Roberta Morosetti

The recovery of platelet cyclooxygenase activity explains interindividual variability in responsiveness to low-dose aspirin in patients with and without diabetes

See also Lordkipanidze M, Harrison P. Aspirin twice a day keeps new COX‐1 at bay. This issue, pp 1217–9.

Therapy related leukemias: susceptibility, prevention and treatment.

Acute leukemia is the most frequent therapy-related malignancy. Together with the increasing use of chemo- and radiotherapy, individual predisposing factors play a key role. Most of secondary leukemias can be divided in two well-defined groups: those secondary to the use of alkylating agents and those associated to topoisomerase inhibitors. Leukemias induced by alkylating agents usually follow a long period of latency from the primary tumour and present as myelodysplasia with unbalanced chromosomal aberrations. These frequently include deletions of chromosome 13 and loss of the entire or of part of chomosomes 5 or 7. The loss of the coding regions for tumor suppressor genes from hematopoietic progenitor cells is a particularly unfavourable event, since the remaining allele becomes susceptible to inactivating mutations leading to the leukemic transformation. The tumorigenic action of topoisomerase inhibitors is on the other hand due to the formation of multiple DNA strand breaks, resolved by chromosomal translocations. Among these, chromosome 11, band q23, where the myeloid-lymphoid leukemia (MLL) gene is located, is often involved. Frequent partners are chromosomes 9, 19 and 4 in the t(9;11), t(19;11) and t(4;11) translocations. Younger age, a mean period of latency of 2 years and monocytic subtypes are characteristic features of this type of leukemia. Among patients at risk for secondary leukemia, those with Hodgkins disease are the most extensively studied, with the major impact of alkylating agents included in the chemotherapy schedule. The same is true for non-Hodgkins lymphoma, while in multiple myeloma and acute lymphoblastic leukemia determinants are the dose of melphalan and of epypodophyllotoxin, respectively. Patients with breast, ovarian and testicular neoplasms are also at risk, in particular if trated with the association of alkylating agents and topoisomerase II inhibitors. According to the EBMT registry, in patients with lymphoma treated with high-dose therapy and autologous stem cell transplantation the cumulative risk of inducing leukemia at 5 years is 2.6%. Among treatment options, supportive therapy is indicated in older patients, while allogeneic stem cell transplantation, related or matched-unrelated, is feasible in younger patients. These data indicate the need for the identification of predisposing factors for secondary leukemia. In particular, frequent follow-up of patients at high-risk should be performed and any peripheral blood cytopenia should be considered suspicious. Whenever possible, the exclusion of drugs known to be leukemogenic from the treatment schedules should be considered, especially in young patients.

MyoD expression restores defective myogenic differentiation of human mesoangioblasts from inclusion-body myositis muscle.

Inflammatory myopathies (IM) are acquired diseases of skeletal muscle comprising dermatomyositis (DM), polymyositis (PM), and inclusion-body myositis (IBM). Immunosuppressive therapies, usually beneficial for DM and PM, are poorly effective in IBM. We report the isolation and characterization of mesoangioblasts, vessel-associated stem cells, from diagnostic muscle biopsies of IM. The number of cells isolated, proliferation rate and lifespan, markers expression, and ability to differentiate into smooth muscle do not differ among normal and IM mesoangioblasts. At variance with normal, DM and PM mesoangioblasts, cells isolated from IBM, fail to differentiate into skeletal myotubes. These data correlate with lack in connective tissue of IBM muscle of alkaline phosphatase (ALP)-positive cells, conversely dramatically increased in PM and DM. A myogenic inhibitory basic helix–loop–helix factor B3 is highly expressed in IBM mesoangioblasts. Indeed, silencing this gene or overexpressing MyoD rescues the myogenic defect of IBM mesoangioblasts, opening novel cell-based therapeutic strategies for this crippling disorder.

NCAM is hyposialylated in hereditary inclusion body myopathy due to GNE mutations

The authors found that the neural cell adhesion molecule (NCAM) is hyposialylated in hereditary inclusion body myopathy (HIBM) muscle, as suggested by its decreased molecular weight by Western blot. This abnormality represented the only pathologic feature differentiating HIBM due to GNE mutations from other myopathies with similar clinical and pathologic characteristics. If further confirmed in larger series of patients, this may be a useful diagnostic marker of GNE-related HIBM.

The ER-bound RING finger protein 5 (RNF5/RMA1) causes degenerative myopathy in transgenic mice and is deregulated in inclusion body myositis.

