FUNCTIONAL EXPRESSION OF GAP JUNCTION GENE CX43 AND THE MYOGENIC DIFFERENTIATION OF RHABDOMYOSARCOMA CELLS

Rhabdomyosarcoma (RD) cells express low levels of the gap junction pro tein connexin 43 (Cx43), and its mRNA, and display very weak gap junct ional intercellular communication (GJIC) as detected by Cx43 immunoflu orescence, slot-blot and dye-transfer methods. These cells grow rapidl y and show aberrant and incomplete myogenic differentiation. To invest igate the role of gap junctions in these cells, the expression of Cx43 with relation to cell growth and myogenic differentiation in RD singl e-cell subclones-RDL3 and RDL6 is studied. The subclone RDL3 grows slo wly and displays better myogenic differentiation. The expression of Cx 43, its mRNA and the GJIC in RDL3 is comparable to that in normal myob lasts. Another subclone RDL6 which grows rapidly, but is poorly differ entiated, expresses very low levels of Cx43 and its mRNA, and very wea k GJIC. By using the calcium phosphate precipitate transfection techni que, a full-length cDNA-encoding Cx43 and a pSV2neo have been introduc ed into the RDL6 cells. Several stably transfected clones have been ob tained. A stable Cx43-transfectant clone RDL6/C4 expresses high level of Cx43 and its mRNA, and results in dramatic increase of GJIC. These cells grow slowly but display the enhanced myogenic differentiation. A correlation between the down-regulation of Cx43 gene expression and a reduced expression of myogenic differentiation in RD cells is demonst rated. Forced expression of Cx43 not only inhibits cell growth but als o correlates with the improved myogenic differentiation of RD cells.