Retinoic acid induces nuclear accumulation of Raf1 during differentiation of HL-60 cells

Abstract

All trans-retinoic acid (RA) is a standard therapeutic agent used in differentiation induction therapy treatment of acute promyelocytic leukemia (APL). RA and its metabolites use a diverse set of signal transduction pathways during the differentiation program. In addition to the direct transcriptional targets of the nuclear RAR and RXR receptors, signals derived from membrane receptors and the Raf-MEK-ERK pathway are required. Raf1 phosphorylation and the prolonged activation of Raf1 persisting during the entire differentiation process are required for RA-dependent differentiation of HL-60 cells. Here we identify a nuclear redistribution of Raf1 during the RA-induced differentiation of HL-60 cells. In addition, the nuclear accumulation of Raf1 correlates with an increase in Raf1 phosphorylated at serine 621. The serine 621 phosphorylated Raf1 is predominantly localized in the nucleus. The RA-dependent nuclear accumulation of Raf1 suggests a novel nuclear role for Raf1 during the differentiation process.

Figure 1. Whole cell image stream analysis for total Raf1 and DRAQ5 localization: Retinoic acid induces Raf1 nuclear redistribution in HL-60 cells

To assess cytoplasmic versus nuclear distribution in RA or TPA treated and untreated cells, ImageStream analysis of FITC anti-Raf1 and DRAQ5-labeled cells was performed. Representative brightfield, FITC, DRAQ5, and FITC/DRAQ5 composite images are shown in (A). The correlation (as measured by the Similarity score) between the nuclear DNA/Raf1image pair was quantified on a per cell basis and plotted for all single cells in (B). Cells with a high degree of nuclear Raf1 are identified with the R4 gate; and R5 is the cytoplasmic Raf1. The percentage of cells that contained significant amounts of cytoplasmic and nuclear staining is plotted in (C) which is a graphical representation of the results of the similarity score statistical analysis for the three different samples (untreated, RA or TPA treated samples presented in (B). The percentage of cells with predominantly nuclear Raf1, defined as a similarity score in R4, and predominantly cytoplasmic Raf1, defined as a similarity score in R5, is shown.

Figure 1. Whole cell image stream analysis for total Raf1 and DRAQ5 localization: Retinoic acid induces Raf1 nuclear redistribution in HL-60 cells

To assess cytoplasmic versus nuclear distribution in RA or TPA treated and untreated cells, ImageStream analysis of FITC anti-Raf1 and DRAQ5-labeled cells was performed. Representative brightfield, FITC, DRAQ5, and FITC/DRAQ5 composite images are shown in (A). The correlation (as measured by the Similarity score) between the nuclear DNA/Raf1image pair was quantified on a per cell basis and plotted for all single cells in (B). Cells with a high degree of nuclear Raf1 are identified with the R4 gate; and R5 is the cytoplasmic Raf1. The percentage of cells that contained significant amounts of cytoplasmic and nuclear staining is plotted in (C) which is a graphical representation of the results of the similarity score statistical analysis for the three different samples (untreated, RA or TPA treated samples presented in (B). The percentage of cells with predominantly nuclear Raf1, defined as a similarity score in R4, and predominantly cytoplasmic Raf1, defined as a similarity score in R5, is shown.

Figure 1. Whole cell image stream analysis for total Raf1 and DRAQ5 localization: Retinoic acid induces Raf1 nuclear redistribution in HL-60 cells

To assess cytoplasmic versus nuclear distribution in RA or TPA treated and untreated cells, ImageStream analysis of FITC anti-Raf1 and DRAQ5-labeled cells was performed. Representative brightfield, FITC, DRAQ5, and FITC/DRAQ5 composite images are shown in (A). The correlation (as measured by the Similarity score) between the nuclear DNA/Raf1image pair was quantified on a per cell basis and plotted for all single cells in (B). Cells with a high degree of nuclear Raf1 are identified with the R4 gate; and R5 is the cytoplasmic Raf1. The percentage of cells that contained significant amounts of cytoplasmic and nuclear staining is plotted in (C) which is a graphical representation of the results of the similarity score statistical analysis for the three different samples (untreated, RA or TPA treated samples presented in (B). The percentage of cells with predominantly nuclear Raf1, defined as a similarity score in R4, and predominantly cytoplasmic Raf1, defined as a similarity score in R5, is shown.

Figure 2. Flow cytometric analysis of HL-60 total (nuclear and cytoplasmic) Raf1 p-259 and p-621 phosphorylation status

48h RA-treatment induces p621Raf1 phosphorylation, but not p259Raf1 phosphorylation.

Figure 3. Whole cell ImageStream analysis for pS621Raf1 localization

Untreated or RA-treated (48 hr) cells were stained with FITC- anti p621Raf1 and DRAQ5 and analyzed for p621 nuclear localization using ImageStream cytometry.– Representative brightfield, FITC, DRAQ5 and merged FITC/DRAQ5 images are shown in (A). As determined by the p621Raf1/DRAQ5 Similarity score, p621 Raf1 is localized to the nucleus in the majority of untreated or RA-treated cells (B).

Figure 3. Whole cell ImageStream analysis for pS621Raf1 localization

Untreated or RA-treated (48 hr) cells were stained with FITC- anti p621Raf1 and DRAQ5 and analyzed for p621 nuclear localization using ImageStream cytometry.– Representative brightfield, FITC, DRAQ5 and merged FITC/DRAQ5 images are shown in (A). As determined by the p621Raf1/DRAQ5 Similarity score, p621 Raf1 is localized to the nucleus in the majority of untreated or RA-treated cells (B).

Figure 4. RA induced nuclear enrichment of phosphorylated S-621 Raf

Cytometric analysis of phosphorylated 621Raf within isolated HL-60 nuclei shows a significant fluorescence intensity shift with RA treatment, an expression of RA-induced pRaf1 enrichment in the nucleus.

Figure 5

Western blot analysis shows that phosphorylated 621Raf is more abundant in nuclear fraction, and the total amount is increased by RA treatment. Western blot analysis of total cell and nuclear extracts from RA untreated or treated cells probed for p621Raf1 and reprobed for β actin and histone 3. pRaf(S621) expression normalized to actin (pRaf(S621)/β actin, middle panel) and to histone 3 (pRaf(S621)/histone 3, lower panel) is shown.

Figure 6. Nuclear localization of phosphorylated S621 Raf. Confocal images of RA treated HL-60 cells and normal mouse bone marrow co-stained with DAPI

In both HL-60 and mouse bone marrow cells, pS621Raf1 co-localizes with DNA. DAPI staining for nuclear DNA (left), FITC staining for p621Raf1 (middle) and merged confocal images (right).