lucifer-yellow and Urinary-Bladder-Neoplasms

The tumor-suppressive property of the connexin gap-junction proteins was postulated from the fact that their function of cell coupling is impaired in most cancer cells. However, in conflict with this notion, certain cancer cells are able to communicate through gap junctions despite their malignancy. To explain this phenomenon, we studied by using a dominant-negative strategy the effect on tumorigenicity of loss of intrinsic gap-junction intercellular communication (GJIC) in the rat bladder carcinoma cell line BC31, which shows both expression of connexin 43 (Cx43) and intercellular communication. In cells transfected with a mutant Cx43 with seven residues deleted from the internal loop at positions 130-136 (Cx43delta), transport of the resulting connexin protein to the plasma membrane occurred normally, but the GJIC of the cells was effectively abolished at the level of permeability of established gap junctions. Dominant-negative inhibition of GJIC by Cx43delta accelerated growth of BC31 cells in nude mice. In contrast, when GJIC in BC31 cells was artificially enforced by transfection of wild-type Cx43, the cells lost the capacity to grow in vivo. Decreased phosphorylation of Cx43delta suggested close interaction of the internal loop of connexin with its commonly phosphorylated domains in the C-terminal tail and involvement of this interaction in gap-junction permeability. Therefore, we conclude that the intrinsic GJIC observed in cancer cells should be considered a tumor-suppressor factor and that its level may influence malignant growth capacity.

Topics: Animals; Carcinoma, Transitional Cell; Cell Communication; Coloring Agents; Connexin 43; Gap Junctions; Genes, Dominant; Isoquinolines; Mice; Mice, Nude; Mutagenesis, Site-Directed; Neoplasm Proteins; Neoplasm Transplantation; Protein Conformation; Rats; Recombinant Fusion Proteins; Transfection; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Connexins make up a gene family encoding proteins that form intercellular channels known as gap junctions. Decreases in connexin expression and loss of intercellular communication have been associated with the malignant phenotype in some animal and human cells. The expression of connexin 26 and 43 mRNA was evaluated in cultured normal and malignant human urothelial cells. The normal urothelial cells were shown by Northern analysis to express both connexins. Increased confluence of the cultured normal human urothelial cells was associated with upregulation of connexin 26 mRNA. Connexin 26 mRNA expression was decreased in the bladder cancer cells. Using a human connexin 26 complementary DNA probe, nuclear run-on assays demonstrated that the decreased expression in the cancer cells was due to a failure of transcription. Southern blot analysis did not reveal any alterations in the genomic DNA. Assessment of gap junction function by scrape loading of lucifer yellow demonstrated dye transfer in normal urothelial cells but not in bladder cancer cells. Downregulation of connexin 26 mRNA was associated with functional loss of intercellular communication in the human bladder cancer cells. Connexin 43 expression varied considerably in the bladder cancer cell lines and did not correlate with dye transfer of lucifer yellow. These data suggest that alterations in the regulation of connexin 26 expression are associated with and may contribute to the malignant phenotype in bladder cancer.

Topics: Cell Communication; Connexins; Fluorescent Dyes; Humans; Isoquinolines; RNA, Messenger; Transcription, Genetic; Tumor Cells, Cultured; Urinary Bladder Neoplasms