Figure 1.
FGF2-induced cell growth inhibition of Y1 and 3T3Ras cells is dependent on FGF receptor activation.
(A) Growth curves with FGF2 (10 ng/mL) and PD173074 (150 nM for Y1 and or 300 nM for 3T3Ras cells) added 5 minutes before FGF2 (104 cells/cm2 were plated). (B) Clonogenic assays of Y1 and 3T3Ras cells treated with FGF2 and/or PD173074 (added 5 minutes before FGF2) for 24 hours. Inset below shows one representative clonogenic assay. (C) Clonogenic assays of Y1 and 3T3Ras cells in the presence of FGF2, added at time 0, and PD173074, added after FGF2 at the indicated times. (D) Microphotographs for morphological analysis of Y1 cells treated with FCS (10%) or FGF2 (10 ng/mL) for 48 hours in the presence, where indicated, of PD173074 (150 nM). Scale bar = 50 µm. (E) MTS Assay (CellTiter 96® AQueous) and CytoTox-One™ Assay (membrane integrity) performed with increasing concentrations of FGF2. RFU, Relative Fluorescent Unit. Control refers to 100% cell lysis. Values are mean ± s.e.m (n = 2–5). FCS, fetal calf serum.
Figure 2.
Knockdown of FGFR1, but not FGFR2 nor FGFR3, reduces FGF2’s cytostatic effects in Y1 cells.
(A) Panel on the left shows the knockdown efficiency of FGFR2 in two independent sublines. Panels on the right are clonogenic assays in sublines with FGFR2 knockdown in the presence of FGF2 (10 ng/mL) for 24 h. Inset on the right shows one representative example of clonogenic assay in the indicated conditions. (B) Panel on the left shows the knockdown efficiency of FGFR3 in two independent sublines. Panels on the right are clonogenic assays in sublines with FGFR3 knockdown in the presence of FGF2 (10 ng/mL) for 24 h. Inset on the right shows one representative example of clonogenic assay in the indicated conditions. (C) Panel on the left shows the knockdown efficiency of FGFR1 by two independent siRNAs sequences. Panels on the right are representative microphotographs for morphological analysis of FGFR1 knockdown under FCS or FGF2 stimulation for 24 h. Scale bar = 50 µm. Values are mean ± s.e.m (n = 3). FCS, fetal calf serum.
Figure 3.
(A) FGF2 exerts its classical mitogenic activity via FGF receptor tyrosine kinase activation.
Balb 3T3, Y1, and 3T3Ras cells were synchronized in G0/G1 by serum starvation and stimulated with FCS (10%) and/or FGF2 (10 ng/mL) for 5–15 minutes in the presence of PD173074 (150 nM). Data are representative of two independent experiments. Phospho-ERK1/2 (phosphorylated at Thr202/Tyr204) was analyzed by immunoblot. Total ERK1/2 was used as a loading control. (B) DNA synthesis stimulated by serum is inhibited in the presence of FGF2. Upper panel: G0/G1-arrested Y1 cells were stimulated with FCS and/or FGF2 for 12 hours. Cells were pulse-labeled for 1 hour with [3H]-thymidine before harvesting, and the amount of incorporated radioactivity was measured. FCS was added at time 0, and FGF2 was added 0–10 hours after FCS stimulation. Lower panel: PD173074 was added at the indicated times (0–10 h) after stimulation with FCS and/or FGF2, both added at time 0. [3H]-thymidine incorporation results are presented as the mean ± s.e.m. (n = 2). SFM, serum-free media; FCS, fetal calf serum.
Figure 4.
FGF2 causes G2/M arrest in Y1 cells.
Flow cytometry histograms show DNA content in Y1 cells after 48 hours of FCS (10%) and FGF2 (10 ng/mL) treatment in G0/G1-starved cells. Cells were treated at time 0 and stained for DNA content and BrdU uptake. (A) Quantification of G0/G1, S and G2/M phases was based on DNA content. (B) Quantification of G0/G1, S and G2/M phases was based on DNA content versus BrdU labeling. Quantification of cell cycle phases was gated from the 2N to the 4N population only. Approximately 104 cells were analyzed. SFM, serum-free media; FCS, fetal calf serum.
