To determine the effects of immunomodulatory agents upon HIV replication in macrophages, cultured monocyte-derived macrophages were treated with various substances and then infected with a macrophage-tropic strain of HIV-1. Pretreatment with rIFN-alpha, IFN-beta, and IFN-gamma, or bacterial LPS prevented viral replication in macrophages. In treated cultures, little or no infectious HIV or p24 core antigen was released into the supernatant, no virions were seen by electron microscopy, no viral RNA or DNA was detectable in the cell lysates, and no cytopathology (as determined by multinucleated giant cell formation) occurred. In contrast, pretreatment with a wide dose range of recombinant IL-1 beta, IL-2, IL-4, IL-6, M-CSF, TNF, or lymphotoxin failed to protect macrophages from productive infection by HIV. A consistent effect of granulocyte/macrophage-CSF on HIV replication in macrophages was not observed. In dose response studies, pretreatment with approximately 100 U/ml of IFN-alpha, approximately 10 U/ml of IFN-beta, or approximately 100 U/ml of IFN-gamma was sufficient to prevent virion release maximally and to prevent cytopathology completely. In kinetic studies, IFN-alpha, IFN-gamma, or LPS were added to the macrophage cultures either before or after infection with HIV. Even when added 3 d after infection with a multiplicity of 1 50% tissue-culture infectious dose per cell, all three treatments markedly reduced virion release, suggesting that these agents act at a point in the viral life cycle beyond the early events of virus binding, penetration, and uncoating. These data indicate that HIV replication in previously uninfected macrophages may be regulated by an inducible host cell mechanism. These findings may explain the restricted replication of HIV in macrophages in vivo and suggest an antiviral role for interferons in the therapy of HIV infection.

Recent evidence supports the existence of a plasma membrane ER. In many cells, E2 activates signal transduction and cell proliferation, but the steroid inhibits signaling and growth in other cells. These effects may be related to interactions of ER with signal-modulating proteins in the membrane. It is also unclear how ER moves to the membrane. Here, we demonstrate ER in purified vesicles from endothelial cell plasma membranes and colocalization of ERalpha with the caveolae structural coat protein, caveolin-1. In human vascular smooth muscle or MCF-7 (human breast cancer) cell membranes, coimmunoprecipitation shows that ER associates with caveolin-1 and -2. Importantly, E2 rapidly and differentially stimulates ER-caveolin association in vascular smooth muscle cells but inhibits association in MCF-7 cells. E2 also stimulates caveolin-1 and -2 protein synthesis and activates a caveolin-1 promoter/luciferase reporter in smooth muscle cells. However, the steroid inhibits caveolin synthesis in MCF-7 cells. To determine a function for caveolin-ER interaction, we expressed caveolin-1 in MCF-7 cells. This stimulated ER translocation to the plasma membrane and also inhibited E2-induced ERK (MAPK) activation. Both functions required the caveolin-1 scaffolding domain. Depending upon the target cell, membrane ERs differentially associate with caveolin, and E2 differentially modulates the synthesis of this signaling-inhibitory scaffold protein. This may explain the discordant signaling and actions of E2 in various cell types. In addition, caveolin-1 is capable of facilitating ER translocation to the membrane.

No abstract

Hypoxia is a hallmark of solid tumors, is associated with local invasion, metastatic spread, resistance to chemo- and radiotherapy, and is an independent, negative prognostic factor for a diverse range of malignant neoplasms. The cellular response to hypoxia is primarily mediated by a family of transcription factors, among which hypoxia-inducible factor 1 (HIF1) plays a major role. Under normoxia, the oxygen-sensitive α subunit of HIF1 is rapidly and constitutively degraded but is stabilized and accumulates under hypoxia. Upon nuclear translocation, HIF1 controls the expression of over 100 genes involved in angiogenesis, altered energy metabolism, antiapoptotic, and pro-proliferative mechanisms that promote tumor growth. A designed transcriptional antagonist, dimeric epidithiodiketopiperazine (ETP 2), selectively disrupts the interaction of HIF1α with p300/CBP coactivators and downregulates the expression of hypoxia-inducible genes. ETP 2 was synthesized via a novel homo-oxidative coupling of the aliphatic primary carbons of the dithioacetal precursor. It effectively inhibits HIF1-induced activation of VEGFA, LOX, Glut1, and c-Met genes in a panel of cell lines representing breast and lung cancers. We observed an outstanding antitumor efficacy of both (±)-ETP 2 and meso-ETP 2 in a fully established breast carcinoma model by intravital microscopy. Treatment with either form of ETP 2 (1 mg/kg) resulted in a rapid regression of tumor growth that lasted for up to 14 days. These results suggest that inhibition of HIF1 transcriptional activity by designed dimeric ETPs could offer an innovative approach to cancer therapy with the potential to overcome hypoxia-induced tumor growth and resistance.