Gain-of-Function STIM1 L96V Mutation Causes Myogenesis Alteration in Muscle Cells From a Patient Affected by Tubular Aggregate Myopathy.

Tubular Aggregate Myopathy (TAM) is a hereditary ultra-rare muscle disorder characterized by muscle weakness and cramps or myasthenic features. Biopsies from TAM patients show the presence of tubular aggregates originated from sarcoplasmic reticulum due to altered Ca2+ homeostasis. TAM is caused by gain-of-function mutations in STIM1 or ORAI1, proteins responsible for Store-Operated-Calcium-Entry (SOCE), a pivotal mechanism in Ca2+ signaling. So far there is no cure for TAM and the mechanisms through which STIM1 or ORAI1 gene mutation lead to muscle dysfunction remain to be clarified. It has been established that post-natal myogenesis critically relies on Ca2+ influx through SOCE. To explore how Ca2+ homeostasis dysregulation associated with TAM impacts on muscle differentiation cascade, we here performed a functional characterization of myoblasts and myotubes deriving from patients carrying STIM1 L96V mutation by using fura-2 cytofluorimetry, high content imaging and real-time PCR. We demonstrated a higher resting Ca2+ concentration and an increased SOCE in STIM1 mutant compared with control, together with a compensatory down-regulation of genes involved in Ca2+ handling (RyR1, Atp2a1, Trpc1). Differentiating STIM1 L96V myoblasts persisted in a mononuclear state and the fewer multinucleated myotubes had distinct morphology and geometry of mitochondrial network compared to controls, indicating a defect in the late differentiation phase. The alteration in myogenic pathway was confirmed by gene expression analysis regarding early (Myf5, Mef2D) and late (DMD, Tnnt3) differentiation markers together with mitochondrial markers (IDH3A, OGDH). We provided evidences of mechanisms responsible for a defective myogenesis associated to TAM mutant and validated a reliable cellular model usefull for TAM preclinical studies.

Cyclooxygenase-1 (COX-1) and COX-1 Inhibitors in Cancer: A Review of Oncology and Medicinal Chemistry Literature.

Prostaglandins and thromboxane are lipid signaling molecules deriving from arachidonic acid by the action of the cyclooxygenase isoenzymes COX-1 and COX-2. The role of cyclooxygenases (particularly COX-2) and prostaglandins (particularly PGE2) in cancer-related inflammation has been extensively investigated. In contrast, COX-1 has received less attention, although its expression increases in several human cancers and a pathogenetic role emerges from experimental models. COX-1 and COX-2 isoforms seem to operate in a coordinate manner in cancer pathophysiology, especially in the tumorigenesis process. However, in some cases, exemplified by the serous ovarian carcinoma, COX-1 plays a pivotal role, suggesting that other histopathological and molecular subtypes of cancer disease could share this feature. Importantly, the analysis of functional implications of COX-1-signaling, as well as of pharmacological action of COX-1-selective inhibitors, should not be restricted to the COX pathway and to the effects of prostaglandins already known for their ability of affecting the tumor phenotype. A knowledge-based choice of the most appropriate tumor cell models, and a major effort in investigating the COX-1 issue in the more general context of arachidonic acid metabolic network by using the systems biology approaches, should be strongly encouraged.

Cell Cycle Regulation by Ca2+-Activated K⁺ (BK) Channels Modulators in SH-SY5Y Neuroblastoma Cells.

The effects of Ca2+-activated K⁺ (BK) channel modulation by Paxilline (PAX) (10-7⁻10-4 M), Iberiotoxin (IbTX) (0.1⁻1 × 10-6 M) and Resveratrol (RESV) (1⁻2 × 10-4 M) on cell cycle and proliferation, AKT1pSer473 phosphorylation, cell diameter, and BK currents were investigated in SH-SY5Y cells using Operetta-high-content-Imaging-System, ELISA-assay, impedentiometric counting method and patch-clamp technique, respectively. IbTX (4 × 10-7 M), PAX (5 × 10-5 M) and RESV (10-4 M) caused a maximal decrease of the outward K⁺ current at +30 mV (Vm) of -38.3 ± 10%, -31.9 ± 9% and -43 ± 8%, respectively, which was not reversible following washout and cell depolarization. After 6h of incubation, the drugs concentration dependently reduced proliferation. A maximal reduction of cell proliferation, respectively of -60 ± 8% for RESV (2 × 10-4 M) (IC50 = 1.50 × 10-4 M), -65 ± 6% for IbTX (10-6 M) (IC50 = 5 × 10-7 M), -97 ± 6% for PAX (1 × 10-4 M) (IC50 = 1.06 × 10-5 M) and AKT1pser473 dephosphorylation was observed. PAX induced a G1/G2 accumulation and contraction of the S-phase, reducing the nuclear area and cell diameter. IbTX induced G1 contraction and G2 accumulation reducing diameter. RESV induced G2 accumulation and S contraction reducing diameter. These drugs share common actions leading to a block of the surface membrane BK channels with cell depolarization and calcium influx, AKT1pser473 dephosphorylation by calcium-dependent phosphatase, accumulation in the G2 phase, and a reduction of diameter and proliferation. In addition, the PAX action against nuclear membrane BK channels potentiates its antiproliferative effects with early apoptosis.

