Tumors build vessels by cooption of pre-existing vasculature and de novo recruitment of bone marrow (BM)-derived endothelial progenitor cells (EPCs). However, the contribution and the functional role of EPCs in tumor neoangiogenesis are controversial. Therefore, by using genetically marked BM progenitor cells, we demonstrate the precise spatial and temporal contribution of EPCs to the neovascularization of three transplanted and one spontaneous breast tumor in vivo using high-resolution microscopy and flow cytometry. We show that early tumors recruit BM-derived EPCs that differentiate into mature BM-derived endothelial cells (ECs) and luminally incorporate into a subset of sprouting tumor neovessels. Notably, in later tumors, these BM-derived vessels are diluted with non-BM-derived vessels from the periphery, which accounts for purported differences in previously published reports. Furthermore, we show that specific ablation of BM-derived EPCs with ␣-particle-emitting anti-VE-cadherin antibody markedly impaired tumor growth associated with reduced vascularization. Our results demonstrate that BM-derived EPCs are critical components of the earliest phases of tumor neoangiogenesis.[Keywords: Bone marrow transplantation; tumor angiogenesis; endothelial progenitor cells; endothelial cells; VE-cadherin; neovascularization] Supplemental material is available at http://www.genesdev.org.

We report the analysis of CPI-613, the first member of a large set of analogs of lipoic acid (lipoate) we have investigated as potential anticancer agents. CPI-613 strongly disrupts mitochondrial metabolism, with selectivity for tumor cells in culture. This mitochondrial disruption includes activation of the well-characterized, lipoate-responsive regulatory phosphorylation of the E1α pyruvate dehydrogenase (PDH) subunit. This phosphorylation inactivates flux of glycolysis-derived carbon through this enzyme complex and implicates the PDH regulatory kinases (PDKs) as a possible drug target. Supporting this hypothesis, RNAi knockdown of the PDK protein levels substantially attenuates CPI-613 cancer cell killing. In both cell culture and in vivo tumor environments, the observed strong mitochondrial metabolic disruption is expected to significantly compromise cell survival. Consistent with this prediction, CPI-613 disruption of tumor mitochondrial metabolism is followed by efficient commitment to cell death by multiple, apparently redundant pathways, including apoptosis, in all tested cancer cell lines. Further, CPI-613 shows strong antitumor activity in vivo against human non-small cell lung and pancreatic cancers in xenograft models with low side-effect toxicity.

RNA interference is a powerful genetic approach for efficiently silencing target genes. The existing method of gene suppression by the constitutive expression of short hairpin RNAs (shRNAs) allows analysis of the consequences of stably silencing genes but limits the analysis of genes essential for cell survival, cell cycle regulation, and cell development. We have developed an inducible U6 promoter for synthesis of shRNAs in both human and murine cells. Cells containing stably integrated shRNA expression constructs demonstrate stringent dosage-and time-dependent kinetics of induction with undetectable background expression in the absence of the inducer ecdysone. Inducible suppression of human p53 in glioblastoma cells shows striking morphological changes and defects in cell cycle arrest caused by DNA damage, as expected. Remarkably, the inducibility is reversible after withdrawal of the inducer, as observed by reappearance of the protein and a restoration of the original cell phenotype. Inducible and reversible regulation of RNA interference has broad applications in the areas of mammalian genetics and molecular therapeutics.

BackgroundTargeting cancer cell metabolism is recognized as a promising arena for development of cancer chemotherapeutics. Moreover, redox metabolism is also systematically altered in tumor cells. Indeed, there is growing reason to believe that tumor-specific alteration of redox control of metabolism will be central to understanding and attacking malignancy. We report here that lipoate analog CPI-613 attacks a gate-keeping, lipoate-using metabolic enzyme, alpha-ketoglutarate dehydrogenase (KGDH), by a redox mechanism selectively in tumors cells.ResultsCPI-613 inhibited KGDH function strongly and rapidly, selectively in tumor cells. Moreover, CPI-613 induced a correspondingly rapid, powerful redox signal in tumor cell mitochondria. This signal was associated with redox modification of KGDH (including extensive enzyme glutathionylation and redox blockage of enzyme lipoate sulfhydryls), correlating with KGDH inactivation. The source of this tumor-specific mitochondrial redox modulatory signal was not electron transport complexes (I or III), but was largely or entirely the E3 (dihydrolipoamide dehydrogenase) component of dehydrogenases, including KGDH. Finally, we demonstrated that KGDH activity was redox regulated (in tumor cells), as expected if a tumor-specific redox process (auto)regulates KGDH.ConclusionsOur data demonstrate that lipoate analog CPI-613 attacks redox control of KGDH activity in tumor cells, perhaps by modulation of an existing lipoate-sensitive allosteric process normally governing tumor cell KGDH activity. Together with its previously reported, mechanistically distinct (non-redox) effects on the other major, lipoate-using mitochondrial metabolic enzyme, pyruvate dehydrogenase, CPI-613’s KGDH effects indicate that this agent simultaneously attacks multiple central, essential components of tumor cell metabolic regulation.

