Intercellular Redistribution of cAMP Underlies Selective Suppression of Cancer Cell Growth by Connexin26
Figure 1
Characterization and growth behavior of HeLa cells expressing different w.t. and mutant connexins.
(A) Immunofluorescence staining, using the appropriate anti-Cx antibody, shows characteristic expression of connexins in plaques between cells. HeLaPar tested negative with all the three anti-Cx antibodies (anti-Cx26 antibody results are shown here) (scale bars, 10μm).
(B) Growth of HeLaPar (squares) with pools of HeLa clones transfected with Cx26 (circles), Cx32 (inverted triangle) or Cx43 (upright triangle), subjected to 3 days of serum starvation (days 0-3) followed by 3 days in 1% serum (days 3-6), reveals selective growth suppression by Cx26. Representative data from one of three experiments is shown, with points representing the mean ± SEM of triplicate platings.
(C) Topology diagram of Cx26, with extracellular surface at the top, depicts the location of two point mutants used in this study that disrupt different aspects of channel function (R75Y and T135A).
(D) Immunoflourescence staining with an anti-Cx26 antibody shows that both mutant connexins form plaques at the cell surface, with R75Y having slightly smaller, less frequent plaques, and T135A having larger and more frequent plaques than w.t. Cx26 (panel A) (scale bars, 10μm).
(E) Hemichannel function was assayed by LY dye uptake in a dye dropping assay (Scout et al., 2002). The left panel shows the phase contrast image (asterisks indicate cells taking up the dye) and the right panel shows the fluorescent image (scale bars, 20μm). Note that some dye-positive cells appear to have received dye through intercellular transfer from the original cell that took up dye in the HeLa26 cultures.
(F) Comparison of the growth in low serum (c.f. panel B) of mutant Cx26 expressing cells (Cx26T135A - diamonds, Cx26R75Y – triangles) with those expressing w.t. HeLa26 (circles) and HeLaPar cells (squares) demonstrates that the functional intercellular channels of w.t. Cx26 are required for growth suppression. Representative data from one of three experiments is shown, with points representing the mean ± SEM of triplicate platings.