Abstract Introduction. Impairment in cell death pathways represents a general characteristic of most cancer cells. The receptor-interacting protein kinase 3 (RIP3) associates with RIP1 in a necrosome complex that can induce necroptosis, apoptosis, or cell proliferation. The role of RIP3 in necroptosis and inflammation has been extensively studied, but its role in cancer remains poorly understood Methods. We analyzed the expression of RIP1 and RIP3 in CD34+ leukemia cells from a cohort of patients with acute myeloid leukemia (AML) and CD34+ cells from healthy donors. To analyze the potential advantages for myeloid malignant cells due to reduced RIP3 expression, we induced the expression of RIP3 in the DA1-3b mouse leukemia cell line. Results. RIP3 expression was significantly reduced in most AML samples, whereas the expression of RIP1 did not differ significantly. When re-expressed in the mouse DA1-3b leukemia cell line, RIP3 induced apoptosis, and necroptosis in the presence of caspase inhibitors. Surprisingly, the re-expression of a RIP3 mutant with an inactive kinase domain (RIP3-KD) induced significantly more and earlier apoptosis than wild-type RIP3 (RIP3 WT), indicating that the RIP3 kinase domain is an essential regulator of apoptosis/necroptosis in leukemia cells. The induced in vivo expression of RIP3-KD, but not RIP3 WT prolonged the survival of mice injected with leukemia cells. RIP3-KD-induced cell death but not RIP3 WT was significantly antagonized by an IKKβSSEE constitutively active mutant, showing that RIP3-KD-induced apoptosis, but not RIP3 WT-induced apoptosis, was dependent on NF-κB activity. The expression of RIP3-KD induced p65/RelA NF-κB subunit caspase-dependent cleavage, and a non-cleavable p65/RelA D361E mutant rescued cells from apoptosis. The protective effect of the p65/RelA D361E mutant against apoptosis was specific to RIP3-KD-induced cell death because no change in cell death was observed when apoptosis was instead induced by treatment with imatinib or DMSO. The p65/RelA D361E mutant was generated by mutating the INFD putative consensus recognition site for caspase-6. The caspase-6 inhibitor Z-VEID-fmk partially reduced the cell death induced by RIP3-KD and slightly reduced p65/RelA cleavage. p65/RelA cleavage appears to be at least partially mediated by caspase-6. Conclusions. These data indicate that RIP3 silencing in leukemia cells results in suppression of the complex regulation of the apoptosis/necroptosis switch and the modulation of the NF-κB pathway through the caspase-mediated cleavage of p65/RelA. Citation Format: Anne-Lucie Nugues, Hassiba Bouafia, Dominique Hetuin, Celine Berthon, Anne Loyens, Elisabeth Bertrand, Nathalie Jouy, Thierry Idziorek, Bruno Quesnel. RIP3 is downregulated in human myeloid leukemia cells and modulates apoptosis and caspase-mediated p65/RelA cleavage. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1342. doi:10.1158/1538-7445.AM2014-1342
There is a therapeutic need for glucocorticoid receptor (GR) ligands that distinguish between the transrepression and transactivation activity of the GR, the later thought to be responsible for side effects. These ligands are known as "dissociated glucocorticoids" (dGCs). The first published dGCs, RU24782 (9α-fluoro-11β-hydroxy-16α-methylpregna-21-thiomethyl-1,4-diene-3,20-dione) and RU24858 (9α-fluoro-11β-hydroxy-16α-methylpregna-21-cyanide-1,4-diene-3,20-dione), do not have the 17α-hydroxyl group that characterizes dexamethasone (Dex; 9α-fluoro-11β,17α,21-trihydroxy-16α-methylpregna-1,4-diene-3,20-dione), and they differ from one another by having C21-thiomethyl and C21-cyanide moieties, respectively. Our aim was therefore to establish the structural basis of their activity. Both RU24782 and RU24858 induced a transactivation activity highly dependent on the GR expression level but always lower than dexamethasone. They also display less ability than dexamethasone to trigger steroid receptor coactivator 1 (SRC-1) recruitment and histone H3 acetylation. Docking studies, validated by mutagenesis experiments, revealed that dGCs are not anchored by Gln642, in contrast to Dex, which is hydrogen bonded to this residue via its 17α-hydroxyl group. This contact is essential for SRC-1 recruitment and subsequent dexamethasone-induced GR transactivation, but not transrepression. The ability of dGCs to make contacts with Ile747, for both RU24858 and RU24782 and with Asn564 for RU24858 are not strong enough to maintain GR in a conformation able to efficiently recruit SRC-1, unless SRC-1 is overexpressed. Overall, our findings provide some structural guidelines for the synthesis of potential new dissociated glucocorticoids with a better therapeutic ratio.
