Conditional transformation mediated via a pertussis toxin-sensitive receptor signalling pathway

Abstract

To determine whether a cloned receptor coupled to pertussis toxin (PTx)-sensitive G-proteins can induce cell proliferation and oncogenic transformation, as observed for receptors that elicit PTx-insensitive enhancement of phosphatidyl inositol (PI)-specific phospholipase-C (PLC) activity, nontransformed murine BALB/C-3T3 cells were transfected with the rat serotonin-1A (5-HT1A) receptor. The 5-HT1A receptor is coupled to PTx-sensitive G-proteins to induce a cell-specific activation of PLC. While 1 μM 5-HT induced no change in PI turnover or cytosolic free calcium levels ([Ca2+],) in receptor-negative nontransfected 3T3 cells, 5-HT induced a 2-fold increase in inositol trisphosphate accumulation and a 2.5-fold increase in [Ca2+], in the 3T3-ZD8 clone, which expressed 0.6 ± 0.2 pmol/mg protein of specific 5-HT1A binding sites. The stimulatory actions of 5-HT on PI turnover and [Ca2+], in 3T3ZD-8 cells displayed the pharmacology of the 5-HT1A receptor and were abolished by pretreatment with PTx. Thus, BALB/C-3T3 fibroblast cells express the PLC-linked pathway of the 5-HT1A receptor. Overnight treatment with 5-HT (1 μM) enhanced incorporation of [3H]thymidine into DNA extracted from serum-starved 3T3ZD-8 cells, an action that was also blocked by pretreatment with pertussis toxin. Long term (1-2 weeks) exposure to 5-HT in the medium led to phenotypic transformation of the cells, including the formation of foci with 1 μM 5-HT. These actions of 5-HT were not observed in untransformed 3T3 cells. We conclude that the PTx-sensitive PLC-linked pathway of the 5-HT1A receptor expressed in nontransformed BALB/C-3T3 cells, in concert with other serum-derived factors, predisposes the cells to enhanced proliferation and transformation.