The aberrant production of nitric oxide (NO) contributes to the pathogenesis of diseases as diverse as cancer and arthritis. Sustained NO production via the inducible enzyme, nitric-oxide synthase 2 (NOS2), requires extracellular arginine uptake. Three closely related cationic amino acid transporter genes (Cat1-3) encode the transporters that mediate most arginine uptake in mammalian cells. Because CAT2 is induced coordinately with NOS2 in numerous cell types, we investigated a possible role for CAT2-mediated arginine transport in regulating NO production. The complexity of arginine transport systems and their biochemically similar transport properties called for a genetic approach to determine the role of CAT2. CAT2-deficient mice were generated and found to be healthy and fertile in contrast to Cat1 animals. Analysis of cytokine-activated macrophages from Cat2؊/؊ mice revealed a 92% reduction in NO production and a 95% reduction in L-Arg uptake. The reduction in NO production was not due to differences in NOS2 protein expression, NOS2 activity, or intracellular L-arginine content. In conclusion, our results show that sustained abundant NO synthesis by macrophages requires arginine transport via the CAT2 transporter.The production and release of nitric oxide (NO) 1 are involved in numerous cellular processes. Overproduction of NO by inflammatory cells is implicated in the pathogenesis of diseases as diverse as cancer, endotoxic shock, atherosclerosis, and arthritis (1-7). NO is synthesized from arginine by three related enzymes. Two of these enzymes, the Ca 2ϩ -dependent neuronal nitric-oxide synthase and endothelial NOS, generate small amounts of NO over short periods of time (7). The Ca 2ϩ -independent inducible NOS (NOS2) produces large amounts of NO over sustained periods of time (1, 2). Activated macrophages produce copious quantities of NO via NOS2 over extended periods of time that contribute to tissue injury (2). Extracellular L-arginine is not required for endothelial NO synthasemediated NO production in human endothelial cells (8). In contrast, extracellular arginine is required for sustained NO production via NOS2 in macrophages (8,9). In addition, increased arginine transport is known to accompany NO production via NOS2 (1, 2, 9, 10).Among the several transport systems that mediate L-arginine uptake (y ϩ , B 0ϩ

Monocytes and macrophages extensively colonize solid tumors, where they are thought to promote tumor angiogenesis. Here, we show that integrin A 4 B 1 (VLA4) promotes the invasion of tumors by myeloid cells and subsequent neovascularization. Antagonists of integrin A 4 B 1 , but not of other integrins, blocked the adhesion of monocytes to endothelium in vitro and in vivo as well as their extravasation into tumor tissue. These antagonists prevented monocyte stimulation of angiogenesis in vivo, macrophage colonization of tumors, and tumor angiogenesis. These studies indicate the usefulness of antagonists of integrin A 4 B 1 in suppressing macrophage colonization of tumors and subsequent tumor angiogenesis. These studies further indicate that suppression of myeloid cell homing to tumors could be a useful supplementary approach to suppress tumor angiogenesis and growth.

Previous work in our laboratory led to the cloning, from the same parent tumor cell line (MDA-MB-435), of two human breast cancer cell lines (M-4A4 and NM-2C5) with opposite metastatic phenotypes. Additional investigations revealed that the nonmetastatic cell line NM-2C5 overexpressed the neutrophil collagenase, matrix metalloproteinase (MMP)-8, relative to its partner. Because other studies have implicated the MMP family in promoting tumor metastasis, we investigated the apparently paradoxical expression of MMP-8 in these cell lines. By genetic engineering, we inverted its relative levels of expression in the two partners and studied the effects on the behavior of the tumors that they generated in athymic mice. Knock-down of expression in NM-2C5 cells by transduction with a sequence encoding a specific ribozyme and overexpression of MMP-8 in M-4A4 cells by retroviral transduction both strikingly changed metastatic performance in opposite directions, indicating that this gene plays a role in the regulation of tumor metastasis.

Interaction between germ cells and the supporting somatic cells guides many of the differentiative processes of gametogenesis. The expression pattern of the Pem homeobox gene suggests that it may mediate specific inductive events in murine reproductive tissues. During gestation, Pem is expressed in migrating and early postmigratory primordial germ cells, as well as in all embryo-derived extraembryonic membranes. Pem expression ceases in the germline after Embryonic Day 14 in both sexes and then reappears postnatally in the supporting cells of the gonad. In mature mice, Pem is produced by testicular Sertoli cells during stages VI-VIII of spermatogenesis and transiently by ovarian granulosa cells lining periovulatory follicles. Despite this tightly regulated reproductive expression pattern, mice with a targeted mutation in Pem have normal fecundity, with no detectable alteration in extraembryonic testicular or ovarian development or function. We also show that Pem is expressed throughout embryonic and adult development in a subset of a tissue-specific class of macrophages, Kupffer cells, as well as in a localized fraction of cells in macrophage cell lines. Although the number of Pem-positive Kupffer cells increases in mice treated with lipopolysaccharide, loss of Pem does not detectably interfere with the cells' ability to induce iNOS expression, demonstrating this Kupffer cell function does not require Pem. No differences were observed between Pem-knockout mice in 129, C57BL6/J, or mixed genetic backgrounds. Together, these data show that Pem is dispensable for embryonic and postnatal development, gonadal function, and Kupffer cell activation, perhaps due to compensatory expression of a similar homeobox gene.