Growing evidence supports the importance of ubiquitin ligases in the pathogenesis of muscular disorders, although underlying mechanisms remain largely elusive. Here we show that the expression of RNF5 (aka RMA1), an ER-anchored RING finger E3 ligase implicated in muscle organization and in recognition and processing of malfolded proteins, is elevated and mislocalized to cytoplasmic aggregates in biopsies from patients suffering from sporadic-Inclusion Body Myositis (sIBM). Consistent with these findings, an animal model for hereditary IBM (hIBM), but not their control littermates, revealed deregulated expression of RNF5. Further studies for the role of RNF5 in the pathogenesis of s-IBM and more generally in muscle physiology were performed using RNF5 transgenic and KO animals. Transgenic mice carrying inducible expression of RNF5, under control of β-actin or muscle specific promoter, exhibit an early onset of muscle wasting, muscle degeneration and extensive fiber regeneration. Prolonged expression of RNF5 in the muscle also results in the formation of fibers containing congophilic material, blue-rimmed vacuoles and inclusion bodies. These phenotypes were associated with altered expression and activity of ER chaperones, characteristic of myodegenerative diseases such as s-IBM. Conversely, muscle regeneration and induction of ER stress markers were delayed in RNF5 KO mice subjected to cardiotoxin treatment. While supporting a role for RNF5 Tg mice as model for s-IBM, our study also establishes the importance of RNF5 in muscle physiology and its deregulation in ER stress associated muscular disorders.

Hyposialylation of neprilysin possibly affects its expression and enzymatic activity in hereditary inclusion-body myopathy muscle.

Autosomal recessive hereditary inclusion‐body myopathy (h‐IBM) is caused by mutations of the UDP‐N‐acetylglucosamine 2‐epimerase/N‐acetylmannosamine kinase gene, a rate‐limiting enzyme in the sialic acid metabolic pathway. Previous studies have demonstrated an abnormal sialylation of glycoproteins in h‐IBM. h‐IBM muscle shows the abnormal accumulation of proteins including amyloid‐β (Aβ). Neprilysin (NEP), a metallopeptidase that cleaves Aβ, is characterized by the presence of several N‐glycosylation sites, and changes in these sugar moieties affect its stability and enzymatic activity. In the present study, we found that NEP is hyposialylated and its expression and enzymatic activity reduced in all h‐IBM muscles analyzed. In vitro, the experimental removal of sialic acid by Vibrio Cholerae neuraminidase in cultured myotubes resulted in reduced expression of NEP. This was most likely because of a post‐translational modification consisting in an abnormal sialylation of the protein that leads to its reduced stability. Moreover, treatment with Vibrio Cholerae neuraminidase was associated with an increased immunoreactivity for Aβ mainly in the form of distinct cytoplasmic foci within myotubes. We hypothesize that, in h‐IBM muscle, hyposialylated NEP has a role in hampering the cellular Aβ clearing system, thus contributing to its abnormal accumulation within vulnerable fibers and possibly promoting muscle degeneration.

Expression of cyclin-dependent kinase inhibitor p15INK4B during normal and leukemic myeloid differentiation

OBJECTIVE Expression of the cyclin-dependent kinase inhibitor p15(INK4B) frequently is altered in myeloid malignancies. We previously demonstrated that p15(INK4B) is expressed in normal myeloid cells. The aim of this study was to investigate whether p15(INK4B) expression is restricted to the granulomonocytic lineage and to evaluate its modulation during normal and leukemic myeloid differentiation. MATERIALS AND METHODS Normal CD34(+) cells were cultured in serum-free media to obtain granulomonocytic, erythroid, or megakaryocytic unilineage differentiation. NB4 promyelocytic cell line and fresh leukemic blasts from seven patients with acute promyelocytic leukemia were cultured with all-trans retinoic acid. At different times of culture, cell samples were collected to evaluate p15(INK4B) by semiquantitative reverse transcriptase polymerase chain reaction. RESULTS p15(INK4B) mRNA was found during granulomonocytic and megakaryocytic, but not erythroid, differentiation. In the granulomonocytic lineage, p15(INK4B) was detectable when the majority of cells were at the promyelocytic stage and increased progressively in more mature elements. In the megakaryocytic lineage, p15(INK4B) was expressed in the early phase of differentiation, before megakaryoblasts had appeared, and was mantained throughout the time of culture. NB4 cell line and five of seven leukemic samples displayed undetectable or very low level of p15(INK4B) that rapidly increased during retinoic acid-induced differentiation. Two leukemic samples (both collected from two patients developing all-trans retinoic acid syndrome) showed high basal levels of p15(INK4B), which was not modified by retinoic acid treatment. CONCLUSIONS p15(INK4B) upregulation occurs specifically during normal granulomonocytic and megakaryocytic commitment. In acute promyelocytic leukemic blasts, p15(INK4B), which is detectable at a very low level, is promptly increased by retinoic acid. In contrast, two acute promyelocytic leukemia samples obtained from patients who developed all-trans retinoic acid syndrome showed high basal levels of p15(INK4B) that did not increase further during all-trans retinoic acid-induced differentiation.

