Cytoskeleton of hairy cells and alterations by IFN-α. HCLL-7876 and Eskol cells were either left untreated or incubated in the presence of IFN-α for 18 hours. Cells were harvested, fixed, and sedimented on poly-l-lysine-coated coverslips to be analyzed for F-actin with rhodamine-phalloidin staining. For both cells lines HCLL-7876 and Eskol and primary cells (PR), most untreated cells exhibited thin and curved microspikes made up of long bundles of F-actin on the entire surface of the cell (A) whereas other cells displayed extreme polarization of their surface projections (B). C: Treatment of hairy cells with IFN-α smoothed cell surface outlines. Images are three-dimensional reconstructions of stacks of confocal images. Scale bar, 4 μm.

Effects of RhoGTPase mutants on hairy cell morphology. HCLL-7876 cells were transiently transfected with a plasmid encoding either control GFP or GFP-tagged versions of RhoGTPase mutants. Twenty-four hours after transfection, cells were harvested, fixed, and analyzed for F-actin with rhodamine-phalloidin staining and examined for morphological alterations by confocal microscopy. Transfection efficiency was found very similar for all plasmids (data not shown). A: Expression of N17Cdc42 or V14RhoA inhibited cell protrusions (each image represents one confocal section of a cell. B: A quantitative analysis of the morphological alterations induced by RhoGTPase mutants is shown. The hairy phenotype was considered suppressed only when F-actin-stained cells displayed no residual protrusions. Loss of cellular protrusions was calculated on the basis of the control response obtained on transfection of the GFP encoding vector (*statistical significance of the loss of protrusions versus control, P < 0.01, **significant difference versus N17Cdc42, P < 0.05). Inhibition of Cdc42 or Rac, or expression of active RhoA, suppressed the hairy phenotype. C: In contrast, expression of dominant-active Cdc42 exaggerated the hairy phenotype by inducing the formation of longer and stronger actin spikes (three-dimensional reconstructions of stacks of confocal images). D: The same experiment performed with plasmids encoding Cdc42 effector-loop mutants revealed that signaling through WASP, and not through Rac1, accentuated the hairy phenotype. Results are normalized based on the control response (*significant difference versus control, P < 0.01). E:Cells transfected with either dominant-negative mutant or constitutively active Cdc42 became insensitive or refractory to IFN-α action. Scale bars, 4 μm.

Immunolocalization of endogenous RhoGTPases in hairy cells and relocalization on IFN-α treatment. HCLL-7876, Eskol, or primary cells were either left untreated or treated with IFN-α for 18 hours. Cells were harvested, fixed, and stained for F-actin (rhodamine-phalloidin) and RhoGTPases (primary antibodies recognizing the RhoGTPases were revealed with FITC-labeled secondary antibody) and observed by confocal microscopy. RhoGTPases (green) co-localizing with F-actin (red) at the cell cortex or within cellular protrusions appeared in yellow (merge). IFN-α induced the relocalization of Cdc42 (A), Rac1 (B), and RhoA (C). Each image is a confocal section. Scale bar, 4 μm.

Distribution of RhoGTPases in cell fractionation experiments. HCLL-7876 and Eskol cells were either left untreated or treated with IFN-α for 18 hours. A: Proteins were fractionated into membrane and cytosolic fractions, and the CD20 protein was taken as a membrane marker. B: Equal amounts of membrane proteins were loaded on the gel and separated by electrophoresis followed by Western blotting using RhoGTPase-specific antibodies (one representative experiment). C: The amount of GTPases found in the membrane fraction was quantified in three experiments and is presented as percentage of the control response obtained in untreated cells. IFN-α induced a depletion of Cdc42 and Rac1 from the membrane fraction and the translocation of RhoA to the membrane fraction.

RhoGTPase activities analyzed by pull-down experiments. Hairy cells were either left untreated or incubated in the presence of IFN-α (18 hours). Cells were then lysed, and active GTPases were affinity precipitated with GST-RBD-rhotekin or GST-CRIB-PAK, eluted from the beads, and analyzed by Western blotting with the relevant antibodies. For each point, a fraction of the cell lysate harvested before precipitation was run to monitor GTPase content in each sample. A: IFN-α down-regulated Cdc42 and Rac1 activities and up-regulated that of RhoA in both cell lines as in primary cells. For each GTPase, quantification was made. The percentage of the GTPase under its GTP-bound form in the presence of IFN-α was divided by the one calculated in the absence of IFN-α. A graph showing these ratios is shown in B. IFN-α down-regulated Cdc42 and Rac1 activities and up-regulated that of RhoA.

Alteration of p53 status by IFN-α in hairy cells. HCLL-7876, Eskol, primary hairy, or BL41 cells were either left untreated or treated with IFN-α for various periods of time and cell lysates were prepared. Equal amounts of proteins (200 μg per lane) were loaded on gels and separated by SDS-PAGE. A: p53 phosphorylation and expression were assessed by Western blot using relevant antibodies (shown is one representative experiment, HCLL-7876, Eskol, and PR blots were exposed for 10 minutes for autoradiography, BL41 was exposed for 1 minute). B: The amount of phosphorylated p53 protein measured 18 hours after transfection was quantified in three independent experiments and is presented as fold increase of the basal response obtained in untreated cells. IFN-α consistently induced a time-dependent increase in p53 phosphorylation on S15 in hairy cells but not in the Burkitt lymphoma cell line.

p53 regulates hairy cell morphology. HCLL-7876 cells were transfected with an empty vector or with a plasmid encoding GFP, GFP-p53, or GFP-p53H175. Twenty-four hours after transfection, cells were harvested, fixed, stained for F-actin with rhodamine-phalloidin, and observed by confocal microscopy. A: Expression of GFP-p53 led to a complete disappearance of cell protrusions in all transfected cells whereas expression of p53H175-GFP did not affect the phenotype. B: Western blot analysis of these cells revealed basal phosphorylation of the fusion protein GFP-p53 on S15, which was further increased by IFN-α. HCLL-7876 cells were transfected with plasmids encoding either GFP or the dominant-negative mutant of p53, GFP-p53H175. Cells were allowed to express the plasmids for 24 hours and then treated or not with IFN-α for 18 hours. Cells were harvested, fixed, stained for F-actin with rhodamine-phalloidin, observed by confocal microscopy, and analyzed as described in . Expression of dominant-negative p53 prevented morphological changes in response to IFN-α. C: Results are normalized based on the control response (*significant difference versus control, P < 0.05). To evaluate the effects of the p53 activator etoposide on cell viability, HCLL-7876 cells were treated with increasing concentrations of etoposide to stimulate p53 activity. After 18 hours, cell viability was determined, and results are presented as percentage of viable cells. D: Etoposide killed hairy cells in a dose-dependent manner within 18 hours of treatment). Scale bar, 4 μm.