(A) Immunohistochemical analysis of Ku70, Ku86, Clusterin, Bax and IL-6 expression in normal colonic mucosa (H), node negative (N0) and node positive (N1) colon carcinomas. In normal mucosa, Ku70, Ku86 are exclusively detected in the nuclei, whereas Bax protein is weakly expressed in the cytoplasm. Clusterin is mostly localized in the nucleus. In node negative carcinomas, (N0) Ku70 localized almost exclusively in the nuclei and a weak cytoplasm staining was also observed. Ku86 is lightly decreased in the nuclei, whereas Bax increases in the cytoplasm, as compared with normal mucosa. Clusterin is strongly increased in the cytoplasm. In node positive colorectal carcinomas Ku70 is detected in the nucleus and in the cytoplasm, whereas Ku 86 is not detectable. Bax and Clusterin are strongly upregulated in the cytoplasm and the nuclear staining for Clusterin is lost. IL-6 expression is weakly detectable in normal mucosa. Conversely, IL-6 staining increases in tumors (N0, N1) respect to the corresponding normal mucosae. The IL-6 increase is higher in the node positive carcinomas (N1). (B) (right) IL-6 expression detected in tumor associated macrophages (TAM) (IL-6), identified by CD68 immunostaining (CD68). (B) (left) Bax and Ku70 co-localization in colon carcinomas. Confocal microscopy analysis of Ku70 and Bax double staining in normal colon mucosa (H) and in node positive colon carcinomas (N1). Double fluorescence with anti-Ku70 (FITC-conjugated, green) and anti-Bax (Texas red-conjugate, red) antibodies was performed as described in Methods section. Yellow and/or light red staining were due to multiple positivity. Ku70 staining is detected in the nucleus of normal mucosa, whereas Bax faint staining is present in the cytoplasm. In the pT3N1 adenocarcinoma Ku70 co-localizes with Bax. Scale bar represents 100 μm.

Effect of IL-6 treatment on Ku86 and Clusterin expression. (A) Immunocytochemistry of Ku86 expression in untreated (Un) and 24 h IL-6 treated Caco2 cells. IL-6 induces a strong inhibition of Ku86 protein expression at 24 h (upper). Western blot analysis with anti-Ku86 antibody on protein extracts from untreated (Un) and Caco2 cells treated with IL-6 for) and 24 h. The filter was reprobed with anti-β-actin to normalize the protein levels (lower). (B) Ku86 mRNA level after IL-6 treatment was determined by RT-PCR as described in the Methods section. M, molecular size marker; lane 1, untreated cells; lane 2, 24 h IL-6 treated Caco-2 cells. The optical densities (O.D.) obtained by scanning densitometric analysis of RT-PCR bands are reported by histograms. The values were normalized for β-2 microglobulin transcripts levels and are means (+/-SD) of three independent experiments. (C) Clusterin mRNA level after IL-6 treatment determined by RT-PCR as described in the Methods section. M, molecular size marker; lane 1, untreated cells; lanes 2 and 3, Caco2 cells treated with IL-6 for 4 h and 24 h. In the lower panel, the corresponding β-2 microglobulin transcript levels are shown. The optical densities (O.D.) obtained by scanning densitometric analysis of bands, normalized for β2-microglobulin levels, are reported by histograms. The values are means (+/-SD) of three independent experiments. (D) Western blot analysis with anti-Clusterin antibody on protein extracts from untreated (Un) and 24 h IL-6 treated Caco2 cells. The optical densities (O.D.) obtained by scanning densitometric analysis and normalized for β-actin protein levels, are reported by histograms. Values are means (+/-SD) of three independent experiments.