α-Dystroglycan does not play a major pathogenic role in autosomal recessive hereditary inclusion-body myopathy

Mutations of the GNE gene are responsible for autosomal recessive hereditary inclusion-body myopathy (HIBM). In this study we searched for the presence of any significant abnormality of alpha-dystroglycan (alpha-DG), a highly glycosylated component of the dystrophin-glycoprotein complex, in 5 HIBM patients which were previously clinically and genetically characterized. Immunocytochemical and immunoblot analysis showed that alpha-DG extracted from muscle biopsies was normally expressed and displayed its typical molecular mass. Immunoblot analysis on the wheat germ lectin-enriched glycoprotein fraction of muscles and primary myotubes showed a reduced amount of alpha-DG in 4 out of 5 HIBM patients, compared to normal and other diseased muscles. However, such altered lectin-binding behaviour, possibly reflecting a partial hyposialylation of alpha-DG, did not affect the laminin binding properties of alpha-DG. Therefore, the subtle changes within the alpha-DG glycosylation pattern, detected in HIBM muscles, likely do not play a key pathogenic role in this disorder.

Neprilysin participates in skeletal muscle regeneration and is accumulated in abnormal muscle fibres of inclusion body myositis.

Neprilysin (NEP, EP24.11), a metallopeptidase originally shown to modulate signalling events by degrading small regulatory peptides, is also an amyloid‐β‐ (Aβ) degrading enzyme. We investigated a possible role of NEP in inclusion body myositis (IBM) and other acquired and hereditary muscle disorders and found that in all myopathies NEP expression was directly associated with the degree of muscle fibre regeneration. In IBM muscle, NEP protein was also strongly accumulated in Aβ‐bearing abnormal fibres. In vitro, during the experimental differentiation of myoblasts, NEP protein expression was regulated at the post‐transcriptional level with a rapid increase in the early stage of myoblast differentiation followed by a gradual reduction thereafter, coincident with the progression of the myogenic programme. Treatment of differentiating muscle cells with the NEP inhibitor dl‐3‐mercapto‐2‐benzylpropanoylglycine resulted in impaired differentiation that was mainly associated with an abnormal regulation of Akt activation. Therefore, NEP may play an important role during muscle cell differentiation, possibly through the regulation, either directly or indirectly, of the insulin‐like growth factor I‐driven myogenic programme. In IBM muscle increased NEP may be instrumental in (i) reducing the Aβ accumulation in vulnerable fibres and (ii) promoting a repair/regenerative attempt of muscle fibres possibly through the modulation of insulin‐like growth factor I‐dependent pathways.

The cellular response to PPARγ ligands is related to the phenotype of neuroblastoma cell lines

Neuroblastoma (NB) is a phenotypically heterogeneous tumor, displaying cells of neuronal, melanocytic, or glial/schwannian lineage. This cellular heterogeneity is also present in vitro, where cells of neuroblastic (N)- or stromal (S)-type may be identified. Ligands of peroxisome proliferator-activated receptor gamma (PPARgamma) have been shown to inhibit growth in different tumor cell lines. The purpose of this study was to determine PPARgamma expression and the response to its ligands in NB cell lines with different phenotypes. We used eight NB cell lines with N-, mixed, and S-phenotype. PPARgamma expression was found in all NB cell lines, regardless of their phenotype. Mutational analysis and transactivation assays showed that PPARgamma is not mutated and remains functional in NB cells. Two PPARgamma ligands, 15-deoxy-delta12,14-prostaglandin J2 (PGJ2) and rosiglitazone, inhibited growth of all cell lines, with PGJ, being the most potent agent. PGJ2, but not rosiglitazone, induced arrest of the cells in the G2/M phase as well as apoptosis. The sensitivity to the two ligands appeared to be more related to the phenotype than PPARgamma expression, with the S-type cells being less sensitive than the N-type, partly because of their lower capability of undergoing apoptosis. No synergistic effect on growth inhibition was observed when all cell lines were co-treated with 9-cis retinoic acid (9-cis RA) and rosiglitazone. Our data indicate that PPARgamma expression and function are maintained in phenotipically different NB cell lines. Activation of PPARgamma by its synthetic ligands might have a therapeutic role in advanced NB.