Figure 7.
ARG and NOS inhibitors restore CTL lytic function. (A) Immunohistochemical detection of TIA-1 in PCa samples (n = 10) cultured 4 d in the presence or in the absence of NOS (l-NMMA; 0.5 mM) plus ARG (NOHA; 0.5 mM) inhibitors. The pictures show that in untreated tumor samples cytotoxic granules are evenly distributed in the cytoplasm of CTL (black arrow). In contrast, in tumor samples cultured with inhibitors, CTL cytotoxic granules appear polarized in a confined region of the cell (green arrow: magnification, ×1,000). In each experimental condition, 500 TIA-1+ cells were analyzed. In untreated samples, we counted 23 TIA-1+ cells showing a polarized phenotype (4.6%), whereas in samples cultured in the presence of ARG and NOS inhibitors we counted 461 polarized cells (92.2%). (B) Similar results were obtained by immunofluorescence analysis of TIA-1+ cells. Bar, 10 μm. (C) Immunohistochemical detection of apoptosis by TUNEL assay in PCa samples (n = 10) cultured 4 d in the presence or in the absence of NOS (l-NMMA; 0.5 mM) plus ARG (NOHA; 0.5 mM) inhibitors. Apoptotic cells are indicated (black arrow). In untreated tissues, apoptotic cells were always less than two/sample, whereas in inhibitor-treated tissues we counted >20 apoptotic cells per sample (magnification, ×600). (D) Immunofluorescence analysis of TIA-1+ cells and apoptosis. PCa samples (n = 10) cultured 4 d in the presence of NOS (l-NMMA; 0.5 mM) plus ARG (NOHA; 0.5 mM) inhibitors stained for TIA-1 (red) and subsequently for DNA fragmentation by TUNEL assay (green). Samples were analyzed by confocal microscopy. Bar, 10 μm. Cell margins were drawn to facilitate the readers.