods. This amendment increases the reliability of the characteristic parameters (e.g.,lag phase, maximal growth rate, stationary phase) derived from a single growth curve. By relating obtained parameters to the respective condition (e.g.,concentration of a compound) a dose response curve can be derived that enables the calculation of descriptive pharma-/toxicological values like half maximum effective concentration (EC50). Bootstrap and cross-validation techniques are used for estimating confidence intervals of all derived parameters.