Arturo Leone

Regulation by heavy metals and temperature of the human BAG‐3 gene, a modulator of Hsp70 activity

In HeLa cells the expression of the BAG‐3 gene, a member of the BAG family, is regulated by heavy metals and temperature, with kinetics of accumulation of mRNAs similar to Hsp70 and metallothioneins. Western blot assays performed with a polyclonal anti‐BAG‐3 antibody confirmed that higher levels of the protein were present in the cells following heat and metal exposure. By immunofluorescence techniques and cell fractionation assays we demonstrated that following stress BAG‐3 protein concentrated in the rough endoplasmic reticulum and associated with the heavy membrane fraction. The role of BAG‐3 protein during apoptosis and cellular stress is discussed.

IKKγ protein is a target of BAG3 regulatory activity in human tumor growth

BAG3, a member of the BAG family of heat shock protein (HSP) 70 cochaperones, is expressed in response to stressful stimuli in a number of normal cell types and constitutively in a variety of tumors, including pancreas carcinomas, lymphocytic and myeloblastic leukemias, and thyroid carcinomas. Down-regulation of BAG3 results in cell death, but the underlying molecular mechanisms are still elusive. Here, we investigated the molecular mechanism of BAG3-dependent survival in human osteosarcoma (SAOS-2) and melanoma (M14) cells. We show that bag3 overexpression in tumors promotes survival through the NF-κB pathway. Indeed, we demonstrate that BAG3 alters the interaction between HSP70 and IKKγ, increasing availability of IKKγ and protecting it from proteasome-dependent degradation; this, in turn, results in increased NF-κB activity and survival. These results identify bag3 as a potential target for anticancer therapies in those tumors in which this gene is constitutively expressed. As a proof of principle, we show that treatment of a mouse xenograft tumor model with bag3siRNA-adenovirus that down-regulates bag3 results in reduced tumor growth and increased animal survival.

BAG3 protein regulates stress- induced apoptosis in normal and neoplastic leukocytes

We express our sincere thanks to Dr Junjiro Tsuchiyama for the NK-YS cell line and all medical and nursing staff in the Department of Medicine, Queen Mary Hospital for the provision of expert medical care. CS Chim KY Wong F Loong G Srivastava University Department of Medicine, Queen Mary Hospital, The University of Hong Kong, Hong Kong; and Department of Pathology, Queen Mary Hospital, The University of Hong Kong, Hong Kong

Evidence for BAG3 modulation of HIV-1 gene transcription†

A family of co‐chaperone proteins that share the Bcl‐2‐associated athanogene (BAG) domain are involved in a number of cellular processes, including proliferation and apoptosis. Among these proteins, BAG3 has received increased attention due to its high levels in several disease models and ability to associate with Hsp70 and a number of other molecular partners. BAG3 expression is stimulated during cell response to stressful conditions, such as exposure to high temperature, heavy metals, and certain drugs. Here, we demonstrate that BAG3 expression is elevated upon HIV‐1 infection of human lymphocytes and fetal microglial cells. Furthermore, BAG3 protein was detectable in the cytoplasm of reactive astrocytes in HIV‐1‐associated encephalopathy biopsies, suggesting that induction of BAG3 is part of the host cell response to viral infection. To assess the impact of BAG3 upregulation on HIV‐1 gene expression, we performed transcription assays and demonstrated that BAG3 can suppress transcription of the HIV‐1 long terminal repeat (LTR) in microglial cells. This activity was mapped to the κB motif of the HIV‐1 LTR. Results from in vitro and in vivo binding assays revealed that BAG3 suppresses interaction of the p65 subunit of NF‐κB with the κB DNA motif of the LTR. Results from binding and transcriptional assay identified the C‐terminus of BAG3 as a potential domain involved in the observed inhibitory effect of BAG3 on p65 activity. These observations reveal a previously unrecognized cell response, that is, an increase in BAG3, elicited by HIV‐1 infection, and may provide a new avenue for the suppression of HIV‐1 gene expression. J. Cell. Physiol. 210: 676–683, 2007.

BAG3 protein regulates cell survival in childhood acute lymphoblastic leukemia cells.

No abstract available.

The Anti–Human Leukocyte Antigen-DR Monoclonal Antibody 1D09C3 Activates the Mitochondrial Cell Death Pathway and Exerts a Potent Antitumor Activity in Lymphoma-Bearing Nonobese Diabetic/Severe Combined Immunodeficient Mice

The fully human anti-HLA-DR antibody 1D09C3 has been shown to delay lymphoma cell growth in severe combined immunodeficient (SCID) mice. The present study was aimed at (a) investigating the mechanism(s) of 1D09C3-induced cell death and (b) further exploring the therapeutic efficacy of 1D09C3 in nonobese diabetic (NOD)/SCID mice. The chronic lymphocytic leukemia cell line JVM-2 and the mantle cell lymphoma cell line GRANTA-519 were used. Generation of reactive oxygen species (ROS) and mitochondrial membrane depolarization were measured by flow cytometry following cell incubation with dihydroethidium and TMRE, respectively. Western blot analysis was used to detect c-Jun-NH(2)-kinase (JNK) phosphorylation and apoptosis-inducing factor (AIF). NOD/SCID mice were used to investigate the activity of 1D09C3 in early- or advanced-stage tumor xenografts. In vitro, 1D09C3-induced cell death involves a cascade of events, including ROS increase, JNK activation, mitochondrial membrane depolarization, and AIF release from mitochondria. Inhibition of JNK activity significantly reduced 1D09C3-induced apoptosis, indicating that 1D09C3 activity involves activation of the kinase. In vivo, 1D09C3 induces long-term disease-free survival in a significant proportion of tumor-bearing mice treated at an early stage of disease. Treatment of mice bearing advanced-stage lymphoma results in a highly significant prolongation of survival. These data show that 1D09C3 (a) exerts a potent antitumor effect by activating ROS-dependent, JNK-driven cell death, (b) cures the great majority of mice treated at an early-stage of disease, and (c) significantly prolongs survival of mice with advanced-stage disease.

