5-Lipoxygenase catalyzes the synthesis of leukotrienes from arachidonic acid. This enzyme can reside either in the cytoplasm or the nucleus; its subcellular distribution is influenced by extracellular factors, and its nuclear import correlates with changes in leukotriene synthetic capacity. To identify sequences responsible for the nuclear import of 5-lipoxygenase, we transfected NIH 3T3 cells and RAW 264.7 macrophages with expression vectors encoding various 5-lipoxygenase constructs fused to green fluorescent protein. Overexpression of wild type 5-lipoxygenase with or without fusion to green fluorescent protein resulted in a predominantly intranuclear pattern of fluorescence, similar to the distribution of native 5-lipoxygenase in primary alveolar macrophages. Within the 5-lipoxygenase protein is a sequence (Arg 638 -Lys 655 ) that closely resembles a bipartite nuclear localization signal. Studies using deletion mutants indicated that this region was necessary for nuclear import of 5-lipoxygenase. Analysis of mutants containing specific amino acid substitutions within this sequence confirmed that it was this sequence that was necessary for nuclear import of 5-lipoxygenase and that a specific arginine residue was critical for this function. As nuclear import of 5-lipoxygenase may regulate leukotriene production, natural or induced mutations in this bipartite nuclear localization sequence may also be important in affecting leukotriene synthesis.Leukotrienes (LTs) 1 are lipid mediators with important roles in immune responsiveness and antimicrobial host defense. However, overproduction of LTs can contribute to a variety of pathophysiological inflammatory processes, including asthma and allergic responses (1). Understanding the regulation of LT synthesis is critical to understanding both normal immune responses and the underlying pathophysiology of a variety of diseases.The synthesis of LTs from arachidonic acid (AA) is initiated by the enzyme 5-lipoxygenase (5-LO). Activation of 5-LO, which is characterized by its translocation to the nuclear envelope (2-5), positions 5-LO close to its substrate and 5-LO activating protein (6 -8) and is essential for LT synthesis. Changes in substrate availability or in the level of expression of proteins essential for AA metabolism (5-LO and/or 5-LO activating protein) have been correlated with the modulation of LT synthetic capacity (9 -14). However, changes in these variables do not account for all observed changes in LT synthetic capacity (15-17).Evidence is accumulating that, even in resting or unstimulated cells, the subcellular distribution of 5-LO is dynamic and may influence LT synthetic capacity. In resting cells, the subcellular distribution of 5-LO is cell type-dependent; 5-LO is predominantly cytoplasmic in peripheral blood neutrophils (18 -20), peripheral blood monocytes (4), and peritoneal macrophages (3), whereas it is found in both the nucleus and cytoplasm of alveolar macrophages (4, 5), mast cells (21), and the mast cell-like rat basophilic leukemia cell line (...

Tumor cell migration and proliferation in new organ environments are critical steps in cancer progression and can be modulated by tumor-and host-secreted molecules. Autocrine motility factor (AMF) is a tumor-secreted cytokine which regulates growth and motility by a receptor-mediated pathway. The AMF receptor, a 78-kDa cell surface glycoprotein (gp78). is regulated by cell contact in normal fibroblastic and bladder cells; however, this mechanism is disrupted during tumor progression. A prostatic carcinoma cell line which is low-to non-metastatic in nude mice (PC-3) and a derived metastatic variant (PC3M) were examined to determine if gp78 cell density regulation is involved in prostate cancer progression. Both cell lines expressed gp78 and, although the basal migration of the parental PC-3 cells was higher than that of the metastatic variant, only the PC-3M cells were capable of responding to tumor-derived AMF with increased motility. Furthermore, these cells exhibited differential patterns of wound closure in an experimental system whereby the lowmetastatic PC-3 cells migrated primarily along the wound edge while individual high-metastatic PC3M cells entered the cellfree wound area directly. Cell surface gp78 distribution distinguished the cell populations with a markedly concentrated display of gp78 in polarized capped regions on the surface of the metastatic cells. Cell-cell contact down-regulated gp78 expression in the parental, but not the metastatic, cells, and mitogenic responses to exogenous AMF differed between these cell lines as well. In this model, metastasis appears to be associated with aberrant regulation of gp78 expression and distribution. coupled with enhanced exploitation of AMF's locomotory and proliferative effects.o 199.5 Wiley-Liss, Inc.Although morphological markers of colorectal tumor malignancy, recurrence and progression have been described in some detail, biomarkers which are fully informative of the events leading to the acquisition of an invasive phenotype are required for concrete prognostic and treatment relevance (Risio et al., 1993). Abnormal cell-cell and cell-extracellular matrix interactions which mediate aspects of invasion and metastasis are the result of multiple genetic aberrations (Fearon and Vogelstein, 1990); for example, the DCC gene located on chromosome 18q encodes a protein with homology to the cell-cell adhesion molecule N-CAM, whose expression is reduced or absent in the majority of colorectal carcinomas (Fearon et al., 1990). This suggests that loss of intercellular adhesiveness and the resulting enhanced potential for migration might be paramount during the invasion of these tumors. Studies centering on the identification of factors which regulate and direct cellular movement have elucidated a group of motility factors whose primary function is thought to be regulation of cellular kinesis (Stoker and Gherardi, 1991).Tumor cell autocrine motility factor (AMF) was originally distinguished by its ability to induce the directed (chemotactic) and random (chemokinetic...

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