No abstract

The activity of NF-B/Rel nuclear factors is known to inhibit apoptosis in various cell types. We investigated whether the subtraction of NF-B/Rel activity influenced the response of 11 AML (M1, M2 and M4) patients' cells to AraC. To this end we used a phosphorothioate double-stranded decoy oligodeoxynucleotide (ODN) carrying the NF-B/Rel− consensus sequence. Cell incubation with this ODN, but not its mutated (scrambled) form used as a control, resulted in abating the NF-B/Rel nuclear levels in these cells, as verified by electrophoretic mobility shift assay (EMSA) of cells' nuclear extracts. We incubated the leukemic cells with AraC (32 or 1 M), in either the absence or presence of the decoy Keywords: decoy oligodeoxynucleotides; NF-B/Rel; human AML; apoptosis; chemotherapy The NF-B/Rel transcription factors 1 are dimers of proteins (p50/p105 or NF-B1, p52/p100 or NF-B2, p65 or RelA, c-Rel and RelB) containing an approximately 300 amino acid REL homology region. In cell cytoplasm, the NF-B/Rel complexes are retained by inhibitors of the IB (␣-⑀) family; cytokines, hormones and other stimuli can induce the phosphorylation and ubiquitin-mediated degradation of the IB proteins, allowing the NF-B/Rel dimers to reach the nucleus (reviewed in Ref. 1). In some cell types, including B cells, thymocytes and neurons, appreciable levels of NF-B/Rel complexes can be detected also in nuclei.1,2 Besides regulating a series of genes involved in inflammatory processes or cell adhesion, 1,2 NF-B/Rel activities (specifically, p65-or cRel-containing dimers) can also inhibit apoptosis. [3][4][5][6][7][8][9][10][11][12][13] Indeed, in a number of lines of different origins, cell transfection with constructs expressing a 'superrepressor' IB␣ (ie, mutated in the phosphorylation sites and hence resistant to degradation), [3][4][5] or the infection with a superrepressor IB␣-carrying adenovirus, 9,11 result in enhancing the apoptosis induced by TNF-␣, ionizing radiations or the scrambled ODN, and analyzed cell apoptosis. The spontaneous cell apoptosis detectable in the absence of AraC (Ͻ25%) was not modulated by the oligonucleotide presence in cell cultures. On the other hand, in 10 of the 11 samples tested, the decoy B, but not the scrambled ODN significantly (P Ͻ 0.01 in a Student's t test) enhanced cell apoptotic response to AraC. Such an effect was particularly remarkable at low AraC doses (1 M). These findings indicate that NF-B/Rel activity influences response to AraC in human primary myeloblastic cells, and suggests that the inhibition of NF-B/Rel factors can improve the effect of chemotherapy in AML. Gene Therapy (2000) 7, 1234-1237. or chemotherapeutic agents. On the other hand, the overexpression of p65 or c-Rel can protect the cells from apoptosis.3,10 The inhibition of apoptosis appears to involve more than one NF-B/Rel-regulated gene, including IAP caspase inhibitors, 14,15 the Bcl2-homolog Bfl-1/A1 12,13 and possibly others. 8,16,17 The apoptosis-controlling activity of NF-B/Rel factors supports anti-tumor strategies bas...

We analyzed the effect of CD40 triggering on the fludarabine-induced apoptosis of B chronic lymphocytic leukemia (B-CLL) cells. Peripheral blood samples obtained from 15 patients were incubated with fludarabine in the absence or the presence of the anti-CD40 monoclonal antibody (MoAb) G28-5. In 12 patients a significant proportion of apoptotic cells, ranging from 22% to 38% (mean ± SE: 28.5 ± 1.6), were detected after 3 days of culture. In 9 of these samples, the addition of G28-5 reduced apoptosis by at least 30.1% and by 57.1% ± 7.8% on average (P = .0077). Because the CD40 antigen activates NF-κB/Rel transcription factors in B cells, and NF-κB/Rel complexes can inhibit cell apoptosis, we investigated whether the antiapoptotic effect of G28-5, in our system, could be related to modulation of NF-κB/Rel activity. As expected, B-CLL cells displayed significant levels of nuclear NF-κB/Rel activity; p50, RelA, and c-Rel components of the NF-κB/Rel protein family were identified in these complexes. After exposure to fludarabine, NF-κB/Rel complexes were decreased in the nuclei. The addition of G28-5 upregulated the NF-κB/Rel levels. To determine the involvement of NF-κB/Rel activity in the G28-5–mediated inhibition of apoptosis, we blocked the transcription factor with a decoy oligonucleotide, corresponding to the NF-κB/Rel consensus sequence. Cells incubated with the anti-CD40 MoAb in the presence of the decoy oligonucleotide but not a control oligonucleotide displayed a complete impairment of the G28-5 antiapoptotic effect, indicating that NF-κB/Rel activity was required for the inhibition of apoptosis. These results suggest that CD40 triggering in vivo could counteract the apoptotic effect of fludarabine on B-CLL cells and that its neutralization, or the use of NF-κB/Rel inhibitors, could improve the therapeutic effect of fludarabine. © 1998 by The American Society of Hematology.

In the present study we investigated whether apoptosis and phagocytosis are regulated by nuclear factor (NF)-kappaB in a model of chronic inflammation. The subcutaneous implant of lambda-carrageenin-soaked sponges elicited an inflammatory response, characterized by a time-related increase of leukocyte infiltration into the sponge and tissue formation, which was inhibited by simultaneous injection of wild-type oligodeoxynucleotide decoy to NF-kappaB. Molecular and morphological analysis performed on infiltrated cells demonstrated: 1) an inhibition of NF-kappaB/DNA binding activity; 2) an increase of polymorphonuclear leukocyte apoptosis correlated either to an increase of p53 or Bax and decrease of Bcl-2 protein expression; and 3) an increase of phagocytosis of apoptotic polymorphonuclear leukocytes by macrophages associated with an increase of transforming growth factor-beta1 and decrease of tumor necrosis factor-alpha as well as nitrite/nitrate production. Our results, showing that blockade of NF-kappaB by oligodeoxynucleotide decoy increases inflammatory cell apoptosis and phagocytosis, may contribute to lead to new insights into the mechanisms governing the inflammatory process.

BAG3 protein has emerged as a key regulator of important cellular processes and its expression is increased in some tumor types; however, despite its potential value for future chemotherapeutics, no selective BAG3 modulators have been yet reported. Here we report the 2,4-thiazolidinedione derivative 28 as the first BAG3 protein modulator.