Citations

Circulating cytokines, hormones, and growth factors control all aspects of cell proliferation, differentiation, angiogenesis, apoptosis, and senescence. These chemical signals are propagated from the cell surface to intracellular processes via sequential kinase signaling, arranged in modules that exhibit redundancy and cross talk. This signal transduction system comprising growth factors, transmembrane receptor proteins, and cytoplasmic secondary messengers is often exploited to optimize tumor growth and metastasis in malignancies. Thus, it represents an attractive target for cancer therapy. This review will summarize current knowledge of selected intracellular signaling networks and their role in cancer therapy. The focus will be on pathways for which inhibitory agents are currently undergoing clinical testing. Original data for inclusion in this review were identified through a MEDLINE search of the literature. All papers from 1966 through March 2005 were identified by the following search terms: “signal transduction,” “intracellular signaling,” “kinases,” “proliferation,” “growth factors,” and “cancer therapy.” All original research and review papers related to the role of intracellular signaling in oncogenesis and therapeutic interventions relating to abnormal cell signaling were identified. This search was supplemented by a manual search of the Proceedings of the Annual Meetings of the American Association for Cancer Research, American Society of Clinical Oncology, and the American Association for Cancer Research (AARC) –European Organisation for Research and Treatment of Cancer (EORTC) –National Cancer Institute (NCI) Symposium on New Anticancer Drugs.

To investigate the basis for the LEDGF/p75 dependence of HIV-1 integrase (IN) nuclear localization and chromatin association, we used cell lines made stably deficient in endogenous LEDGF/p75 by RNAi to analyze determinants of its location in cells and its ability to interact with IN. Deletion of C-terminal LEDGF/p75 residues 340-417 preserved nuclear and chromatin localization but abolished the interaction with IN and the tethering of IN to chromatin. Transfer of this IN-binding domain (IBD) was sufficient to confer HIV-1 IN interaction to GFP. HRP-2, the only other human protein with an identifiable IBD domain, was found to translocate IN to the nucleus of LEDGF/p75(–) cells. However, in contrast to LEDGF/p75, HRP-2 is not chromatin bound and does not tether IN to chromatin. A single classical nuclear localization signal (NLS) in the LEDGF/p75 N-terminal region (146RRGRKRKAEKQ156) was found by deletion mapping and was shown to be transferable to pyruvate kinase. Four central basic residues in the NLS are critical for its activity. Strikingly, however, stable expression studies with NLS(+/–) and IBD(+/–) mutants revealed that the NLS, although responsible for LEDGF/p75 nuclear import, is dispensable for stable, constitutive nuclear association of LEDGF/p75 and IN. Both wild-type LEDGF/p75 and NLS-mutant LEDGF/p75 remain entirely chromatin associated throughout the cell cycle, and each tethers IN to chromatin. Thus, these experiments reveal stable nuclear sequestration of a transcriptional regulator by chromatin during the nuclear-cytosolic mixing of cell division, which additionally enables stable tethering of IN to chromatin. LEDGF/p75 is a multidomain adaptor protein that interacts with the nuclear import apparatus, lentiviral IN proteins and chromatin by means of an NLS, an IBD and additional chromatin-interacting domains.

ABSTRACT Human immunodeficiency virus type 1 (HIV-1), feline immunodeficiency virus (FIV), and Moloney murine leukemia virus (MoMLV) integrases were stably expressed to determine their intracellular trafficking. Each lentiviral integrase localized to cell nuclei in close association with chromatin while the murine oncoretroviral integrase was cytoplasmic. Fusions of pyruvate kinase to the lentiviral integrases did not reveal transferable nuclear localization signals. The intracellular trafficking of each was determined instead by the transcriptional coactivator LEDGF/p75, which was required for nuclear localization. Stable small interfering RNA expression eliminated detectable LEDGF/p75 expression and caused dramatic, stable redistribution of each lentiviral integrase from nucleus to cytoplasm while the distribution of MoMLV integrase was unaffected. In addition, endogenous LEDGF/p75 coimmunoprecipitated specifically with each lentiviral integrase. In vitro integration assays with preintegration complexes (PICs) showed that endogenous LEDGF/p75 is a component of functional HIV-1 and FIV PICs. However, HIV-1 and FIV infection and replication in LEDGF/p75-deficient cells was equivalent to that in control cells, whether cells were dividing or growth arrested. Two-long terminal repeat circle accumulation in nondividing cell nuclei was also equivalent to that of LEDGF/p75 wild-type cells. Virions produced in LEDGF/p75-deficient cells had normal infectivity. We conclude that LEDGF/p75 fully accounts for cellular trafficking of diverse lentiviral, but not oncoretroviral, integrases and is the main lentiviral integrase-to-chromatin tethering factor. While lentiviral PIC nuclear import is unaffected by LEDGF/p75 knockdown, this protein is a component of functional lentiviral PICs. A role in HIV-1 integration site distribution merits investigation.

ABSTRACT Caveolin-1 (Cav-1) is a major protein constituent of caveolae, a type of plasma membrane raft. We observed that coexpression of human Cav-1 with human immunodeficiency virus type 1 (HIV-1) blocked virion production from cells that are ordinarily highly permissive. Further investigation showed that this effect is specific, occurs at low ratios of Cav-1 to HIV-1 DNA, depends on expression of Cav-1 protein, and involves severely impaired expression of HIV-1 proteins. Cav-1 also blocked HIV-2 expression. In contrast, Cav-1 did not inhibit protein expression by a paramyxovirus and did not induce apoptosis or affect cellular morphology, cell viability, or cell cycle progression. Although only small amounts of HIV-1 virions were released from Cav-1-transfected cells, these were fully infectious. Deletion mutagenesis showed that the C-terminal 78 residues were as active as the full-length (178-amino-acid) protein in producing the block. In contrast, the 100 most N-terminal amino acids of Cav-1, which include the previously identified oligomerization and scaffolding domains, were shown to be dispensable. Study of single-amino-acid-exchange mutants of Cav-1 established that palmitoylation was not required. Additional deletion mutants then identified the hydrophobic, membrane-associated domain (residues 101 to 135) as the main determinant. Cellular distribution of wild-type and mutant proteins correlated with ability to block HIV-1 expression. Finally, Cav-2 also blocked HIV-1 expression. These data show that coexpression of caveolins can markedly inhibit expression of HIV proviral DNA and establish that the inhibition is mediated by the hydrophobic, membrane-associated domain.