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Conditional transformation mediated via a pertussis toxin-sensitive receptor signalling pathway
H Abdel-Baset1, V Bozovic, M Szyf
1Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada.
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 microM 5-HT induced no change in PI turnover or cytosolic free calcium levels ([Ca2+]i) 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+]i 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+]i in 3T3ZD8 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 microM) 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 microM 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.
Insights
The serotonin-1A (5-HT1A) receptor, when coupled to pertussis toxin (PTx)-sensitive G-proteins, can trigger cell proliferation and oncogenic transformation in BALB/c-3T3 cells. This occurs via a phospholipase-C (PLC) pathway, leading to enhanced DNA synthesis and phenotypic changes.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Receptor signaling pathways can influence cell growth and transformation.
- Pertussis toxin (PTx)-sensitive and insensitive G-proteins mediate distinct cellular responses.
- The serotonin-1A (5-HT1A) receptor is known to couple with PTx-sensitive G-proteins.
Purpose of the Study:
- To investigate if a cloned 5-HT1A receptor, coupled to PTx-sensitive G-proteins, can induce cell proliferation and oncogenic transformation.
- To compare the transforming potential of PTx-sensitive PLC activation with PTx-insensitive pathways.
- To elucidate the role of the 5-HT1A receptor-mediated PLC pathway in cellular transformation.
Main Methods:
- Transfection of murine BALB/c-3T3 cells with the rat 5-HT1A receptor gene.
- Measurement of phosphatidyl inositol (PI) turnover and cytosolic free calcium ([Ca2+]i) levels.
- Assessment of [3H]thymidine incorporation into DNA to evaluate proliferation.
- Long-term cell culture to observe phenotypic transformation and focus formation.
- Pharmacological characterization using 5-HT and PTx treatment.
Main Results:
- The 5-HT1A receptor successfully expressed in 3T3 cells activated PLC, increasing PI turnover and [Ca2+]i in a PTx-sensitive manner.
- 5-HT treatment significantly enhanced [3H]thymidine incorporation in transfected cells, indicating increased DNA synthesis.
- Long-term exposure to 5-HT induced phenotypic transformation, including focus formation, in the transfected 3T3 cells.
- These proliferative and transformative effects were dependent on the PTx-sensitive PLC pathway and were blocked by PTx pretreatment.
Conclusions:
- The PTx-sensitive PLC-linked pathway mediated by the 5-HT1A receptor can promote cell proliferation.
- Expression of the 5-HT1A receptor coupled to PTx-sensitive G-proteins can predispose cells to oncogenic transformation.
- These findings highlight the role of specific G-protein coupled receptor signaling pathways in cellular transformation, in conjunction with other factors.