Figure 5
Figure 5. MAPK-mediated synergistic proliferation is mediated in part by unique modulation of the G1/S cell-cycle checkpoint. (A) The NK-cell line DERL-7 is more than 95% viable by trypan blue staining and is entirely in G0 after 48 hours in SFM (left panel). After 24 hours in SCF, IL-2, or SCF + IL-2, increasing proportions of cells transit beyond G1/S. SCF + IL-2–stimulated cells (right panel) have the highest proportion beyond the G1/S checkpoint (P < .05). Statistically significant results are representative of 3 independent experiments. (B) Starved DERL-7 cells were stimulated with SCF, IL-2, or SCF + IL-2 for 18 hours in the presence of DMSO (carrier) or an inhibitor of MAPK. Top: Immunoblotting for p27 in the top left panel reveals that, in the presence of DMSO, loss of p27 is greater in response to SCF + IL-2 than in response to IL-2 or SCF alone. The bottom panel shows actin as a loading control. Immunoblotting for p27 in the top right panel reveals that, in the presence of the MAPK inhibitor UO126, there is no change in p27 expression. Bottom left: The bar graph summarizes band densitometry data shown as the composite mean (± SEM) from 3 independent immunoblotting experiments such as that shown on the top left in the presence of DMSO. It confirms that these differences in p27 expression are statistically significant (comparisons indicated by * in panel B, P < .05 for all pairwise comparisons). Bottom right: The bar graph summarizes band densitometry data shown as the composite mean (± SEM) from 3 independent immunoblotting experiments such as that shown on the top right in the presence of the MAPK inhibitor UO126. It confirms that these differences in p27 expression are not statistically significant (P = n/s). (C) A similar phenomenon is observed in primary human NK cells that show the greatest loss of p27 expression in the top panel is in response to SCF + IL-2 compared with SCF or IL-2 alone after 18 hours. Band densitometry in the bottom panel again shown as composite mean (± SEM) from 2 independent experiments in 2 different donors confirms statistical significance of differences (for comparisons indicated by * in panel C, P < .05 for all pairwise comparisons). (D) The y-axis represents the mean percentage proliferation (± SEM) above that measured for untreated controls as measured by the MTS assay, of NK-cell line DERL-7 in response to cytokine stimulation preincubated in a selective CDK4 inhibitor (PD0332991) compared with vehicle (DMSO) alone. Statistically significant differences were observed in proliferation in all conditions as a function of CDK4 inhibition (*P < .05, **P < .01, and ***P < .001). Results shown are combined from 3 independent experiments.

MAPK-mediated synergistic proliferation is mediated in part by unique modulation of the G1/S cell-cycle checkpoint. (A) The NK-cell line DERL-7 is more than 95% viable by trypan blue staining and is entirely in G0 after 48 hours in SFM (left panel). After 24 hours in SCF, IL-2, or SCF + IL-2, increasing proportions of cells transit beyond G1/S. SCF + IL-2–stimulated cells (right panel) have the highest proportion beyond the G1/S checkpoint (P < .05). Statistically significant results are representative of 3 independent experiments. (B) Starved DERL-7 cells were stimulated with SCF, IL-2, or SCF + IL-2 for 18 hours in the presence of DMSO (carrier) or an inhibitor of MAPK. Top: Immunoblotting for p27 in the top left panel reveals that, in the presence of DMSO, loss of p27 is greater in response to SCF + IL-2 than in response to IL-2 or SCF alone. The bottom panel shows actin as a loading control. Immunoblotting for p27 in the top right panel reveals that, in the presence of the MAPK inhibitor UO126, there is no change in p27 expression. Bottom left: The bar graph summarizes band densitometry data shown as the composite mean (± SEM) from 3 independent immunoblotting experiments such as that shown on the top left in the presence of DMSO. It confirms that these differences in p27 expression are statistically significant (comparisons indicated by * in panel B, P < .05 for all pairwise comparisons). Bottom right: The bar graph summarizes band densitometry data shown as the composite mean (± SEM) from 3 independent immunoblotting experiments such as that shown on the top right in the presence of the MAPK inhibitor UO126. It confirms that these differences in p27 expression are not statistically significant (P = n/s). (C) A similar phenomenon is observed in primary human NK cells that show the greatest loss of p27 expression in the top panel is in response to SCF + IL-2 compared with SCF or IL-2 alone after 18 hours. Band densitometry in the bottom panel again shown as composite mean (± SEM) from 2 independent experiments in 2 different donors confirms statistical significance of differences (for comparisons indicated by * in panel C, P < .05 for all pairwise comparisons). (D) The y-axis represents the mean percentage proliferation (± SEM) above that measured for untreated controls as measured by the MTS assay, of NK-cell line DERL-7 in response to cytokine stimulation preincubated in a selective CDK4 inhibitor (PD0332991) compared with vehicle (DMSO) alone. Statistically significant differences were observed in proliferation in all conditions as a function of CDK4 inhibition (*P < .05, **P < .01, and ***P < .001). Results shown are combined from 3 independent experiments.

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