IL-6 influences Bax-Ku70 interaction. Ku70 and Bax protein levels in the cytoplasmic fraction of untreated (Un) and IL-6 treated Caco2 cells (A and B). (A) Ku70 level after 4 and 24 h of IL-6 treatment. The cytokine does not affect Ku70 cytoplasmic level. However, the acid extracti: Ku70u70 acid ext.) shows an increase of Ku70 presence in the cytoplasm of treated cells. In the lower gel, the cytoplasmic extracts immunoprecipitated with anti-Ku70 antibody (Ku70 IP) probed with anti-acetyl-lysine antibody to evidence the increase of the Ku70 acetylated fraction, following IL-6 treatment. (B) Bax level in untreated Caco-2 cells (Un) and after 4 h and 24 h of treatment with IL-6. The cytokine does not affect Bax cytoplasmic levels. β-actin levels were detected on the same filter to normalize the protein extracts. (C) Western blot analysis of Bax- and Ku70-immunoprecipitated cytoplasmic extracts from untreated (Un) and IL-6 treated Caco-2 cells. Bax co-immunoprecipitated fraction were probed with Ku70, acetyl-lysine and phosphoserine antibodies. IL-6 induces a significant increase of Ku70 fraction bound to Bax in the cytoplasm. This fraction is lysine-acetylated and serine-phosphorylated, as shown by the signal detected at 70 kDa molecular weight, in the corresponding lower filters. The lower panel shows the same extracts immunoprecipitated with anti-Ku70 antibody (Ku70 IP) and detected with anti-phosphoserine. IL-6 increases the Ku70 serine-phosphorylation at 24 hr (1.8 times) correlated to Ku70 phosphorylated fraction bound to Bax (BaxIP). (D) shows the same extracts from untreated (Un) or 4 h and 24 h IL-6 treated cells immunoprecipitated with Bax antibody (Bax IP) and probed with anti-Ku86 and Bax antibodies. Immunoblotting with anti-Ku86 MoAb shows a significant increase of Ku86 fraction bound to Bax after 24 h of IL-6 treatment. An additional and specific band of 96 kDa is evident in the gel. The Bax immunoblotting on the same IP filter is shown as loading control. The lower panel shows the cytoplasmic extracts from untreated (Un) or 4 h and 24 h treated cells immunoprecipitated with anti-Clusterin antibody (Clu IP) and probed with anti-Bax antibody. IL-6 treatment increases the sClu binding to Bax.

(A) Western blot analysis on protein extracts from Caco-2 untreated (Un)or 1, 4 and 24 h somatostatin (SST) treated cells. The filters were probed with Ku86, Ku70, Clu and Bax antibodies. β-actin protein levels were detected as loading control. Ku86 protein is induced after 4 h of IL6 treatment, whereas Ku70 levels is not affected. Increased levels of nClu (50–55 kDa) and Bax are evident after 1 h, and 4 h of SST treatment. Bax decreases to the basal levels after 4 h of treatment. Cytoplasmic proteins were extracted from Caco-2 untreated or treated with SST for 4 h and 24 h and immunoprecipitated with anti-Ku70 (Ku70 IP) (B) and anti-Clu Abs (Clu IP) (C). In (B), no interaction with Ku86 is found in untreated cells, whereas a strong binding with Bax are detected in Ku70 co-immunoprecipitated proteins from untreated (Un) cells. Moreover an interaction with nCLU was also evident. After 4 h of SST treatment, Bax is partially released (6 folds of reduction) from Ku70. Conversely, Ku70 interacts with Ku86 and a significant increase in nCLU-Ku70 binding is evident. After 24 h, Ku70-Ku86 complex decreases. In (C) the cytoplasmic extracts were co-immunoprecipitated with anti-Clusterin antibody directed (Clu IP). A significant increase of Ku70 protein is evident after 4 h of SST treatment. nCLU and Ku70 interaction is still evident 24 h after treatment. The non-immunoprecipitated levels of Ku70 corresponding cytoplasmic extracts are shown as Input. β-actin was detected on the Input filter, as loading control. (D) Confocal microscopy analysis of Ku70, Clusterin and Bax staining in Caco-2 cells treated with somatostatin for 24 h. Panels show a 2D reconstruction triple stain for Bax (blue), Ku70 (red) and Clu (green) (2000× magn.), detected as described in Materials and Methods, in Caco-2 cells. Merging of triple stain in untreated Caco-2 cell (UnMerge) and after 24 h of SST treatment (SST Merge) is shown. Yellow, light blue and white areas are due to multiple positivity. The blue areas demonstrate the Bax release from Ku70 and CLU after SST treatment (arrow).