We previously demonstrated that long term treatment of the Ag-specific CD4+ T cell clone P28D with soluble HIV envelope glycoprotein gp120 results in a marked impairment of CD3/TCR-mediated responses. In this report, to further understand these inhibitory effects, the binding properties and internalization of gp120 have been investigated, in parallel with functional studies, in long term incubations of P28D cells with gp120. Immunofluorescence studies show that surface-bound gp120 level is maximal within 1 h of incubation at 37 degrees C and then gradually decreases. This decrease is accompanied by a progressive down-modulation of membrane CD4 (30-35% loss over a 18-h incubation period) without concomitant alteration of the CD4 mRNA steady-state level. Similar experiments performed with 125I-labeled gp120 demonstrate that the glycoprotein is progressively internalized (up to 35% internalized material after 18 h) and that it accumulates inside the cells. Confocal microscopy studies show that internalized gp120 is concentrated in localized intracellular compartments. CD4 also accumulates in compartments with a similar localization and is stained with mAb OKT4 but not with mAb OKT4a. Concomitantly to internalization of gp120 and disappearance of membrane CD4, a correlated loss of the CD4-associated tyrosine kinase p56lck is evidenced. Interestingly, a progressive impairment of the P28D responses to specific Ag or to anti-CD3 mAb is also observed. Inhibitions of T cell proliferation increase with the degree of both CD4 and p56lck down-modulation. Removal of exogenous gp120 results in a rapid and spontaneous release of internalized gp120 into a degraded form. A progressive restoration of CD4 and p56lck levels is also noticed. In parallel, CD3/TCR-mediated responses of clone P28D are fully recovered. Altogether, our results suggest that HIV-1 glycoprotein gp120 is able to down-modulate membrane CD4 presumably by a cointernalization process and to further down-modulate the associated p56lck. This dual phenomenon is presumably involved in the direct immunosuppressive effect of gp120 on the CD3/TCR-mediated activation pathway.
We have previously reported the detection of anti-CD4 autoantibodies in the sera of approximately 10% of HIV-1-infected patients. These antibodies recognized epitopes located on the V3V4 domains of the soluble recombinant CD4 molecule but did not recognize CD4 in its native conformation on the cell membrane. To analyse further the biological properties of these antibodies, we have purified them from the sera of six patients using affinity chromatography. We demonstrate that purified and concentrated anti-CD4 antibodies still cannot recognize CD4 either in its native conformation or after the binding of HIV gp120. They also cannot mediate antibody-dependent cellular cytotoxicity on CD4+ or HIV-coated CD4+ cells. In one patient, however, we could detect a minor fraction of anti-CD4 antibodies directed to the V1V2 domains of CD4.
Ferula elaeochytris Korovin (Apiaceae) is a species found in Turkey and Syria. A previous phytochemical work led to the isolation of several compounds [1], among which ferutinin was shown later to have antiproliferative and cytotoxic properties, on estrogene-dependent [2] or independent [3,4] tumor cell lines. We undertook a new phytochemical investigation of a root methanolic extract that lead to the isolation of three new compounds: two sesquiterpene esters, elaeochytrin A (6-anthraniloyljaeschkeanadiol) and elaeochytrin B (4β-hydroxy-6α-(p-hydroxybenzoyloxy)dauc-9-ene) and a saponin, ferulasaponin (3-O-[β-D-glucopyranosyl-(1→2)-β-D-glucopyranuronyl-(1→2)-β-D-glucopyranosyl]-28-O-glucopyranosyl oleanolic acid).
