Arnaldo A. Arbini

Tumor Necrosis Factor-α Increases Circulating Osteoclast Precursor Numbers by Promoting Their Proliferation and Differentiation in the Bone Marrow through Up-regulation of c-Fms Expression

Osteoclasts are essential cells for bone erosion in inflammatory arthritis and are derived from cells in the myeloid lineage. Recently, we reported that tumor necrosis factor-α (TNFα) increases the blood osteoclast precursor (OCP) numbers in arthritic patients and animals, which are reduced by anti-TNF therapy, implying that circulating OCPs may have an important role in the pathogenesis of erosive arthritis. The aim of this study is to investigate the mechanism by which TNFα induces this increase in OCP frequency. We found that TNFα stimulated cell division and conversion of CD11b+/Gr-1-/lo/c-Fms- to CD11b+/Gr-1-/lo/c-Fms+ cells, which was not blocked by neutralizing macrophage colony-stimulating factor (M-CSF) antibody. Ex vivo analysis of monocytes demonstrated the following: (i) blood CD11b+/Gr-1-/lo but not CD11b-/Gr-1- cells give rise to osteoclasts when they were cultured with receptor activator NF-κB ligand and M-CSF; and (ii) TNF-transgenic mice have a significant increase in blood CD11b+/Gr-1-/lo cells and bone marrow proliferating CD11b+/Gr-1-/lo cells. Administration of TNFα to wild type mice induced bone marrow CD11b+/Gr-1-/lo cell proliferation, which was associated with an increase in CD11b+/Gr-1-/lo OCPs in the circulation. Thus, TNFα directly stimulates bone marrow OCP genesis by enhancing c-Fms expression. This results in progenitor cell proliferation and differentiation in response to M-CSF, leading to an enlargement of the marrow OCP pool. Increased marrow OCPs subsequently egress to the circulation, forming a basis for elevated OCP frequency. Therefore, the first step of TNF-induced osteoclastogenesis is at the level of OCP genesis in the bone marrow, which represents another layer of regulation to control erosive disease.

Mitochondrial DNA depletion reduces PARP-1 levels and promotes progression of the neoplastic phenotype in prostate carcinoma

Mitochondrial dysfunction resulting from mitochondrial DNA (mtDNA) mutations and/or depletion has been correlated with cancer progression and drug resistance. To investigate the role of mtDNA in prostate cancer progression, we used LNCaP and PC-3 prostate carcinoma cells as experimental model. Compared to minimally invasive androgen-dependent LNCaP cells, highly invasive androgen-independent PC-3 cells, as well as androgen-independent DU145 and C4-2 cells, exhibited significantly reduced mtDNA content. In PC-3 cells, reduction of mtDNA was accompanied by decreased mitochondrial membrane potential (ΔΨm), increased migration onto the basement membrane protein laminin-1, reduced chemosensitivity to paclitaxel (IC50=110 nM vs. 22 nM) and decreased expression of poly(ADP-ribose) polymerase (PARP)-1. To investigate the relationship between mtDNA depletion and these phenotypic characteristics, we established mtDNA-depleted LNCaP cells [Rho(−)] by long-term exposure to ethidium bromide or treated wild-type LNCaP cells with a mitochondrial ionophore, carbonyl cyanide m-chlorophenylhydrazone. Both manipulations resulted in ΔΨm loss, acquisition of invasive cytology, increased motility onto laminin-1, reduced sensitivity to paclitaxel (IC50=~100 nM) and ~75% reduction in PARP-1 protein levels, resembling PC-3 cells. Overall, these results provide novel evidence demonstrating that mtDNA depletion in early prostate carcinoma may contribute to the acquisition of a more invasive phenotype that is less sensitive to paclitaxel-induced apoptosis.

Multiparameter Flow Cytometric Analysis Reveals Low Percentage of Bone Marrow Hematogones in Myelodysplastic Syndromes

Diagnosis of myelodysplastic syndromes (MDS) could be difficult. We explored the usefulness of the enumeration of maturing B-lineage precursors (hematogones) by multiparameter flow cytometric analysis in the diagnosis of MDS in bone marrow (BM) specimens. We evaluated 111 MDS, 120 non-MDS (most with cytopenias; control group 1), and 41 noncytopenic lymphoma staging BM (control group 2) specimens. The percentage of total hematogones was significantly lower in MDS (median, 0%; mean, 0.10%) compared with non-MDS (control group 1, median, 0.38%, and mean, 0.91%; control group 2, median, 0.38%, and mean, 0.60%; P < .0001), as was the percentage of the most immature (stage I) hematogones. Thus, hematogone enumeration may serve as a biomarker to aid in the diagnosis of MDS. Interestingly, the percentage of hematogones was not significantly different between MDS subgroups or patients with MDS with and without chromosomal abnormalities, implying that a defect in maturing B-cell precursors may be an early event in the pathogenesis of MDS.

