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Circulating polymorphonuclear cell (PMN) levels rise in proportion to the metastatic potential of the tumor in 13762NF mammary adenocarcinoma tumor-bearing rats. These tumor-elicited PMNs (tcPMNs) secrete high levels of the basement-membrane-degrading enzymes, type IV collagenase and heparanase, suggesting that metastatic tumor cells stimulate neutrophilia so that the tcPMNs might assist tumor cell extravasation during metastasis. To test this hypothesis, purified proteose peptone-elicited PMNs from peritoneal exudate, circulating normal PMNs, and tcPMNs were evaluated for their effects on in vitro invasive and in vivo metastatic potentials of syngeneic 13762NF mammary adenocarcinoma tumor cells. tcPMNs caused a dose-dependent increase in invasion through a reconstituted basement membrane barrier in an in vitro invasion assay. At PMN:tumor cell ratios of 30:1, invasion potential significantly (P < 0.05) rose to 26-fold, 40-fold, and 37-fold for poorly metastatic MTLn2 cells, higly metastatic MTLn3 cells, and moderately metastatic MTF7 cells, respectively. In contrast, purified proteose peptone-elicited PMNs and circulating normal PMNs did not sgificantly alter invasive potential. Intravenous coij'ections of purified proteose peptone-elicited PMNs did not change the number of experimental lung metastases, but tcPMNs at ratios to 50:1 significantly raised the mean number of metasass 23-fold for MTLn2, 3-to 4-fold for MTLn3, and 1.6-to 1.8-fold for MTF7. These results demonstrate that tcPMNs contribute to the metastatic propensity of mammary adeocarcinoma clones by increasing efficiency of invasion through basement membrane. Although polymorphonuclear cells (PMNs) are not the predominant circulating leukocyte population in normal rats, PMNs are the predominant population in humans. Neutrophils have been seen in close association with metastatic human and animal tumor cells in vivo at the primary tumor (10) and within the vasculature (17). The observation that the level of circulating PMNs increases to 50-fold as the primary tumor proliferates and the observation that tumor-elicited PMNs (tcPMNs) secrete high levels of type IV collagenase and heparanase and are noncytotoxic and noncytostatic (9), combine to suggest that tcPMNs may enhance the ability of tumor cells to extravasate, hence to metastasize.The results presented here demonstrate that tcPMNs, but not normal circulating PMNs (cPMNs), augment the ability of a tumor cell to penetrate a basement membrane-like matrix in vitro and to form lung colonies in vivo.MATERIALS AND METHODS Animals. Cell Lines and Tissue Culture. 13762NF rat mammary adenocarcinoma clones MTLn2, MTLn3, and MTF7 were grown and maintained as described (10, 18). Briefly, cells were grown in a-modified minimum essential medium (aMEM) supplemented with 5% fetal bovine serum. Cells were subcultured when the plates became 70-80% confluent by using 0.25% trypsin solution.Isolation and Purification of PMN. Purified proteose peptone-elicited PMNs (ppPMNs) were obtained by injecting syngen...

The phosphodiesterases (PDEs) are metal ion-dependent enzymes that regulate cellular signaling by metabolic inactivation of the ubiquitous second messengers cAMP and cGMP. In this role, the PDEs are involved in many biological and metabolic processes and are proven targets of successful drugs for the treatments of a wide range of diseases. However, because of the rapidity of the hydrolysis reaction, an experimental knowledge of the enzymatic mechanisms of the PDEs at the atomic level is still lacking. Here, we report the structures of reaction intermediates accumulated at the reaction steady state in PDE9/crystal and preserved by freeze-trapping. These structures reveal the catalytic process of a PDE and explain the substrate specificity of PDE9 in an actual reaction and the cation requirements of PDEs in general.crystallography ͉ enzyme mechanism ͉ reaction intermediates ͉ freeze-trapping T he phosphodiesterases (PDEs) are a superfamily of enzymes that metabolically inactivate the ubiquitous intracellular messengers cAMP and cGMP. This function involves the PDEs in a broad range of important cellular functions, such as immune response, memory, and vision (1-4). The human genome encodes for 21 PDEs that are categorized into 11 families (2). Alternative splicing results in the generation of Ͼ60 identified isoforms. These enzymes share a conserved catalytic domain of approximately 300 aa that is located in the C-terminal region of the protein. The N-terminal regions, which vary among different PDEs, serve regulatory functions including autoinhibition of the catalytic domains or control of subcellular localization (2). The PDEs have different substrate preferences: PDE 4, -7, and -8 preferentially hydrolyze cAMP; PDE5, -6, and -9 are cGMP specific. PDE1, -2, -3, -10, and -11 can hydrolyze both cyclic nucleotides (2). The different substrate preferences, combined with different expression profiles, cellular compartmentalization, and regulation, allow the PDEs to play a very versatile role in cell signal transduction (5).It is becoming increasingly clear that the physiological role of PDEs is the temporal and spatial control of cyclic nucleotide signaling, not simply inactivation (1, 2, 6). The clearest example of this control is in the fast action of PDE6 required for the temporal resolution of human vision (4). However, a complete understanding of the catalytic mechanism of these enzymes that accounts for the substrate specificity and reaction kinetics at the atomic level is lacking. Crystal structures of the catalytic domains of PDE1B, -2, -3, -4, -5, -7, -9, and -10, by themselves or in complex with inhibitors, substrates, or products, have been reported (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). Generally, the active sites of PDEs can be divided into 2 parts: a nucleotide recognition pocket and a hydrolysis center (7,19). Each PDE has its unique nucleotide recognition pocket, resulting in different substrate specificity and inhibition profiles (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). Based on t...

