Previous studies with U937 cells, a human monocyte cell line, have shown that the activity of cyclic nucleotide phosphodiesterase 4 (PDE4) is increased by agents that elevate cyclic AMP content. The present experiments were conducted to determine 1) whether an increase in PDE4 steady-state message and/or protein accompanies the up-regulation of PDE4 activity and 2) whether the up-regulation changes the functional responses of U937 cells to activators of adenylyl cyclase. To up-regulate PDE4 activity, U937 cells were treated for 4 h with a combination of 1 M salbutamol, a -adrenoceptor agonist, and 30 M rolipram, a PDE4 inhibitor. Cells were washed extensively to remove drugs and used immediately in various experimental protocols. Reverse transcriptase-polymerase chain reactions conducted with primers specific for the four PDE4 subtypes suggested that pretreatment with salbutamol and rolipram increased steady-state mRNA levels of PDE4A and PDE4B, but not PDE4C or PDE4D. Immunoblot analyses using two rabbit polyclonal antibodies, one directed against human recombinant PDE4A and PDE4D and a second directed against human recombinant PDE4B, revealed bands of immunoreactivity corresponding to ϳ125 kDa (PDE4A) and ϳ70 kDa (PDE4B), respectively, that increased in intensity after cells were treated with salbutamol and rolipram. As demonstrated in both time course and concentration-response studies with prostaglandin E 2 (PGE 2 ), an agent that activates adenylyl cyclase by a non--adrenoceptor-mediated mechanism, cAMP accumulation was substantially decreased in cells in which PDE4 activity had been up-regulated. The difference in PGE 2 -stimulated cAMP accumulation between control and PDE4 up-regulated cells was greatly reduced in the presence of rolipram, consistent with the notion that an increase in PDE4 activity was responsible for the heterologous desensitization. Functionally, upregulation of PDE4 markedly decreased the ability of PGE 2 to inhibit LTD 4 -induced Ca 2؉ mobilization in intact cells. A hypothetical implication of these results is that increasing PDE4 activity in vivo by administering -adrenoceptor agonists could exacerbate inflammatory processes by decreasing the activity of endogenous antiinflammatory agents such as PGE 2 .
1 Of the four major phosphodiesterase 4 (PDE4) subtypes, PDE4A, PDE4B and PDE4D are widely expressed in human in¯ammatory cells, including monocytes and T lymphocytes. We explored the functional role of these subtypes using ten subtype-selective PDE4 inhibitors, each belonging to one of two classes: (i) dual PDE4A/PDE4B inhibitors or (ii) PDE4D inhibitors. 2 These compounds were evaluated for their ability to inhibit antigen-stimulated T-cell proliferation and bacterial lipopolysaccharide (LPS)-stimulated tumour necrosis factor a (TNFa) release from peripheral blood monocytes. 3 All compounds inhibited T-cell proliferation in a concentration-dependent manner; with IC 50 values distributed over an approximately 50 fold range. These compounds also inhibited TNFa release concentration-dependently, with a wider (*1000 fold) range of IC 50 values. 4 In both sets of experiments, mean IC 50 values were signi®cantly correlated with compound potency against the catalytic activity of recombinant human PDE4A or PDE4B when analysed by either linear regression of log IC 50 values or by Spearman's rank-order correlation. The correlation between inhibition of in¯ammatory cell function and inhibition of recombinant PDE4D catalytic activity was not signi®cant in either analysis. 5 These results suggest that PDE4A and/or PDE4B may play the major role in regulating these two in¯ammatory cell functions but do not rule out PDE4D as an important mediator of other activities in mononuclear leukocytes and other immune and in¯ammatory cells. Much more work is needed to establish the functional roles of the PDE4 subtypes across a broader range of cellular functions and cell types.
CNTO 95 is a fully human monoclonal antibody that recognizes alphav integrins. Previous studies have shown that CNTO 95 exhibits both anti-tumor and anti-angiogenic activities (Trikha M et al., Int J Cancer 110:326-335, 2004). In this study we investigated the biological activities of CNTO 95 on breast tumor cells both in vitro and in vivo. In vitro treatment with CNTO 95 decreased the viability of breast tumor cells adhering to vitronectin. CNTO 95 inhibited tumor cell adhesion, migration, and invasion in vitro. CNTO 95 treatment also induced tyrosine dephosphorylation of focal adhesion kinase (FAK), and the docking protein paxillin that recruits both structural and signaling molecules to focal adhesions (Turner CE, Int J Biochem Cell Biol 30:955-959, 1998; O'Neil GM et al., Trends Cell Biol 10:111-119, 2000). These results suggest that CNTO 95 inhibits breast tumor cell growth, migration and invasion by interruption of alphav integrin mediated focal adhesions and cell motility signals. In vivo studies of CNTO 95 were conducted in an orthotopic breast tumor xenograft model. Treatment with CNTO 95 resulted in significant inhibition of both tumor growth and spontaneous metastasis of MDA-MB-231 cells to the lungs. CNTO 95 also inhibited lung metastasis in a separate experimental (tail vein injection) model of metastasis. The results presented here demonstrate the anti-tumor and anti-metastatic activities of CNTO 95 in breast cancer models and provide insight into the cellular and molecular mechanisms mediating its inhibitory effects on metastasis.
