Hepatocyte nuclear factor-4alpha (HNF4alpha) exists in multiple isoforms that are generated by alternative promoter (P1 and P2) usage and splicing. Here we establish monoclonal antibodies (mAbs) for detecting P1 and P2 promoter-driven HNF4alpha, and evaluate their expression in normal adult human tissues and surgically resected carcinomas of different origins. Using immunohistochemical analysis, we demonstrate that, while P1 promoter-driven HNF4alpha is expressed in hepatocytes, small intestine, colon, kidney and epididymis, P2 promoter-driven HNF4alpha is expressed in bile duct, pancreas, stomach, small intestine, colon and epididymis. Altered expression patterns of P1 and P2 promoter-driven HNF4alpha were observed in gastric, hepatocellular and colorectal carcinomas. HNF4alpha was expressed in lung metastases from renal cell, hepatocellular and colorectal carcinoma but was not observed in lung tumours. The P1 and P2 promoter-driven HNF4alpha expression pattern of tumour metastases correlated with the primary site of origin. P1 promoter-driven HNF4alpha was also found in intestinal metaplasia of the stomach. These data provide evidence for the tissue distribution of P1 and P2 promoter-driven HNF4alpha at the protein level and suggest that HNF4alpha may be a novel diagnostic marker for metastases of unknown primary. We propose that the dysregulation of alternative promoter usage of HNF4alpha is associated with the pathogenesis of certain cancers.

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In murine ontogeny, macrophage precursor cells develop in the yolk sac and fetal liver. Primitive macrophages also appear in the yolk sac, migrate to various tissues, and differentiate into several fetal macrophage populations. Because the development of the monocytic cell lineage is incomplete in the early stage of fetal hematopoiesis, primitive/fetal macrophages are considered to originate from granulocyte-macrophage colony forming cells or earlier macrophage precursors, bypassing the early monocytic cell series. In adult mice rendered severely monocytopenic by administration of strontium-89, resident macrophages are maintained by self-renewal. In contrast, administration of liposome-encapsulated dichloromethylene diphosphonate (clodronate) results in the elimination of various tissue macrophage populations. The repopulation of affected macrophages is dependent on the increase of precursors in the liver and spleen during the period of macrophage depletion. Such precursors reconstitute heterogeneous macrophage subpopulations. In mice homozygous for the osteopetrosis (op) mutation, the absence of macrophage colony-stimulating factor (M-CSF) activity results in a deficiency of monocytes and monocyte-derived macrophages. However, immature macrophages are present in various tissues. Administration of M-CSF to op/op mice induces the increased proliferative capacity and the morphological maturation of macrophages. However, the responses of individual tissue macrophage subpopulations to M-CSF are different. These results indicate that macrophage development, differentiation, and proliferation are regulated by the tissue microenvironment including the in situ production of macrophage growth factors in both fetal and adult life.

Dichloromethylene diphosphonate (MDPCl2) encapsulated in multilamellar liposomes was selectively incorporated by macrophages, immediately transferred to lysosomes, then released from liposomes into lysosomes by enzymatic digestion of the liposomal lipid layers. From 4 h after ingesting liposome-encapsulated MDPCL2 murine macrophages in vivo and in vitro acquired the ultrastructural features of apoptosis, such as condensed nuclear chromatic, nuclear fragmentation, cell shrinkage, and blebbing of the plasma membrane. Murine peritoneal macrophages and isolated rat Kupffer cells incubated in the medium containing liposome-encapsulated MDPCl2 increased DNA fragmentation in a dose-dependent manner. Electrophoretic analysis of extracted DNA from the isolated Kupffer cells showed DNA fragmentation. Another diphosphonate, Alendronate (4-amino-1-hydroxy-butylidene-1,1-diphosphonate) had less potent macrophage cytotoxicity. However, MDPCl2, Alendronate, and gadolinium chloride in solution were not cytotoxic to macrophages. These results implied that the intralysosomal accumulation of MDPCl2 generates signals to induce macrophage apoptosis.