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Nitric oxide (NO) and its metabolites are implicated in carcinogenesis and metastasis. Both stimulatory and inhibitory effects of NO have been reported in relation to breast cancer and its role in the development of malignancies and metastasis remains uncertain. We have used the polyomavirus middle T antigen (PyV-mT) targeted to the mouse mammary gland and bred into an inducible NO synthase (iNOS)-deficient C57Bl/6 strain to examine a role for nitric oxide in modulating tumors that develop in the complex environment of the whole animal. The development of hyperplasias was delayed to the extent that the earliest palpable tumors arose 2-4 weeks later in PyV-mT/iNOS ؊/؊ mice compared with PyV-mT/iNOS ؉/؉ mice, identifying a role for iNOS in early events in mammary tumor formation. Tumors that did develop in PyV-mT/iNOS ؊/؊ mice were characteristically well differentiated and had a cribriform pattern. Key words: iNOS; breast; cancer; polyomavirus middle T antigen; mouseA multitude of biologic effects are attributed to nitric oxide (NO) released by NO synthases (NOS) as a byproduct of the reaction converting the amino acid L-arginine to L-citrulline. 1,2 Of the 3 NOS isoforms found, endothelial NOS (eNOS) and neuronal NOS (nNOS) are constitutively produced, while inducible NOS (iNOS) is modulated in response to inflammatory cytokines and bacterial endotoxins. Chronic inflammation and release of NO by macrophages and epithelial cells resulting in DNA damage and nitrosylation of proteins have been implicated in carcinogenesis, particularly in the gastrointestinal tract. 3,4 The role of iNOS in promoting breast cancer is less certain due to reports of stimulatory and inhibitory effects in human tumors and in different model systems; however, an increasing body of evidence supports a role for NO in promoting tumorigenesis. 5 Breast cancer cell lines, human breast cancer cells, and mouse mammary tumor cell lines produce NO in amounts that correlate with tumor grade and metastasis. 6 -9 Mechanisms by which NO may enhance mammary tumor development and metastases include increasing DNA damage, 10 angiogenesis 11 and migration of tumor cells. 12 In contrast, other studies have demonstrated an inverse correlation between iNOS expression and human breast cancer progression and metastasis. [13][14][15] These effects may be explained by a reduction in cell proliferation, 16 increase in apoptosis 17 or enhanced tumoricidal activity 18 in the presence of NO.Mouse models of breast cancer have provided valuable information regarding the molecular basis of carcinogenesis. The bestcharacterized transgenic models express an oncogene under the control of the mouse mammary tumor virus long terminal repeat (MMTV-LTR) or whey acid protein (WAP) promoters. 19 Interestingly, the initiating oncogene defines a specific pattern of signal transduction events 20 that results in recognizable tumor phenotypes. 21,22 The MMTV-LTR-driven polyomavirus middle T antigen (PyV-mT) mice used in this study can be grouped genetically with mice carrying the ...

The mKAT-2 gene encodes a Na+-independent cationic amino acid (AA) transporter that is inducibly expressed in a tissue-specific manner in various physiological conditions. When mCAT-2 protein is expressed in Xenopus oocytes, the elicited AA transport properties are similar to the biochemically defined transport system y+. The mCAT-2 protein sequence is closely related to another cationic AA transporter (mCAT-1); these related proteins elicit virtually identical cationic AA (3). Interest in cationic AA transport regulation was stimulated by the discovery that arginine is the exclusive precursor of NO, and "de novo" arginine transport is required for NO production in some tissues (e.g., refs. 4-6).The mCAT genes were, to our knowledge, the first mammalian AA transporters cloned (reviewed in ref. 7). Their isolation permits a molecular and genetic analysis of cationic AA transporter expression and regulation (8-11). The mCAT proteins share substantial sequence (9), structural (11), and functional (12, 13) similarity when expressed and assessed in Xenopus oocytes (8,10,12,13). Based on their transport characteristics, the mCAT genes are considered to encode the y+ transport system (8,10,(12)(13)(14). Although the two genes share many similarities, they differ in their chromosomal location (9, 15) and expression patterns (7,12). Their apparent functional redundancy raises questions about mCAT gene regulation. Although little molecular information is available regarding the mechanism of AA transporter gene regulation, it is well established that AA transport systems are responsive to metabolic demands (3). We have shown that mCAT-2 mRNA accumulates during T-cell activation (9, 12) and in other tissues in response to surgical trauma (16). In contrast, mCAT-1 mRNA levels are more constant in parallel test conditions (7,12,16), although the mRNA levels are altered in response to glycogen and during liver regeneration (17).Here we report evidence that mCAT-2 gene transcription is initiated from multiple promoters. Sequence analysis of several cDNA clones revealed distinct 5' untranslated regions (UTRs) that could result from multiple promoters, alternative splicing, or trans-splicing. To analyze this further, the entire coding region of the structural gene was isolated. Moreover, the regions surrounding three of the 5' UTRs were sequenced and found to contain classical promoter and enhancer elements. We present evidence that these promoters are utilized and result in regulated gene expression in response to surgical stress. Finally, we document the differential utilization of these putative promoters in different cell types. Our data provide strong evidence that mCAT-2 transcription initiates from several distinct, widely spaced promoters.t MATERIALS AND METHODS Animals and Cell Culture. Six-to 8-week-old female AKR/J mice were purchased from The Jackson Laboratories. Tissues were harvested for RNA preparation in accordance with University of California and National Institutes of Health guidelines. The SL12.3 a...