Effect of mofezolac-galactose distance in conjugates targeting cyclooxygenase (COX)-1 and CNS GLUT-1 carrier.

Neuroinflammation is the earliest stage of several neurological and neurodegenerative diseases. In the case of neurodegenerative disorders, it takes place about 15-20 years before the appearance of specific neurodegenerative clinical symptoms. Constitutive microglial COX-1 is one of the pro-inflammatory players of the neuroinflammation. Novel compounds 3, 14 and 15 (Galmof0, Galmof5 and Galmof11, respectively) were projected, and their synthetic methodologies developed, by linking by an ester bond, directly or through a C5 or C11 unit linker the highly selective COX-1 inhibitor mofezolac (COXs selectivity index > 6000) to galactose in order to obtain substances capable to cross blood-brain barrier (BBB) and control the CNS inflammatory response. 3, 14 and 15 (Galmofs) were prepared in good to fair yields. Galmof0 (3) was found to be a selective COX-1 inhibitor (COX-1 IC50 = 0.27 µM and COX-2 IC50 = 3.1 µM, selectivity index = 11.5), chemically and metabolically stable, and capable to cross Caco-2 cell monolayer, resembling BBB, probing that its transport is GLUT-1-mediated. Furthermore, Galmof0 (3) powerfully inhibits PGE2 release higher than mofezolac (1) in LPS-stimulated mouse BV2 microglial cell line, a worldwide recognized neuroinflammation model. In addition, Fingerprints for Ligands and Proteins (FLAP) was used to explain the different binding interactions of Galmofs with the COX-1 active site.

Investigating Structural Requirements for the Antiproliferative Activity of Biphenyl Nicotinamides.

A number of trimethoxybenzoic acid anilides, previously studied as permeability glycoprotein (P-gp) modulators, were screened with the aim of identifying new anticancer agents. One of these compounds, which showed antiproliferative activity against resistant MCF-7 cell line, was selected as the hit structure. Replacement of the trimethoxybenzoyl moiety with a nicotinoyl group, in order to overcome solubility issues, led to a new series of N-biphenyl nicotinoyl anilides, among which a nitro derivative, N-(3',5'-difluoro-3-nitro-[1,1'-biphenyl]-4-yl)nicotinamide (3), displayed antiproliferative activity against MCF-7 and MDA-MB-231 cells in the nanomolar range. The search for a bioisostere of the nitro group led to nitrile analogue N-(3-cyano-4'-fluoro-[1,1'-biphenyl]-4-yl)nicotinamide (36), which shows a strong increase in activity against MCF-7 and MDA-MB-231 cells. Compound 36 induced a dose-dependent accumulation of G2 - and M-phase MCF-7 cell populations, and a decrease in S-phase cells. Relative to vinblastine, a well-known potent antimitotic agent, compound 36 also induced G1 -phase arrest at low doses (20-40 nm), but did not inhibit in vitro tubulin polymerization.

Effect of cisplatin containing liposomes formulated by unsaturated chain-containing lipids on gynecological tumor cells.

Gynecological tumors are major therapeutic areas of platinum-based anticancer drugs. Here, we report the characterization and in vitro biological assays of cisplatin-containing Egg L-α-phosphatidylcholine liposomes with different amounts of cholesterol. Dynamic light scattering estimated sizes of all obtained liposomes in the 100 nm range that are suitable for in vivo use. On the basis of these data and of the drug loading values, the best formulation has been selected. Stability and drug release properties of platinum-containing liposomes have been verified in serum. The growth inhibitory effects of both liposomal and free drug in a panel of ovarian and breast human cancer cell lines, characterized by a different drug sensitivity, give comparable or better results with respect to free cisplatin drug.

The biological characteristics of CD34+ CD2+ adult acute promyelocytic leukemia and the CD34 CD2 hypergranular (M3) and microgranular (M3v) phenotypes.