Quassinoids can induce mitochondrial membrane depolarisation and caspase 3 activation in human cells.

Quassinoids can induce mitochondrial membrane depolarisation and caspase 3 activation in human cells

1-Methoxy-canthin-6-one induces c-Jun NH2-terminal kinase-dependent apoptosis and synergizes with tumor necrosis factor-related apoptosis-inducing ligand activity in human neoplastic cells of hematopoietic or endodermal origin.

We investigated the effects of 1-methoxy-canthin-6-one, isolated from the medicinal plant Ailanthus altissima Swingle, on apoptosis in human leukemia (Jurkat), thyroid carcinoma (ARO and NPA), and hepatocellular carcinoma (HuH7) cell lines. Cultures incubated with the compound showed >50% of sub-G1 (hypodiploid) elements in flow cytometry analysis; the apoptosis-inducing activity was evident at <10 micromol/L and half-maximal at about 40 micromol/L 1-methoxy-canthin-6-one. The appearance of hypodiploid elements was preceded by mitochondrial membrane depolarization, mitochondrial release of cytochrome c, and Smac/DIABLO and procaspase-3 cleavage. We subsequently investigated the effect of 1-methoxy-canthin-6-one in combination with human recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in the four cell lines. Suboptimal concentrations (10 micromol/L 1-methoxy-canthin-6-one and 0.25 ng/mL TRAIL, respectively) of the two agents, unable to elicit apoptosis when used alone, induced mitochondrial depolarization, activation of caspase-3, and 45% to 85% of sub-G1 elements when added together to the cells. The synergism seemed to rely partly on the enhanced expression of TRAIL receptor 1 (TRAIL-R1; DR4), analyzed by immunofluorescence, by 1-methoxy-canthin-6-one. Cell incubation with 1-methoxy-canthin-6-one resulted in activating c-Jun NH2-terminal kinase (JNK), as revealed by Western blotting; induction of apoptosis and TRAIL-R1 up-regulation by 1-methoxy-canthin-6-one were >80% prevented by the addition of the JNK inhibitor (JNKI) SP600125JNKI, indicating that both effects were almost completely mediated by JNK activity. On the other hand, synergism with TRAIL was reduced by about 50%, suggesting that besides up-regulating TRAIL-R1, 1-methoxy-canthin-6-one could influence other factor(s) that participated in TRAIL-induced apoptosis. These findings indicate that 1-methoxy-canthin-6-one can represent a candidate for in vivo studies of monotherapies or combined antineoplastic therapies.

Regulation of ZiRF1 and basal SP1 transcription factor MRE-binding activity by transition metals.

The metal‐dependent activation of metallothionein (MT) genes requires the interaction of positive trans‐activators (MRFs) with metal‐regulatory (MRE) regions of MT promoters. In this report, we examined the role of transition metals in modulating the MRE‐binding activities of two different MRE‐binding proteins: the metal‐regulated factor ZiRF1 and the basal factor SP1. We showed the ability of both proteins to interact with a similar sequence specificity with the cognate target site (MRE‐S) of another known MRE‐binding protein, mMTF1. We next evaluated the role of metal ions in modulating the MRE‐binding activity of recombinant ZiRF1 and basal SP1 proteins by measuring the effect of different metal chelators on DNA interaction. We observed a dose‐dependent inhibition of the GST‐ZiRF1/MRE‐binding activity using three different metal chelators: EDTA, 1,10 PHE and TPEN. Interestingly, EDTA treatment failed to inhibit the recombinant SP1 MRE‐binding activity while the effect of 1,10 PHE was comparable to that obtained analyzing 1,10 PHE‐treated GST‐ZiRF1. The MRE‐binding complexes detected in cell extracts showed a response to metal chelator treatment very similar to that displayed by the recombinant ZiRF1 and SP1 proteins. The hypothesis of mutual interactions of both basal and metal‐regulated transcription factors with the same metal‐regulatory regions is discussed.

Copper-binding activity of Trefoil factor 1 (TFF1): a new perspective in the study of the multifunctional roles of TFFs.

Trefoil factors (TFFs) are gastrointestinal peptides playing an essential role in the epithelial restitution. Among the three known TFF peptides, TFF1 is characterized by three disulfide bonds producing a compact globular structure and an extended and disordered tail formed by amino- and carboxy-termini. The presence of a cysteine surrounded by several negatively charged residues in this region of the protein, highly conserved in different species, suggests the possible formation of a metal-binding site. Affinity chromatography and mass spectrometric analyses allowed us to demonstrate a selective binding affinity of TFF1 for copper. The binding induces conformational changes in the tertiary structure as demonstrated by circular dichroism experiments, while limited proteolysis revealed an altered access to the cleavage sites in the amino- and carboxy-termini. The results of this study reveal a new property of TFF1 and suggest that copper could influence its biological activities by interfering with the dimerization of the peptide and/or the interaction with mucins or putative TFF receptors.