Developing thymocytes and some T-cell hybridomas undergo activation-dependent programmed cell death. Although recent studies have identified some critical regulators in programmed cell death, the role of cell cycle regulation in activation-induced cell death in T cells has not been addressed. We demonstrate that synchronized T-cell hybridomas, irrespective of the point in the cell cycle at which they are activated, stop cycling shortly after they reach G2/M. These cells exhibit the diagnostic characteristics of apoptotic cell death. Although p34cdc2 levels are not perturbed after activation of synchronously cycling T cells, cyclin B- and p34cdc2-associated histone H1 kinase activity is persistently elevated. This activation-dependent induction of H1 kinase activity in T cells is associated with a decrease in the phosphotyrosine content of p34cdc2. We also demonstrate that transient inappropriate coexpression of cyclin B with p34cdc2 induces DNA fragmentation in a heterologous cell type. Finally, in T cells, cyclin B-specific antisense oligonucleotides suppress activation-induced cell death but not cell death induced by exposure to dexamethasone. We therefore conclude that a persistent elevation of the level of cyclin B kinase is required for activation-induced programmed T-cell death.

Cell cycle progression is regulated by cyclin-dependent kinases. Using in vitro replication of SV40 origin containing DNA as a model system, we have performed a detailed analysis of the dependence on cyclin-associated kinases of mammalian DNA replication. Complete immunodepletion of cyclin A from human S phase cell extracts decreases replication, and replication activity of cyclin A-depleted S phase extracts can subsequently be restored by the addition of purified CDK2-cyclin A kinase. Addition of cyclin A alone reconstitutes both kinase activity and DNA replication, whereas addition of cyclin E or cyclin B reconstitutes neither. We therefore conclude that reconstitution of DNA replication specifically correlates with an increase in kinase activity. By comparison, depletion of cyclin E from S phase cell extracts does not have any significant inhibitory effect on DNA replication. Moreover, specific p21Waf1 mutants that bind to CDK2-cyclin and inhibit both cyclin A and cyclin E kinase activities, but do not bind to proliferating cell nuclear antigen, inhibit DNA replication to the same extent as cyclin A depletion. Together, these results show that the kinase activity associated with cyclin A, but not with cyclin E, is primarily responsible for activating SV40 plasmid replication in mammalian S phase cell extracts. Finally, we present evidence that the cyclin-dependent kinase does not influence the assembly of initiation complexes but acts at a stage prior to elongation.Replication of DNA is a strictly regulated event that occurs at a discrete period during the cell cycle. Cell cycle progression is regulated by distinct cyclin-dependent kinases that activate at different times in the cell cycle (reviewed in Ref. 1). In mammalian cells, cyclin E-dependent kinase is activated at the G 1 to S phase transition, after the D type cyclins but prior to A type cyclins (reviewed in Ref.2). Microinjection of either anticyclin A antibody (3, 4) or antisense cyclin A plasmid (3, 5) into exponentially growing cells prevents the entry of cells into S phase. Similarly, microinjection of anti-cyclin E antibody also prevents the entry of cells into S phase (6). However, unlike cyclin E-dependent kinase activity, the timing of activation of cyclin A-associated kinase activity in human cells coincides with the onset of DNA synthesis in S phase, which occurs several hours subsequent to the commitment to S phase (7-9). Cyclin A may therefore have a direct role in DNA replication in S phase.Cyclin-dependent kinases have been implicated as inducers of DNA replication using systems for in vitro replication of DNA. p13 suc1 , the product of Schizosaccharomyces pombe suc1 gene, binds avidly to active forms of CDC2 and CDK2 (10 -12). The removal of Cdk2 and Cdc2 proteins from an in vitro Xenopus egg extract replication system, using p13 suc1 affinity matrices, has been shown to decrease its ability to replicate sperm DNA (13,14). Specific depletion of Cdk2 protein, but not of Cdc2 protein from Xenopus egg extracts, has been shown to correlat...

