Parmender P. Mehta

Growth inhibition of transformed cells correlates with their junctional communication with normal cells.

The growth of various chemically and virally transformed cell types in culture is inhibited when they are in contact with normal cell types. We show that this growth inhibition is contingent on the presence of junctional communication between the normal and transformed cells (heterologous communication), as probed with a 443 dalton microinjected fluorescent tracer. In cell combinations where heterologous communication is weak or absent there is no detectable growth inhibition; the inhibition appears when communication is induced by cyclic AMP-dependent phosphorylation, and only then. In cell combinations where heterologous communication is spontaneously strong, the growth inhibition is present, but it is abolished when the communication is blocked by retinol or retinoic acid. The cell-to-cell membrane channels of gap junctions are the likely conduits of the signals for this growth control.

Incorporation of the gene for a cell-cell channel protein into transformed cells leads to normalization of growth.

SummaryIncorporation of the gene for connexin 43, a cell-cell channel protein of gap junction, into the genome of communication-deficient transformed mouse 10T1/2 cells restored junctional communication and inhibited growth. Growth was slowed, saturation density reduced and focus formation suppressed, and these effects were contingent on overexpression of the exogenous gene and the consequent enhancement of communication. In coculture with normal cells the growth of the connexin overexpressors was completely arrested, as these cells established strong communication with the normal ones. Thus, in culture by themselves or in coculture, the connexin overexpressor cells grew like normal cells. These results demonstrate that the cell-cell channel is instrumental in growth control; they are the expected behavior if the channel transmits cytoplasmic growth-regulatory signals.

GAP-JUNCTIONAL COMMUNICATION IN NORMAL AND NEOPLASTIC PROSTATE EPITHELIAL CELLS AND ITS REGULATION BY CAMP

Gap‐junctional communication and expression of gap junction‐forming proteins were investigated in normal human prostate epithelial cells and in several malignant prostate cell lines. In comparison with normal cells, gap‐junctional communication in malignant cells, as assayed by the transfer of 443‐Da fluorescent tracer Lucifer yellow, was either reduced or not detected. Malignant cells expressed mRNA transcripts for connexin (Cx) 43, whereas normal cells expressed mRNA transcripts for Cx32 and Cx40. In both normal and malignant cells, gap‐junctional communication was enhanced twofold to fivefold by treatment with forskolin, an agent known to increase intracellular levels of cAMP. Immunocytochemical staining with a Cx43‐specific antibody revealed that in malignant cells this enhancement correlated with the number of gap junctions and occurred without any qualitative or quantitative alteration in Cx43 mRNA or protein. Moreover, western blot analyses showed that both control and forskolin‐treated malignant cells expressed only one form of Cx43. Our data suggest that gap‐junctional communication in both normal and malignant prostate cells may be regulated by hormones that work via a cAMP‐dependent signal transduction pathway. Thus, both normal and malignant cells offer a new experimental model system in which interactions between a hormonal form of cellular communication and intercellular communication mediated via gap junctions can be studied.

Neoplastic phenotype of gap-junctional intercellular communication-deficient WB rat liver epithelial cells and its reversal by forced expression of connexin 32.

Gap‐junctional intercellular communication (GJIC) is involved in cellular growth control and is often reduced in neoplastic cells. In this study, four GJIC‐deficient rat liver epithelial cell lines (WB‐aB1, WB‐bA2, WB‐cD6, and WB‐dA2) were examined for altered growth and tumorigenicity in comparison with their GJIC‐competent parental cell line, WB‐F344. WB‐aB1 cells were also forced to express connexin 32 (C×32) by transduction with a C×32 cDNA retroviral expression vector to help determine whether the restoration of GJIC could reverse their neoplastic phenotype. WB‐aB1 and WB‐bA2 cells had faster population doubling times (PDTs) and higher saturation densities (SDs) than did WB‐F344 cells. In contrast, the growth of WB‐cD6 and WB‐dA2 cells was not significantly different from that of WB‐F344 cells. WB‐aB1 and WB‐bA2 cells formed tumors in male F344 rats, but WB‐cD6 and WB‐dA2 cells did not. After transduction of WB‐aB1 cells with C×32, four stable clones (WB‐a/32‐3, ‐8, ‐9, and ‐10) were isolated that had GJIC levels of 5.2%, 44.5%, 69.8%, and 90.5%, respectively. The growth of poorly coupled clones 3 and 8 was similar to that of parental WB‐aB1 cells, but the growth of well‐coupled clones 9 and 10 was similar to that of WB‐F344 cells. The tumorigenicity of WB‐a/32‐9 and WB‐a/32‐10 cells was also significantly lower than that of WB‐aB1 cells. Our results suggest that reduced GJIC contributes to neoplastic transformation of WB cells, that additional changes are necessary, and that restoration of GJIC by forced C×32 protein expression can suppress the neoplastic phenotype of these cells. Mol. Carcinog. 22:120–127, 1998.

INHIBITION OF GLYCOSYLATION INDUCES FORMATION OF OPEN CONNEXIN-43 CELL-TO-CELL CHANNELS AND PHOSPHORYLATION AND TRITON X-100 INSOLUBILITY OF CONNEXIN- 43

We transfected the cDNA for the cell-to-cell channel protein connexin-43 (Cx43) into Morris hepatoma H5123 cells, which express little Cx43 and lack gap junctional communication (open cell-to-cell channels). We found that cells overexpressing Cx43 nonetheless lacked open cell-to-cell channels, but that inhibition of glycosylation by tunicamycin induced open channels in these cells. Tunicamycin also induced biochemical changes in Cx43 protein; the level increased, and a considerable fraction became phosphorylated and Triton X-100 insoluble, in contrast to untreated cells where Cx43 was non-phosphorylated and Triton X-100 soluble. Although tunicamycin caused the formation of open channels, channels were not found aggregated into gap junctional plaques, as they are when they have been induced by elevation of intracellular cAMP. The results suggest that although Cx43 itself is not glycosylated, other glycosylated proteins influence Cx43 posttranslational modification and the formation of Cx43 cell-to-cell channels.