CCR5 regulates p53 transcriptional activity. (A) Time course induction of p53 (dotted line, ▴), p21^WAF1 (solid line, ▪), and Mdm2 (dashed line, •) in CCL5-stimulated MCF-7 cells. Western blots from four independent experiments were quantified by densitometry and the values were normalized using the tubulin loading control. Data points are plotted relative to mean values obtained before chemokine addition (n = 5). (B) CCL5-induced p53 targets in MCF-7 cells expressing the p53^175H mutant. One representative experiment of three is shown. (C and D) MCF-7 cells transfected with control or p53-specific siRNA oligonucleotides were stimulated with CCL5 (C) and incubated at 37°C for the times indicated or γ irradiated (30 Gy; D) and incubated for 3 h at 37°C. Cell lysates were analyzed by Western blot. One representative experiment of three is shown. (E) CCR5 was detected in live mock- or KDELΔ32-expressing MCF-7 cells by FACS^®. Surface CCR5 expression in mock- and KDELΔ32-expressing cells was estimated by multiplying average fluorescence intensity by the percentage of CCR5⁺ cells (n = 3). (F and G) Mock- and KDELΔ32-expressing cells were stimulated with CCL5 (F) or γ irradiated (30 Gy; G) and incubated at 37°C for the times indicated. Cell lysates were analyzed by Western blot. One representative experiment of three is shown.

Cell surface levels of CCR5 specifically affect the proliferation of breast tumor xenografts with functional p53. BrdU (A and B) and TUNEL (C and D) staining of mock- and KDELΔ32-expressing MCF-7 (A and C) or MDA-MB-231 (B and D) xenografts. Counterstaining was done with hematoxylin and eosin for BrdU and nuclei were DAPI stained for TUNEL. ×40. Data points at the right represent the percentage of BrdU⁺ or TUNEL⁺ nuclei determined in four random fields for each tumor analyzed (two-tailed Mann-Whitney test). *, P < 0.05.

The Δ32 polymorphism affects human breast cancer progression. Kaplan-Meier DFS curves for the 547 breast cancer patients evaluated according to p53 expression and the Δ32 polymorphism, as indicated. CCR5 wild-type (+/+) patients, solid line; Δ32/+ and Δ32/Δ32 patients, dashed line.

CCR5 regulates p53 activity through in a G_i-, JAK2-, and p38 MAPK–dependent manner. (A) Measurement of p21^WAF1 and Mdm2 induction analyzed by Western blot after CCL5 stimulation of MCF-7 cells pretreated with the indicated chemical inhibitors. Data points represent the mean ± SD of densitometric values obtained in two independent experiments for each inhibitor. Data are plotted relative to those obtained in DMSO-treated cells before CCL5 addition. (B and C) Mock- and KDELΔ32-expressing cells were stimulated with CCL5 (B) or UV irradiated (30 J/m², C), and then incubated at 37°C for the times indicated (for CCL5) or for 30 min (for UV irradiation). Cell lysates were analyzed by Western blot with anti–phospho-p38 (pp38) or anti-p38–specific antibodies. One representative experiment of three is shown. (D and E) Mock-, dnMKK3-, and dnMKK6-expressing cells and cells coexpressing dnMKK3 and dnMKK6 were stimulated with CCL5 (D) or UV irradiated (30 J/m², E), and then incubated at 37°C as above. CCL5-induced p21^WAF1 mdm2 up-regulation was visualized by Western blot (D). In the case of UV irradiation (E), cell lysates were analyzed by Western blot with anti–phospho-p38 (pp38) or anti-p38–specific antibodies. In all cases, dnMKK3 and dnMKK6 expression was detected using an anti-Flag antibody. One representative experiment of two is shown.

p21^WAF1 and phospho-p38-MAPK detection in xenografts from mock- and KDELΔ32-expressing tumor cells. (A and B) Lysates from mock- and KDELΔ32-expressing MCF-7 (A) or MDA-MB-231 (B) xenografts were analyzed sequentially with anti-p21^WAF1, anti-Mdm2, anti-p53, and anti-tubulin antibodies by Western blot. (C) Cryosections of xenografts derived from mock- and KDELΔ32-expressing MCF-7 and MDA-MB-231 cells, as indicated, were stained with anti-p21^WAF1 antibody followed by a peroxidase-labeled second antibody. Counterstaining was performed with hematoxylin and eosin. (D) Paraffin sections from mock- and KDELΔ32-expressing MCF-7 xenografts were stained with an anti–phospho-p38 antibody followed by a peroxidase-labeled second antibody, and were hematoxylin and eosin counterstained. For C and D, the background staining with the second antibody was also analyzed (not depicted). ×40.