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Inhibition of DNA synthesis by protein kinase C-activating phorbol esters in NIH/3T3 cells
T Yamamoto1, T Tsuda, Y Hamamori
1Department of Biochemistry, Kobe University School of Medicine, Hyogo.
Abstract:
Fibroblast growth factor (FGF) plus insulin induced DNA synthesis in and proliferation of NIH/3T3 cells. The protein kinase C-activating phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), inhibited both the DNA synthesis and cell proliferation induced by FGF plus insulin. The concentration of TPA required for 50% inhibition of the DNA synthesis was about 5 nM. Phorbol-12,13-dibutyrate, another protein kinase C-activating phorbol ester, also inhibited the DNA synthesis but 4 alpha-phorbol-12,13-didecanoate, known to be inactive for this enzyme, was ineffective. DNA synthesis started at about 12 h after the addition of FGF plus insulin. The inhibitory action of TPA on the DNA synthesis was observed when it was added within 12 h after the addition of FGF plus insulin. These results suggest that phorbol esters exhibit an antiproliferative action through protein kinase C activation in NIH/3T3 cells, and that this action of phorbol esters is due to inhibition of the progression from the late G1 to the S phase of the cell cycle.
Insights
Fibroblast growth factor (FGF) and insulin stimulate NIH/3T3 cell growth. Phorbol esters, activating protein kinase C, inhibit this proliferation by blocking cell cycle progression.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Fibroblast growth factor (FGF) and insulin are key regulators of cell growth.
- Protein kinase C (PKC) is involved in various cellular signaling pathways.
- Understanding cell cycle regulation is crucial for cancer research and drug development.
Purpose of the Study:
- To investigate the effect of phorbol esters on FGF- and insulin-induced NIH/3T3 cell proliferation.
- To determine the role of protein kinase C activation in mediating the antiproliferative effects of phorbol esters.
- To elucidate the specific phase of the cell cycle affected by phorbol esters.
Main Methods:
- NIH/3T3 cells were treated with FGF and insulin to induce DNA synthesis and proliferation.
- The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) and phorbol-12,13-dibutyrate on cell proliferation were assessed.
- The inactive phorbol ester 4 alpha-phorbol-12,13-didecanoate was used as a control.
- The timing of TPA addition relative to FGF/insulin stimulation was varied to determine its inhibitory window.
Main Results:
- FGF plus insulin significantly induced DNA synthesis and proliferation in NIH/3T3 cells.
- TPA, a PKC activator, inhibited both DNA synthesis and proliferation in a dose-dependent manner (IC50 ~5 nM).
- Another PKC activator, phorbol-12,13-dibutyrate, also inhibited DNA synthesis, while the inactive analog 4 alpha-phorbol-12,13-didecanoate had no effect.
- TPA's inhibitory effect was observed when added within 12 hours of FGF plus insulin stimulation, coinciding with the onset of DNA synthesis.
- The inhibition occurred during the progression from the late G1 to the S phase of the cell cycle.
Conclusions:
- Phorbol esters exert an antiproliferative effect on NIH/3T3 cells via protein kinase C activation.
- PKC activation by phorbol esters inhibits cell cycle progression at the G1/S transition.
- These findings highlight a specific mechanism by which PKC signaling can regulate cell proliferation.