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Tumor-associated macrophages (TAMs) exhibit an M2 macrophage phenotype that suppresses anti-tumor immune responses and often correlates with poor outcomes in patients with cancer. Patients with ovarian cancer frequently present with peritoneal carcinomatosis, but the mechanisms that induce naïve peritoneal macrophages into TAMs are poorly understood. In this study, we found an increased abundance of TAMs in mouse i.p. xenograft models of ovarian cancer that expressed HOXA9, a homeobox gene that is associated with poor prognosis in patients with ovarian cancer. HOXA9expression in ovarian cancer cells stimulated chemotaxis of peritoneal macrophages and induced macrophages to acquire TAM-like features. These features included induction of the M2 markers, CD163 and CD206, and the immunosuppressive cytokines, IL-10 and chemokine ligand 17, and down-regulation of the immunostimulatory cytokine, IL-12. HOXA9-mediated induction of TAMs was primarily due to the combinatorial effects of HOXA9-induced, tumor-derived transforming growth factor-β2 and chemokine ligand 2 levels. High HOXA9 expression in clinical specimens of ovarian cancer was strongly associated with increased abundance of TAMs and intratumoral T-regulatory cells and decreased abundance of CD8+tumor-infiltrating lymphocytes. Levels of immunosuppressive cytokines were also elevated in ascites fluid of patients with tumors that highly expressed HOXA9. HOXA9 may, therefore, stimulate ovarian cancer progression by promoting an immunosuppressive microenvironment via paracrine effects on peritoneal macrophages.

Homeobox genes control developmental patterning and are increasingly being found to be deregulated in tumors. The DLX4homeobox gene maps to the 17q21.3-q22 region that is amplified in some epithelial ovarian cancers. Because amplification of this region correlates with poor prognosis, we investigated whether DLX4overexpression contributes to aggressive behavior of this disease. DLX4 was not detected in normal ovary and cystadenomas, whereas its expression in ovarian carcinomas was strongly associated with high tumor grade and advanced disease stage. Overexpression of DLX4in ovarian cancer cells promoted growth in low serum and colony formation. Imaging of mice bearing intraperitoneal tumors revealed that DLX4overexpression substantially increased tumor burden. Tumors that overexpressed DLX4were more vascularized than vector-control tumors. Conditioned medium of DLX4-overexpressing tumor cells was more effective than medium conditioned by vector-control cells in stimulating endothelial cell growth. These observations were associated with the ability of DLX4 to induce expression of vascular endothelial growth factor as well as intracellular and secreted isoforms of fibroblast growth factor-2. Moreover, increased levels of these fibroblast growth factor-2 isoforms induced vascular endothelial growth factor expression in tumor cells. This study reveals a novel role for a homeobox gene in ovarian tumorigenicity by its induction of a proangiogenic, growth-stimulatory molecular program.

Rat bone marrow-derived macrophages formed multinucleated giant cells spontaneously when cultured in slide glass chambers or when induced with the polyanion acetyl lignin. Nuclei in such cells tended to cluster in distinct rings. DNA fragmentation appeared to occur in multinucleated cells, as detected by 3′ end-labeling. Southern blot analyses, using probes specific for nucleolar and non-nucleolar genes, indicated that chromatin DNA was fragmented whereas nucleolar DNA was relatively intact. Autoradiography revealed preservation, in multinucleated cells, of nucleoli into which radiolabeled uridine was incorporated. Multinucleated macrophages appeared to eventually fragment. Preserved integrity of nucleoli seems to be a feature of macrophage multinucleation, a process which apparently culminates in cell death.

The mechanisms that regulate the mRNA levels of interleukin-5 (IL-5) were compared with those regulating the mRNA levels of two other coordinately expressed lymphokines in the murine T lymphoma EL4.23. Our results indicate that IL-5 mRNA levels are independently regulated from those of IL-2 and granulocyte-macrophage colony-stimulating factor IGM-CSF) mRNAs. The induction of IL-5 mRNA by phorbol 12-myristate 13-acetate (PMA) stimulation was found to be cyclosporin A-resistant, in contrast to the induction of IL-2 and GM-CSF mRNAs. Although the three lymphokine mRNAs were not detected in unstimulated cells by Northern blot analysis, the GM-CSF gene was found by nuclear runoff analysis to be constitutively transcribed. However, the IL-2 and IL-5 genes were transcriptionally inactive in the absence of PMA stimulation. The induction of IL-5 mRNA by PMA stimulation primarily involved increased transcriptional activity. In contrast, GM-CSF mRNA induction predominantly involved enhanced mRNA stability. Both transcriptional and mRNA stabilization mechanisms appeared to regulate IL-2 mRNA induction. The activation of IL-2 and IL-5 gene transcription was dependent on de novo protein synthesis. Cellular treatment with cycloheximide enhanced IL-2 gene transcription once activation was initiated, implicating the involvement of a labile repressor(s). Furthermore, IL-5 mRNA was more stable than IL-2 and GM-CSF mRNAs. These latter two species were stabilized by cycloheximide, suggesting that a labile mechanism may regulate their degradation.

Skin renewal is a typical example of the active participation of a cell in its own death process. Cells arising from mitotic activity in the stratum germinativum of the epidermis continuously migrate upwards to the stratum corneum, where dead cells are eventually desquamated. Recent studies have suggested that apoptosis is involved in the dynamic process of skin renewal. However, this still remains to be further elucidated. In this paper, we investigated the involvement of apoptosis in the skin renewal process. Changes in the morphology of cells in different epidermal layers were compared with histochemical analyses of the extent of DNA fragmentation, as determined by nick end-labelling, and of the reactivities to a monoclonal antibody directed to Ley-antigen, difucosylated type 2 chain determinant, which has a close association with apoptosis, and to a monoclonal antibody directed to the proliferating cell nuclear antigen. The results show that apoptosis proceeds concomitantly with cell movement in the epidermis. It seems likely that commitment of a cell to death by apoptosis occurs in the epidermal tissue immediately after completion of cell proliferation, and that Ley-antigen expression may be involved in the entire apoptotic process including this early event.