Circulating neutrophil (PMN) levels can increase in rats bearing subcutaneously growing clones of the 13762NF mammary adenocarcinoma and the level of increase correlates with the metastatic potential of the clone. In rats with poorly metastatic MTC tumors, numbers of circulating PMN did not rise, whereas PMN levels rose 50-fold in rats bearing highly metastatic MTLn3, 12-fold in rats with weakly metastatic MTLn2, and 14-fold in those with moderately metastatic MTF7 tumors. Neutrophilia was caused partly by tumor size, but metastatic potential was a stronger determinant, suggesting that PMNs may play a role in the metastatic process. To determine whether circulating PMNs indeed contribute to cellular metastatic potential, we examined effects of PMN on various aspects of the metastatic process. Experimental metastasis assays involving i.v. co-injections of PMNs yielded a dose-dependent increase in extrapulmonary metastases for MTLn3, but no change in lung colonization potential for any of the clones examined. The change in the metastatic profile was not due to any modification in in vivo distribution of i.v. injected tumor cells or in adhesion to endothelial monolayers in vitro. PMNs also had no effect on in vitro DNA, RNA or protein synthesis and were not cytolytic (E:T 100:1). However, PMNs collected from high-passage MTLn3 tumor-bearing rats had a 50% increase in heparanase and type-IV collagenolytic activity as compared to unstimulated PMNs isolated from normal rats. These results indicate that polymorphonuclear cells may contribute to the metastatic potential of highly metastatic clones from the 13762NF mammary adenocarcinoma cells by assisting in the degradation of basement membrane during extravasation.

The Membrane Invasion Culture System (MICS) assay was adapted for relatively rapid screening of compounds and used to identify anti-invasive drugs that inhibit human and murine tumor cell migration through a reconstituted basement membrane in vitro. Cell lines demonstrating low and high invasive and metastatic potentials were tested with all compounds for tumoricidal effects prior to evaluation in MICS at non-cytotoxic doses. The effect on invasive potential in the MICS assay was determined in 3 categories: (1) 48 hr drug pre-treatment prior to seeding in the MICS (exceptions: 90 min pre-treatment with pertussis toxin and, for some studies, continuous exposure for 2-7 days); (2) peptide or prostaglandins 2 hr after seeding and attachment to the membranes in MICS followed by continuous exposure; and (3) cells receiving neither drug nor peptide treatment and serving as controls in each MICS chamber. Since invasion involves cellular motility and deformability, some cytoskeleton disrupting agents were selected. Of these, vincristine, colcemid and colchicine inhibited invasion but taxol did not. Pre-treatment with cAMP agonists produced conflicting results: dibutyryl cAMP and 8-(4-chloro-phenylthio) cAMP resulted in 50% and 38% reduction in invasion, respectively, whereas 8-bromo cAMP stimulated invasive potential by 30%. Forskolin and cholera toxin both significantly reduced invasiveness. Pre-treatment with 5-azacytidine and araC, to consider the role of methylation and proliferations decreased invasive ability. Anti-metastatic drugs such as gamma-interferon and razoxane inhibited invasive potential but to varying degrees. Treatment of cells with prostaglandins E2, F2 alpha, A2, and D2 were ineffectual; however, indomethacin mildly inhibits invasion (less than 30%).(ABSTRACT TRUNCATED AT 250 WORDS)