No abstract

No abstract

Marine natural products provide a rich source of chemical diversity that can be used to design and develop new, potentially useful therapeutic agents. We report here that scytonemin, a pigment isolated from cyanobacteria, is the first described small molecule inhibitor of human polo-like kinase, a serine/threonine kinase that plays an integral role in regulating the G 2 /M transition in the cell cycle. Scytonemin inhibited polo-like kinase 1 activity in a concentration-dependent manner with an IC 50 of 2 M against the recombinant enzyme. Biochemical analysis showed that scytonemin reduced GST-polo-like kinase 1 activity in a time-independent fashion, suggesting reversibility, and with a mixed-competition mechanism with respect to ATP. Although scytonemin was less potent against protein kinase A and Tie2, a tyrosine kinase, it did inhibit other cell cycle-regulatory kinases like Myt1, checkpoint kinase 1, cyclin-dependent kinase 1/cyclin B, and protein kinase C␤2 with IC 50 values similar to that seen for polo-like kinase 1. Consistent with these effects, scytonemin effectively attenuated, without chemical toxicity, the growth factor-or mitogen-induced proliferation of three cell types commonly implicated in inflammatory hyperproliferation. Similarly, scytonemin (up to 10 M) was not cytotoxic to nonproliferating endotoxin-stimulated human monocytes. In addition, Jurkat T cells treated with scytonemin were induced to undergo apoptosis in a non-cell cycle-dependent manner consistent with its activities on multiple kinases. Here we propose that scytonemin's dimeric structure, unique among natural products, may be a valuable template for the development of more potent and selective kinase inhibitors used for the treatment of hyperproliferative disorders.

No abstract

Activation of lymphocytes induces blastogenesis and cell division which is accompanied by membrane lipid metabolism such as increased fatty acid turnover. To date little is known about the enzymatic mechanism(s) regulating this process. Release of fatty acids such as arachidonic acid requires sn-2-deacylation catalyzed by a class of enzymes known as phospholipases A 2 (PLA 2 , EC 3.1.1.4). Herein, we confirm that human peripheral blood B or T lymphocytes (PBL) do not possess measurable levels of 85-kDa PLA 2 as assessed by Western immunoblot. Low levels of 14-kDa PLA 2 protein and activity were detectable in the particulate fraction of PBL and Jurkat cells. Western immunoblot analysis indicates that PBLs possess the calcium-independent PLA 2 (iPLA 2 ) protein. Calcium-independent sn-2-acylhydrolytic activity was measurable in PBL cytosols and could be inhibited by the selective iPLA 2 inhibitor bromoenol lactone. Mitogen activation of PBLs resulted in maintenance of activity levels which remained constant over 72 h suggesting an important role for iPLA 2 in this proliferative process. Indeed, evaluation of iPLA 2 activity in cell cycle-arrested Jurkat T cell fractions revealed the highest iPLA 2 levels occurring at the G 2 /M phase. Addition of the iPLA 2 inhibitors, bromoenol lactone, or arachidonyl trifluoromethyl ketone (AAOCF 3 ), inhibited both mitogen-induced PBL as well as Jurkat T cell proliferation. Moreover, specific depletion of iPLA 2 protein by antisense treatment also resulted in marked suppression of cell division. Inhibition of Jurkat cell proliferation was not associated with arrest at a particular phase of the cell cycle nor was it associated with apoptosis as assessed by flow cytometry. These findings provide the first evidence that iPLA 2 plays a key role in the lymphocyte proliferative response.Activation of resting lymphocytes occurs by ligation of antigen-specific co-receptors or artificially through mitogen stimulation. This in turn results in the formation of blast cells which undergo cellular proliferation, differentiation, and clonal expansion. Preparation for cell division requires not only synthesis of nucleotide pools and new protein but also increases in phospholipid (PL) 1 content and remodeling of existing PL in support of new membrane formation (1). Several studies have demonstrated that changes in membrane PL (1, 2) and arachidonic acid (AA) metabolism (3, 4) occur during the cell cycle. More specifically, mitogen activation of lymphocytes is accompanied by an increase in fatty acid turnover and an enrichment of PL with AA, the major substrate of proinflammatory eicosanoids (3-6). The AA comes from internal pools since the enrichment occurs in serum-free conditions (3, 4). The fact that this phenomenon, requiring deacylation-reacylation of PL, is AA specific and targeted toward selected PL classes (e.g. phosphatidylethanolamine and phosphatidylinositol) suggests regulation through specific enzyme systems (4). Indeed, the lysophosphatide acyltransferase, a reacylating enzyme, pr...