Differential, Tissue-specific, Transcriptional Regulation of Apolipoprotein B Secretion by Transforming Growth Factor β

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Antimyeloma Effects of the Heat Shock Protein 70 Molecular Chaperone Inhibitor MAL3-101

Multiple myeloma (MM) is the second most common hematologic malignancy and remains incurable, primarily due to the treatment-refractory/resistant nature of the disease. A rational approach to this compelling challenge is to develop new drugs that act synergistically with existing effective agents. This approach will reduce drug concentrations, avoid treatment resistance, and also improve treatment effectiveness by targeting new and nonredundant pathways in MM. Toward this goal, we examined the antimyeloma effects of MAL3-101, a member of a new class of non-ATP-site inhibitors of the heat shock protein (Hsp) 70 molecular chaperone. We discovered that MAL3-101 exhibited antimyeloma effects on MM cell lines in vitro and in vivo in a xenograft plasmacytoma model, as well as on primary tumor cells and bone marrow endothelial cells from myeloma patients. In combination with a proteasome inhibitor, MAL3-101 significantly potentiated the in vitro and in vivo antimyeloma effects. These data support a preclinical rationale for small molecule inhibition of Hsp70 function, either alone or in combination with other agents, as an effective therapeutic strategy for MM

Endothelial progenitor cells display clonal restriction in multiple myeloma

BACKGROUND: In multiple myeloma (MM), increased neoangiogenesis contributes to tumor growth and disease progression. Increased levels of endothelial progenitor cells (EPCs) contribute to neoangiogenesis in MM, and, importantly, covary with disease activity and response to treatment. In order to understand the mechanisms responsible for increased EPC levels and neoangiogenic function in MM, we investigated whether these cells were clonal by determining X-chromosome inactivation (XCI) patterns in female patients by a human androgen receptor assay (HUMARA). In addition, EPCs and bone marrow cells were studied for the presence of clonotypic immunoglobulin heavy-chain (IGH) gene rearrangement, which indicates clonality in B cells; thus, its presence in EPCs would indicate a close genetic link between tumor cells in MM and endothelial cells that provide tumor neovascularization. METHODS: A total of twenty-three consecutive patients who had not received chemotherapy were studied. Screening in 18 patients found that 11 displayed allelic AR in peripheral blood mononuclear cells, and these patients were further studied for XCI patterns in EPCs and hair root cells by HUMARA. In 2 patients whose EPCs were clonal by HUMARA, and in an additional 5 new patients, EPCs were studied for IGH gene rearrangement using PCR with family-specific primers for IGH variable genes (V(H)). RESULTS: In 11 patients, analysis of EPCs by HUMARA revealed significant skewing (≥ 77% expression of a single allele) in 64% (n = 7). In 4 of these patients, XCI skewing was extreme (≥ 90% expression of a single allele). In contrast, XCI in hair root cells was random. Furthermore, PCR amplification with V(H )primers resulted in amplification of the same product in EPCs and bone marrow cells in 71% (n = 5) of 7 patients, while no IGH rearrangement was found in EPCs from healthy controls. In addition, in patients with XCI skewing in EPCs, advanced age was associated with poorer clinical status, unlike patients whose EPCs had random XCI. CONCLUSION: Our results suggest that EPCs in at least a substantial subpopulation of MM patients are related to the neoplastic clone and that this is an important mechanism for upregulation of tumor neovascularization in MM

Chalazia Development in Multiple Myeloma: A New Complication Associated with Bortezomib Therapy

Multiple myeloma (MM) is a neoplasm of plasma cells within the bone marrow. A major impact on improving survival in MM has been the use of the boronic acid-derived proteasome inhibitor bortezomib, a first-in-class selective inhibitor of the 26S proteasome. Ocular side effects of bortezomib are rare. In this report, we present 2 patients with active MM in whom persistent chalazia became a therapy-interfering complication of treatment with bortezomib. Both patients had relapsed ISS III B kappa light chain myeloma, and they were responding to treatment with bortezomib until chalazia−which caused intolerable discomfort−started. In both patients discontinuation of bortezomib was necessary for chalazia to heal, and restarting of bortezomib was associated with relapse of chalazia