Cell growth control by interferons (IFNs) involves upregulation of the tumor suppressor interferon regulatory factor 1 (IRF1). To exert its anti-proliferative effects, this factor must ultimately control transcription of several key genes that regulate cell cycle progression. Here we show that the G 1 /S phase-related cyclindependent kinase 2 (CDK2) gene is a novel proliferationrelated downstream target of IRF1. We find that IRF1, but not IRF2, IRF3, or IRF7, selectively represses CDK2 gene transcription in a dose-and time-dependent manner. We delineate the IRF1-responsive repressor element between nt ؊68 to ؊31 of the CDK2 promoter. For comparison, the tumor suppressor p53 represses CDK2 promoter activity independently of IRF1 through sequences upstream of nt ؊68, and the CDP/cut/Cux1 homeodomain protein represses transcription downstream of ؊31. Thus, IRF1 repression represents one of three distinct mechanisms to attenuate CDK2 levels. The ؊68/؊31 segment lacks a canonical IRF responsive element but contains a single SP1 binding site. Mutation of this element abrogates SP1-dependent enhancement of CDK2 promoter activity as expected but also abolishes IRF1-mediated repression. Forced elevation of SP1 levels increases endogenous CDK2 levels, whereas IRF1 reduces both endogenous SP1 and CDK2 protein levels. Hence, IRF1 represses CDK2 gene expression by interfering with SP1-dependent transcriptional activation. Our findings establish a causal series of events that functionally connect the anti-proliferative effects of interferons with the IRF1-dependent suppression of the CDK2 gene, which encodes a key regulator of the G 1 /S phase transition.Interferon regulatory factors (IRFs) 1 are activated by the anti-proliferative actions of interferons through JAK/STATmediated signaling mechanisms to inhibit cell growth (1-3).Genetic evidence suggests IRF1, the prototypical member of the IRF class of transcription factors, functions as a tumor suppressor (4, 5) presumably by regulating cell growth-related target genes (6). There are few experimentally validated IRF1 target genes, and only a subset of these may contribute to the cell growth inhibitory potential of IRF1 (1, 6 -10). To clarify the biological functions of IRF1 as a tumor suppressor, it is necessary to define additional cell growth regulatory genes that are IRF1-responsive.Our laboratory has shown that IRF1, as well as the closely related protein IRF2 (also known as histone nuclear factor-M (HiNF-M)), can function in the activation of histone H4 gene transcription at the G 1 /S phase transition through a phylogenetically conserved cell cycle regulatory element (9 -16), which encompasses a canonical IRF consensus sequence (17). A characteristic N-terminal DNA binding domain spanning a winged helix-turn-helix motif (18 -20) mediates the interaction of IRF factors with their cognate sites. The C-terminal region supports transcriptional enhancement or repression (21-23). The transcriptional activity of IRF factors is influenced by posttranslational modifications, ...

DNA replication in eukaryotic cells is restricted to the S-phase of the cell cycle. In a cell-free replication model system, using SV40 origin-containing DNA, extracts from G1 cells are inefficient in supporting DNA replication. We have undertaken a detailed analysis of the subcellular localization of replication proteins and cell cycle regulators to determine when these proteins are present in the nucleus and therefore available for DNA replication. Cyclin A and cdk2 have been implicated in regulating DNA replication, and may be responsible for activating components of the DNA replication initiation complex on entry into S-phase. G1 cell extracts used for in vitro replication contain the replication proteins RPA (the eukaryotic single-stranded DNA binding protein) and DNA polymerase alpha as well as cdk2, but lack cyclin A. On localizing these components in G1 cells we find that both RPA and DNA polymerase alpha are present as nuclear proteins, while cdk2 is primarily cytoplasmic and there is no detectable cyclin A. An apparent change in the distribution of these proteins occurs as the cell enters S-phase. Cyclin A becomes abundant and both cyclin A and cdk2 become localized to the nucleus in S-phase. In contrast, the RPA-34 and RPA-70 subunits of RPA, which are already nuclear, undergo a transition from the uniform nuclear distribution observed during G1, and now display a distinct punctate nuclear pattern. The initiation of DNA replication therefore most likely occurs by modification and activation of these replication initiation proteins rather than by their recruitment to the nuclear compartment.