Angiopoietin-2 (Ang2) exhibits broad expression in the remodeling vasculature of human tumors but very limited expression in normal tissues, making it an attractive candidate target for antiangiogenic cancer therapy. To investigate the functional consequences of blocking Ang2 activity, we generated antibodies and peptide-Fc fusion proteins that potently and selectively neutralize the interaction between Ang2 and its receptor, Tie2. Systemic treatment of tumor-bearing mice with these Ang2-blocking agents resulted in tumor stasis, followed by elimination of all measurable tumor in a subset of animals. These effects were accompanied by reduced endothelial cell proliferation, consistent with an antiangiogenic therapeutic mechanism. Anti-Ang2 therapy also prevented VEGF-stimulated neovascularization in a rat corneal model of angiogenesis. These results imply that specific Ang2 inhibition may represent an effective antiangiogenic strategy for treating patients with solid tumors.

The mechanism of BRCA1 tumor suppression in human breast and ovarian cells is the focus of intense investigation. In this report, full length BRCA1 (230 kDa) introduced into cells with CMV promoter constructs was nuclear when transgene expression was low whereas high expression resulted in cytoplasmic accumulation, aberrant nuclear and cell morphology. A nuclear localization signal (NLS) was mapped to BRCA1 amino acid positions 262 ± 570. We describe a splice variant, BRCA1-D11b, missing the majority of exon 11 including the NLS. Exogenous BRCA1-D11b (110 kDa) was cytoplasmic and, unlike the full-length protein, overexpression of the protein encoded by the variant did not appear to be toxic. RNA probe titrations and RT ± PCR demonstrated that BRCA1 and D11b transcripts are coexpressed in a wide variety of cells and tissues. Interestingly, BRCA1-D11b message was greatly reduced or absent in several breast and ovarian tumor lines relative to exon 11 transcripts and a D9,10 splice variant. Taken together our results suggest that fulllength BRCA1 and BRCA1-D11b may have distinct roles in cell growth regulation and tumorigenesis.

A novel cyclin gene was discovered by searching an expressed sequence tag database with a cyclin box profile. The human cyclin E2 gene encodes a 404-amino-acid protein that is most closely related to cyclin E. Cyclin E2 associates with Cdk2 in a functional kinase complex that is inhibited by both p27Kip1 and p21 Cip1 . The catalytic activity associated with cyclin E2 complexes is cell cycle regulated and peaks at the G 1 /S transition. Overexpression of cyclin E2 in mammalian cells accelerates G 1 , demonstrating that cyclin E2 may be rate limiting for G 1 progression. Unlike cyclin E1, which is expressed in most proliferating normal and tumor cells, cyclin E2 levels were low to undetectable in nontransformed cells and increased significantly in tumor-derived cells. The discovery of a novel second cyclin E family member suggests that multiple unique cyclin E-CDK complexes regulate cell cycle progression.The eukaryotic cell cycle is regulated by a family of cyclindependent kinases (CDKs) that are cyclically activated to trigger the different phases of the cell cycle in the correct order and at the right time. CDKs are regulated by a number of different proteins, including the cyclins that bind and activate the CDKs to form a serine/threonine kinase holoenzyme complex. In mammals, D-type cyclins in conjunction with cyclins E and A are required for cells to traverse G 1 and enter S phase (19,25,29). There are three members of the D-type cyclins, two members of the cyclin A family, and, to date, one mammalian cyclin E gene (33). The formation of distinct G 1 cyclin-CDK complexes regulates the temporal phosphorylation of specific substrates that drive cells through G 1 and into S phase. Cyclin-CDK complexes are negatively regulated by two families of CDK inhibitors (34). The Ink4 family of CDK inhibitors exclusively regulate D-type cyclin-CDK complexes, while the Kip/Cip family regulate D-type cyclins as well as CDK complexes comprised of cyclin E or A. p27Kip1 and p21 Cip1 bind and inhibit cyclin E-Cdk2 complexes by acting as competitive inhibitors for substrates and by preventing cyclin-activating kinase (CAK)-mediated phosphorylation and activation of Cdk2 (23,34).Cyclin E was originally discovered by its ability to complement the G 1 cyclins in budding yeast (16,18). This observation suggested that cyclin E regulated the progression of cells through the cell cycle. It was later shown that cyclin E-Cdk2 complexes stimulate mammalian cells to traverse G 1 and enter S phase by the temporal phosphorylation of specific substrates such as the retinoblastoma tumor suppressor protein (Rb) (17,20,33). In fission yeast, a critical size must be reached prior to entry into mitosis, and this is normally regulated by coupling Cdk1 activation to cell growth. Premature activation of Cdk1 in fission yeast causes cells to enter mitosis at a reduced size. A similar effect occurs when cyclin E is overexpressed three-to fourfold in mammalian cells; the cells have a shorter G 1 and enter mitosis at a reduced size (25, 29). These resul...

In mammalian cells, the aurora kinases (aurora-A, -B, and -C) play essential roles in regulating cell division.

In eukaryotic cells, cyclin-dependent kinase (CDK) complexes regulate the temporal progression of cells through the cell cycle. Deregulation in the cell cycle is an essential component in the evolution of cancer. Here, we validate CDK1 and CDK2 as potential therapeutic targets using novel selective smallmolecule inhibitors of cyclin B1/CDK1 and cyclin E2/CDK2 enzyme complexes (CDKi). Flow cytometry-based methods were developed to assess intracellular retinoblastoma (Rb) phosphorylation to show inhibition of the CDK pathway. Tumor cells treated with CDK inhibitors showed an overall decrease in cell proliferation, accumulation of cells in G 1 and G 2 , and apoptosis in a cell line-specific manner. Although CDK inhibitors activate p53, the inhibitors were equipotent in arresting the cell cycle in isogenic breast and colon tumor cells lacking p53, suggesting the response is independent of p53. In vivo, the CDK inhibitors prevented the growth of colon and prostate tumors, blocked proliferation of tumor cells, and inhibited Rb phosphorylation. The discovery and evaluation of novel potent and selective CDK1 and CDK2 inhibitors will help delineate the role that CDK complexes play in regulating tumorigenesis. (Cancer Res 2006; 66(8): 4299-308)