Segmental Tandem Triplication of the MLL Gene in an Intravascular Large B-Cell Lymphoma With Multisystem Involvement: A Comprehensive Morphologic, Immunophenotypic, Cytogenetic, and Molecular Cytogenetic Antemortem Study

An association between intravascular large B-cell lymphoma (IVLBCL) and the mixed lineage leukemia (MLL) gene has never been demonstrated. Here, we report an IVLBCL in a 47-year-old Asian man. Morphologically, the atypical lymphoid infiltrate was entirely confined in the lumina of capillaries, small vessels, and sinusoidal space. Within the kidney, the neoplastic lymphoid cells exhibited both the glomerular and peritubular capillary distribution pattern. Conventional cytogenetic analysis from the bone marrow aspirates displayed a complex karyotype, with a notable triple tandem repeat at band segment q22-q25 of chromosome 11. Fluorescence in situ hybridization with an MLL probe set, performed on both interphase cells and metaphase spreads, confirmed the presence of 3 copies of the MLL gene on the derivative chromosome 11. From this finding and 3 other IVLBCL cases reported in the literature, we conclude that MLL may play an important role in the lymphomagenesis of IVLBCL at least in a subset of cases.

Unusual presentation of myeloid sarcoma in a case of acute promyelocytic leukemia with a cryptic PML–RARA rearrangement involving multiple sites including the atrium

Myeloid sarcoma is an extramedullary tumor mass composed of immature myeloid cells. Myeloid sarcoma may develop de novo, concurrently with acute myeloid leukemia (AML), or at relapse. Although myeloid sarcoma can occur at any site, myeloid sarcoma involving the heart is extremely rare. Reported here is the case of a 30-year-old man, initially diagnosed with acute promyelocytic leukemia (APL) in bone marrow, who presented later with myeloid sarcoma at multiple anatomical sites (left scapula, thoracic vertebra, right atrium, and supraclavicular mass) in multiple relapses. Conventional cytogenetic studies performed on the atrial sample revealed a karyotype with additional material on the short arm of chromosome 7, at 7p22. Fluorescence in situ hybridization studies confirmed a cryptic PML-RARA fusion on the short arm of chromosome 7, as well as a second fusion on one copy of chromosome 15. With the fourth and latest relapse, molecular cytogenetic studies performed on interphase nuclei of the myeloid sarcoma specimen (a supraclavicular mass) showed evidence of six related abnormal clones with a PML-RARA fusion, suggesting clonal evolution. This represents a rare case of APL with a cryptic PML-RARA rearrangement presenting as myeloid sarcoma at multiple relapses and involving multiple anatomical sites, including cardiac atrium.

Aromatase Deficiency Inhibits the Permeability Transition in Mouse Liver Mitochondria

Lack of estrogens affects male physiology in a number of ways, including severe changes in liver metabolism that result in lipid accumulation and massive hepatic steatosis. Here we investigated whether estrogen deficiency may alter the functionality and permeability properties of liver mitochondria using, as an experimental model, aromatase knockout (ArKO) male mice, which cannot synthesize endogenous estrogens due to a disruption of the Cyp19 gene. Liver mitochondria isolated from ArKO mice displayed increased activity of the mitochondrial respiratory complex IV compared with wild-type mice and were less prone to undergo cyclosporin A-sensitive mitochondrial permeability transition (MPT) induced by calcium loading. The altered permeability properties of the mitochondrial membranes were not due to changes in reactive oxygen species, ATP levels, or mitochondrial membrane potential but were associated with increased content of the phospholipid cardiolipin, structural component of the mitochondrial membranes and regulator of the MPT pore, and with increased mitochondrial protein levels of Bcl-2 and the adenine nucleotide translocator (ANT), regulator and component of the MPT pore, respectively. Real-time RT-PCR demonstrated increased mRNA levels for Bcl-2 and ANT2 but not for the ANT1 isoform in ArKO livers. Supplementation of 17beta-estradiol retrieved ArKO mice from massive hepatic steatosis and restored mitochondrial permeability properties, cardiolipin, Bcl-2, and ANT2 levels. Overall, our findings demonstrate an important role of estrogens in the modulation of hepatic mitochondrial function and permeability properties in males and suggest that estrogen deficiency may represent a novel positive regulator of Bcl-2 and ANT2 proteins, two inhibitors of MPT occurrence and powerful antiapoptotic molecules.