Response to hypoxia involves smad proteins in human endothelial cells

Smad proteins are the downstream substrates of membrane ser/thr kinase TGF-β receptors, and mediate responses of endothelial cells (EC) to mechanical and metabolic stress. Oxygen deprivation (Hypoxia: Hx) is a consistent component of ischémie vascular disorders and induces an inflammatory response in vascular endothelium. This study, performed in human umbilical vein endothelial cells (HUVEC), examined the effect of Hx on activity of Smads and their target gene TGF-β2, a cytokine regulator of inflammation in EC. RNase protection studies showed that exposure to Hx (1% O2) increased mRNA levels of TGF-β2 by 10-fold in a time-dependent fashion (P \u3c .01). Parallel increases in active and latent TGF-β2 protein were found by measuring the response of a TGF-βresponsive luciferase construct in a bioassay. Hx stimulated TGF-β2 transcription in HUVEC, determined by activity of a TGF-β2 CAT promoter construct, by 3-fold; Hxinduced transcription was increased by a further 4-fold (P \u3c .01) after cotransfection of HUVEC with Smad 3/4 expression vectors. Hx also increased transcription from a known TGF-β-responsive promoter, 3TP-lux, by 10-fold (P \u3c .01); cotransfection of Smad 3/4 vectors increased the activity of 3TP-lux by a further 3-fold ( P \u3c .01). Smad association with DNA was shown with EMSA using a Smad-binding oligonucleotide, SEE, as probe. Binding to SBE occurred only with nuclear extracts from hypoxic but not normoxic HUVEC, was not competed by oligonucleotides corresponding to DNA-binding sites of SP1 or HIF-1, and was supershifted with antibody to Smad 3 and 4 but not to HIF-1 or preimmune sera. To further examine the effects of Hx on EC signal transduction and function, total RNA obtained from HUVEC exposed to Hx for increasing time periods was used for mRNA transcript profiling by cDNA arrays (Clonetech, Atlas 1.2). Results were normalized to β-actin and show that mRNA of TGF-β l, -β2, and their upstream regulators such as TGF-β RI and Ang II Rl were induced at 18h after Hx compared to normoxia. Furthermore, Hx induced mRNAs of Smad co-factors SP1 and -2, which cooperate to inhibit Gl cell cycle progression. mRNA levels of Smads, Jak-Stat proteins, or NF-KB were unchanged, but HIF-1 mRNA was increased. In sum, Hx-induced increases in TGF-β2, TGF-β RI, and Smad 3/4 function in EC suggest that this pathway plays an important role in endothelial response to hypoxic stress

Adult glucocorticoid receptor mRNA expression volatility in response to an acute stressor and juvenile CSF corticotropin-releasing factor: A pilot neurodevelopmental study

Early life stress (ELS) has been shown to play a role in establishing persistent maladaptive HPA axis modifications that may contribute to the pathogenesis of mood and anxiety disorders. Central glucocorticoid receptor (GR) messenger RNA (mRNA) expression may facilitate (mal)adaptive responsivity to ELS. The role of adult monocytic GR mRNA expression, a putative CNS proxy, during acute stress exposure was explored as well as the ELS marker, juvenile cerebrospinal fluid (CSF) corticotropin-releasing factor. Six adult macaques (three of which were exposed to variable foraging demand, a form of ELS) underwent acute restraint. Baseline GR expression and plasma cortisol concentrations were separately measured followed by subsequent measurements following stress completion (t=0min, 4h, 5 days and 7 days). Juvenile CSF CRF concentrations were available in five subjects to determine their developmental association with GR expression in response to stress. As expected acute restraint stress produced a significant increase in plasma cortisol concentrations most robustly observed at 4h post-stress time point. There was a significant juvenile CSF CRF concentration x time interaction in predicting adult GR mRNA expression in response to stress (partial η =0.80). During acute stress juvenile CRF concentrations negatively predicted GR expression and during recovery, "flipped" to positively predict expression. Juvenile CSF CRF concentrations positively correlated with the volatility of adult GR mRNA expression. During acute stress, relatively high CSF CRF concentrations are associated with relatively rapid reductions in GR expression. Return to an ambient post-stress state was characterized by a direct relationship, consistent with increased HPA axis restraint in high CRF subjects. An ELS-associated allostatic adaptation suggests relative elevations of juvenile CSF CRF concentration set the stage for a relative hyper-volatility of adult GR mRNA expression in response to acute stress with potential long-term implications for HPA axis regulation