Local immune responses are thought to play an important role in the development of atherosclerosis. Histological studies have shown that human atherosclerotic lesions contain T lymphocytes throughout all stages of development, many of which are in an activated state. A number of novel CC chemokines have been described recently, which are potent chemoattractants for lymphocytes: PARC (pulmonary and activation-regulated chemokine), ELC (EBI1-ligand chemokine), LARC (liver and activation-regulated chemokine), and SLC (secondary lymphoid-tissue chemokine). Using reverse transcriptase-polymerase chain reaction and in situ hybridization, we have found gene expression for PARC and ELC but not for LARC or SLC in human atherosclerotic plaques. Immunohistochemical staining of serial plaque sections with specific cell markers revealed highly different expression patterns of PARC and ELC. PARC mRNA was restricted to CD68+ macrophages (n = 14 of 18), whereas ELC mRNA was widely expressed by macrophages and intimal smooth muscle cells (SMC) in nearly all of the lesions examined (n = 12 of 14). ELC mRNA was also found to be expressed in the medial SMC wall of highly calcified plaques (n = 4). Very low levels of ELC mRNA expression could also be detected in normal mammary arteries but no mRNA expression for PARC was detected in these vessels (n = 4). In vitro, ELC mRNA was found to be up-regulated in aortic SMC stimulated with tumor necrosis factor-a and interferon-gamma but not in SMC stimulated with serum. Both PARC and ELC mRNA were expressed by monocyte-derived macrophages but not monocytes. The expression patterns of PARC and ELC mRNA in human atherosclerotic lesions suggest a potential role for these two recently described CC chemokines in attracting T lymphocytes into atherosclerotic lesions.
Purpose: Targeted delivery of cytotoxic agents to solid tumors through cell surface antigens can potentially reduce systemic toxicity and increase the efficacy of the targeted compounds. The purpose of this study was to show the feasibility of treating solid tumors by targeting a v integrins with antibody-maytansinoid conjugates and to test the relative in vivo activities of several linkermaytansinoid chemistries. Experimental Design: CNTO 364, CNTO 365, and CNTO 366 are targeted cytotoxic agents created by conjugating the CNTO 95 anti-a v integrin antibody with three distinct maytansinoidlinker structures.These structures were designed to have varying degrees of chemical substitution surrounding the disulfide bond linking the cytotoxic agent to the antibody. A model conjugate was shown to be specifically cytotoxic in vitro and highly active against established human tumor xenografts in immunocompromised rats. The in vivo antitumor activities of CNTO 364, CNTO 365, and CNTO 366 were compared in rat xenograft models. Results: CNTO 365, with a linker chemistry of expected intermediate stability, was shown to be substantially more active than the other two conjugates with lesser or greater substitution around the disulfide linkage. Conclusion: CNTO 95^maytansinoid immunoconjugates are potent antitumor agents against a v integrin^expressing human carcinomas. These studies show for the first time the feasibility of targeting a v integrins on solid tumors with tumor-activated prodrugs. The DM4 linker-maytansinoid configuration of CNTO 365 was substantially more active in the models tested here when compared with alternative configurations with greater or lesser chemical substitution surrounding the linker.The a v integrin subfamily consists of at least five members, including a v h 1 , a v h 3 , a v h 5 , a v h 6 , and a v h 8 (1). a v Integrins a v h 3 , a v h 5 , and a v h 6 have been implicated in angiogenesis and tumor progression (2 -5). Up-regulation of a v integrins has been observed in various types of human cancer, including melanoma (6), renal (7), ovarian (8), gastric (9), breast (10), and colorectal carcinoma (11). The frequent overexpression of a v integrins by tumors, their more limited expression by healthy tissues, and their importance in tumor growth and progression make them attractive targets for cancer therapies. CNTO 95, a fully human monoclonal antibody, inhibits a v integrins and has in vivo antitumor and antiangiogenic activity (12). It is one of several a v integrin inhibitors, including both monoclonal antibodies and small molecules, that have been tested both preclinically and clinically for the treatment of solid tumors (4).Immunoconjugates are bifunctional molecules that combine the specificity of monoclonal antibodies to tumor antigens with the potency of cytotoxic agents (13 -15). To take advantage of the a v integrin specificity of CNTO 95, we generated the antibody-drug conjugates CNTO 364, CNTO 365, and CNTO 366. These molecules are tumor-activated prodrugs in which derivativ...
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