Evidence for a functional link between Dd-STATa and Dd-PIAS, a Dictyostelium PIAS homologue.

Several mammalian protein families inhibit the activity of signal transducer and activator of transcription (STAT) proteins. The protein inhibitor of activated STAT (PIAS) was initially identified through its ability to interact with human STAT proteins. We isolated a gene (pisA) encoding a Dictyostelium orthologue of PIAS, Dd-PIAS, which possesses almost all the representative motifs and domains of mammalian PIAS proteins. A Dd-PIAS null mutant strain displays a normal terminal morphology but with accelerated development once cells are aggregated. In contrast, Dd-PIAS overexpressor strains demonstrate delayed aggregation, almost no slug phototaxis, impaired slug motility, and a prolonged slug migration period. This strain is a near phenocopy of the Dd-STATa null mutant, although it eventually forms a fruiting body, albeit inefficiently. The expression of several Dd-STATa-activated genes is upregulated in the Dd-PIAS null mutant and there is ectopic expression of pstAB makers. The concentration of a PIAS-green fluorescent protein (GFP) fusion protein, expressed under the PIAS promoter, is greatest in the pstO cells and gradually decreases with proximity to the tip of the slug and culminant: a pattern diametrically opposite to that of Dd-STATa. Our results suggest a functional interrelationship between Dd-PIAS and Dd-STATa that influences gene expression and development.

Role of an expansin-like molecule in Dictyostelium morphogenesis and regulation of its gene expression by the signal transducer and activator of transcription protein Dd-STATa.

Expansins are proteins involved in plant morphogenesis, exerting their effects on cellulose to extend cell walls. Dictyostelium is an organism that possesses expansin-like molecules, but their functions are not known. In this study, we analyzed the expL7 (expansin-like 7) gene, which has been identified as a putative target of Dd-STATa, a Dictyostelium homolog of the metazoan signal transducer and activator of transcription (STAT) proteins. Promoter fragments of the expL7 were fused to a lacZ reporter and the expression patterns determined. As expected from the behavior of the endogenous expL7 gene, the expL7/lacZ fusion gene was downregulated in Dd-STATa null slugs. In the parental strain, the expL7 promoter was activated in the anterior tip region. Mutational analysis of the promoter identified a sequence that was necessary for expression in tip cells. In addition, an activator sequence for pstAB cells was identified. These sequences act in combination with the repressor region to prevent ectopic expL7 expression in the prespore and prestalk regions of the slug and culminant. Although the expL7 null mutant showed no phenotypic change, the expL7 overexpressor showed aberrant stalk formation. These results indicate that the expansin-like molecule is important for morphogenesis in Dictyostelium.

Immunohistochemical Analysis of WT1 Antigen Expression in Various Solid Cancer Cells.

For a peptide-pulsed dendritic cell (DC) vaccine to work effectively in cancer treatment, it is significant that the target protein is expressed in cancer cells. Wilms' tumor 1 (WT1) has been identified as a molecular target for immune cell therapy of cancer. We evaluated the protein expression levels of WT1 in various solid tumors, as well as mucin 1 (MUC1) or major histocompatibility complex (MHC) class l molecules. Seven hundred and thirty-eight patients whose tissue samples were examined by immunohistochemical analysis agreed to undergo DC vaccine therapy. The positive staining of WT1 in tumor cells was observed in 25.3% of patients, with only 8.5% of them showing moderate to strong expression; moreover, WT1 tended to localize in the nucleus and cytoplasm. A positive staining of tumor cells by an anti-MHC class l monoclonal antibody was observed in 98.6% and by an anti-MUC1 monoclonal antibody in 76.8% of the patients. In relation to the application of cancer-specific immunotherapy, these findings provide useful information for determining the efficacy of MUC1- and WT1-targeted therapy.

Improvement of Impaired Immunological Status of Patients with Various Types of Advanced Cancers by Autologous Immune Cell Therapy.

We evaluated the immunological status of patients with various solid tumors by flow cytometry of immune cell populations and their frequencies in peripheral blood samples. The change in immunological status was also analyzed in patients given autologous immune cell therapy, such as αßT cell, γδT cell, NK cell or DC vaccine therapy. The frequency of regulatory T-cells (Tregs) was shown to be high in patients with cancers of the lung (squamous carcinoma cells), head and neck, esophagus and uterus, although there were no significant differences in effector cell population or Th1/2 ratio between various types of cancers except for a few. The cellular immunological status was impaired in most patients with advanced solid tumors before immune cell therapy and the impaired T-cell immune status was restored by infusion of effector cells, such as αßT cells or γδT cells, although the number of NK cells in the peripheral blood did not always increase after autologous NK cell therapy. The concurrent αßT cell therapy and DC vaccine therapy could successfully increase the number of CD8(+) T-cells in the peripheral blood of patients with various types of cancers. Two or three injections of αßT cells could potentially reduce Tregs frequency prior to DC vaccine, as well as the concurrent αßT cell and DC vaccine therapy. However, an increase in the Tregs frequency was observed in some patients who received NK cell therapy. These findings suggest that it is necessary to include or combine certain types of immune cell therapy when the Tregs frequency of cancer patients is high before or after autologous immune cell therapy.