Temozolomide is an alkylating cytostatic drug that finds increasing application in the treatment of melanoma, anaplastic astrocytoma and glioblastoma multiforme. The compound is a prodrug that decomposes spontaneously, independent of an enzymatic activation step. DNA methylation induces futile mismatch repair cycles and depletion of the DNA repair enzyme O 6 -methylguanine-DNA methyltransferase should then initiate programmed cell death. We show drug-dependent inhibition of tumour growth in a threedimensional cell culture model of the glioma cell lines U87MG and GaMG. Migrational behaviour of the glioblastoma cells remained unaltered. However, coincubation of tumour spheroids with primary brain aggregates showed reduced tumour cell invasion into brain tissue in the presence of temozolomide. This was not achieved by slowing cellular migration, as temozolomide-treated cells displayed no reduced motility. By transferase-mediated dUTP nick-end labelling (TUNEL) of apoptotic nuclei, we found that the drug was able to induce apoptosis throughout the tumour cell spheroids. Apoptosis was highest in the core region of the spheroids. Repetitive application of sublethal doses of temozolomide to multicellular spheroids resulted in the development of drug resistance in GaMG cells. We suggest that temozolomide is a strong initiator of apoptosis in glioblastoma tumour cells in a spheroid cell culture system, when cells are already in a stressful environment.

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A defining characteristic of HIV-1 infection is the ability of the virus to persist within the host. Specifically, MHC-I downregulation by the HIV-1 accessory protein Nef is of critical importance in preventing infected cells from cytotoxic T-cell mediated killing. Nef downregulates MHC-I by modulating the host membrane trafficking machinery, resulting in the endocytosis and eventual sequestration of MHC-I within the cell. In the current report, we utilized the intracellular protein-protein interaction reporter system, bimolecular fluorescence complementation (BiFC), in combination with super-resolution microscopy, to track the Nef/MHC-I interaction and determine its subcellular localization in cells. We demonstrate that this interaction occurs upon Nef binding the MHC-I cytoplasmic tail early during endocytosis in a Rab5-positive endosome. Disruption of early endosome regulation inhibited Nef-dependent MHC-I downregulation, demonstrating that Nef hijacks the early endosome to sequester MHC-I within the cell. Furthermore, super-resolution imaging identified that the Nef:MHC-I BiFC complex transits through both early and late endosomes before ultimately residing at the trans-Golgi network. Together we demonstrate the importance of the early stages of the endocytic network in the removal of MHC-I from the cell surface and its re-localization within the cell, which allows HIV-1 to optimally evade host immune responses.

Efferocytosis is essential for homeostasis and prevention of the inflammatory and autoimmune diseases resulting from apoptotic cell lysis. CD93 is a transmembrane glycoprotein previously implicated in efferocytosis, with mutations in CD93 predisposing patients to efferocytosis‐associated diseases. CD93 is a cell surface protein, which is proteolytically shed under inflammatory conditions, but it is unknown how CD93 mediates efferocytosis or whether its efferocytic activity is mediated by the soluble or membrane‐bound form. Herein, using cell lines and human monocytes and macrophages, we demonstrate that soluble CD93 (sCD93) potently opsonizes apoptotic cells but not a broad range of microorganisms, whereas membrane‐bound CD93 has no phagocytic, efferocytic, or tethering activity. Using mass spectrometry, we identified αxβ2 as the receptor that recognizes sCD93, and via deletion mutagenesis determined that sCD93 binds to apoptotic cells via its C‐type lectin‐like domain and to αxβ2 by its EGF‐like repeats. The bridging of apoptotic cells to αxβ2 markedly enhanced efferocytosis by macrophages and was abrogated by αxβ2 knockdown. Combined, these data elucidate the mechanism by which CD93 regulates efferocytosis and identifies a previously unreported opsonin‐receptor system utilized by phagocytes for the efferocytic clearance of apoptotic cells.

BackgroundThe HIV-1 accessory proteins Nef and Vpu alter cell surface levels of multiple host proteins to modify the immune response and increase viral persistence. Nef and Vpu can downregulate cell surface levels of the co-stimulatory molecule CD28, however the mechanism of this function has not been completely elucidated.ResultsHere, we provide evidence that Nef and Vpu decrease cell surface and total cellular levels of CD28. Moreover, using inhibitors we implicate the cellular degradation machinery in the downregulation of CD28. We shed light on the mechanisms of CD28 downregulation by implicating the Nef LL165 and DD175 motifs in decreasing cell surface CD28 and Nef DD175 in decreasing total cellular CD28. Moreover, the Vpu LV64 and S52/56 motifs were required for cell surface CD28 downregulation, while, unlike for CD4 downregulation, Vpu W22 was dispensable. The Vpu S52/56 motif was also critical for Vpu-mediated decreases in total CD28 protein level. Finally, the ability of Vpu to downregulate CD28 is conserved between multiple group M Vpu proteins and infection with viruses encoding or lacking Nef and Vpu have differential effects on activation upon stimulation.ConclusionsWe report that Nef and Vpu downregulate cell surface and total cellular CD28 levels. We identified inhibitors and mutations within Nef and Vpu that disrupt downregulation, shedding light on the mechanisms utilized to downregulate CD28. The conservation and redundancy between the abilities of two HIV-1 proteins to downregulate CD28 highlight the importance of this function, which may contribute to the development of latently infected cells.Electronic supplementary materialThe online version of this article (10.1186/s12977-018-0388-3) contains supplementary material, which is available to authorized users.