Figure 5.
FGF2’s cytostatic mechanisms do not involve MEK, PI3K and PKC pathways.
(A) Clonogenic assays of Y1 and 3T3Ras cells treated with FGF2 and/or U0126, LY294002 or Gö6983 inhibitors (added 1 hour before FGF2) for 24 hours. Values are mean ± s.e.m. (n = 2). (B) Microphotographs for morphological analysis of Y1 cells under serum or FGF2 stimulation for 24 h, in the presence or absence of inhibitors: U0126, LY294002, Gö6983 or PD173074, added 1 h before the stimulation with serum or FGF2. The FGF receptor inhibitor (PD173074) was used as a positive control. Scale bar = 50 µm. FCS, fetal calf serum.
Figure 6.
Src activation is necessary for FGF2’s cytostatic mechanisms.
(A) Growth curves with FGF2 (10 ng/mL) and PP1 or PP2 (5 µM) added 1 hour before FGF2 (104 cells/cm2 were plated). (B) Clonogenic assays of Y1 cells treated with FGF2 and/or PP1 (10 µM) or PP2 (5 µM) (added 30 minutes before FGF2) for 24 hours. Insets on the bottom show one representative clonogenic assay in the indicated conditions. Values are mean ± s.e.m. (n = 3). (C) Microphotographs for morphological analysis of Y1 cells under FCS or FGF2 stimulation for 48 hours, in the presence, where indicated, of PP2 (10 µM). (D) Y1 cells were synchronized in G0/G1 by serum starvation and stimulated with FCS (10%) and/or FGF2 (10 ng/mL) for 30 minutes in the presence of PP1 or PP2 (10 µM). Data are representative of four independent experiments. Phospho-Src (phosphorylated at Tyr416) and total Src were analyzed by immunoblot. Hprt was used as a loading control. (E) G0/G1-arrested Y1 cells were stimulated with FCS and/or FGF2 for 12 hours. Cells were pulse-labeled for 1 hour with [3H]-thymidine before harvesting, and the amount of incorporated radioactivity was measured. FCS and FGF2 were added at time 0. PP1 or PP2 were added 1 hour before stimulation with FCS and/or FGF2. [3H]-thymidine incorporation results are presented as the mean ± s.e.m. (n = 2). (F) Flow cytometry (FC) histograms show DNA content in Y1 cells after 24 and 48 hours of FCS and FGF2 treatment in G0/G1-starved cells. Cells were treated at time 0 and stained for DNA content and BrdU. FC histograms of DNA content (upper panels) and DNA/BrdU scatterplots (lower panels) of Y1 cells with or without PP2 (- PP2 and+PP2, respectively). Cells were stained with propidium iodide to assess DNA content. Biparametric flow cytometry analysis of DNA content and BrdU incorporation was performed on the same samples as described in the text. BrdU-positive cells were quantified by gates in the BrdU/DNA scatterplots. The upper panel shows the quantification of G0/G1, S and G2/M phases based on DNA content. The lower panel shows the quantification of G0/G1, S and G2/M phases based on DNA content versus BrdU labeling. Quantification of cell cycle phases, gated from the 2N to the 4N population only. Approximately 2×104 cells were analyzed. (G) Y1 cells were synchronized in G0/G1 by serum starvation and stimulated with FCS (10%) and/or FGF2 (10 ng/mL) for 30 minutes in the presence of PP1 or PP2 (10 µM). Data are representative of two independent experiments. phospho-ERK1/2 (phosphorylated at Thr202/Tyr204) and total ERK 1/2 were analyzed by immunoblot. Hprt was used as a loading control. Scale bar = 50 µm. SFM, serum-free media; FCS, fetal calf serum.
Figure 7.
Proposed mechanism for the cytostatic effects of FGF2 in malignant cells transformed by Ras.
FGF2 normally activates FGF receptors that trigger mitogenic responses. However, in Ras-transformed cells, FGF2 activates anti-proliferative mechanisms that are dependent on the Ras oncogene, active RhoA and Src tyrosine kinases, which ultimately trigger G2/M cell cycle arrest and senescence.