BACKGROUND AND OBJECTIVES: Acute promyelocytic leukemia (APL) is characterized by leukemic cells blocked at the promyelocytic stage of granulocytic differentiation. To date, it is still not clear whether CD34 expression identifies a subset of APL patients with peculiar characteristics. We, therefore, conducted a detailed analysis of CD34 expression at diagnosis in 136 adults with de novo APL. DESIGN AND METHODS: We investigated 136 newly diagnosed APL patients from four Italian Institutions. All 136 cases were tested for CD34 and CD2 expression: 124 (91%) cases were classified as hypergranular (M3) and 12 (9%) as the hyporgranular M3 variant (M3v). The parameters considered were white blood cell (WBC) and platelet counts, hemoglobin levels, percentage of peripheral blood leukemic promyelocytes (PBLP), CD15, CD56 and HLA-DR expression, and the PML/RARalpha isoform, to assess their relationship with CD34 and CD2 expression. RESULTS: CD34 expression was associated with the M3v subtype and higher proportion of HLA-DR+ and CD2+ cases. Moreover, compared with CD34- APL patients, CD34+ APL patients had a significantly higher percentage of PBLP at presentation, were more frequently female and had a higher proportion of bcr3 expression. Among the 136 APL cases, 24 (17.6%) and 80 (58.8%) were identified as CD34+CD2+ and CD34-CD2-, respectively. The two groups showed statistically significant differences in terms of M3vfrequency, WBC and platelet counts, percentage of PBLP, and bcr3 expression. Moreover, the CD34+CD2+ group showed a higher proportion of CD34+ and bcr3 isoforms compared to the M3v cases. There were no differences between the two groups in terms of complete remission, overall survival and disease-free survival. INTERPRETATION AND CONCLUSIONS: Our findings suggest that immunophenotypic analysis can distinguish a subset of APL patients with different biological characteristics.

Molecular cytogenetic study of instability at 1q21 approximately q32 in adult acute lymphoblastic leukemia.

In the present paper, we report a molecular cytogenetic study of 1q abnormalities associated with t(8;14)(q24;q32) in an adult common B acute lymphoblastic leukemia case with FAB-L2 morphology. The use of appropriate molecular cytogenetic probes allowed us to detect 13 different subclones showing heterogeneous chromosome 1 abnormalities. A complex pattern of rearrangements consisting of translocations, duplications, and inversions was observed. Breakage-fusion-bridge cycle and jumping translocation are hypothesized to have been involved in generating the large number of aberrations we detected.

Molecular detection of minimal residual disease is associated with early relapse in adult acute lymphoblastic leukemia.

Several studies in childhood acute lymphoblastic leukemia (ALL) have documented that molecular detection of minimal residual disease (MRD) based on screening for T-cell receptor and immunoglobulin gene rearrangements can identify patients at a high risk of relapse. In our experience, evaluation of MRD in adult ALL can help to identify high risk patients.

Derivative chromosome 9 deletions in chronic myeloid leukemia are associated with loss of tumor suppressor genes.

It has recently been postulated that the absence of a single tumor suppressor gene (TSG) allele can provide a selective advantage for an emerging tumor cell. We have characterized the precise extension of the deletion on der(9) in 20 chronic myeloid leukemia (CML) cases using FISH analysis with an appropriate set of BAC/PAC probes to attempt a better definition of TSGs encompassed by these genomic deletions. Chromosome 9 deletions on the der(9) were detected in 15 (75%) cases; the TSG PTGES gene was lost in 11 (73%) cases. Chromosome 22 deletions on der(9) were found in 18 (90%) of the analysed cases; two TSGs were found located inside the deleted sequences of chromosome 22: SMARCB1 and GSTT1. These TSGs were found deleted in 16 (89%) cases bearing deletions of chromosome 22. Fourteen (70%) patients were treated with IFN-alpha therapy: 12 did not obtain complete haematologic remission (CHR) and 2 were not evaluable for response. Therefore, the patients did not respond to the IFN-alpha treatment started Glivec obtaining CHR and major cytogenetic response (MCR). The observation that deletions on der(9) are associated with the loss of TSGs suggests their possible involvement in the CML outcome, mediated by a haplo-insufficiency mechanism.

Non-treatment-related chronic myeloid leukemia as a second malignancy.

The characteristics of the very rare non-treatment-related chronic myeloid leukemia (nTr-CML) cases have never before been analyzed. The literature up to December 2002 was screened using the Medline database to identify cases of Tr-CML and nTr-CML. We considered five cases with nTr-CML identified among 270 newly diagnosed CML at our Department. Our report thus considers nine cases with nTr-CML compared to 77 affected by Tr-CML as a secondary neoplasm. The median age at the appearance of the first tumor was higher in nTr-CML patients compared to that of the Tr-CML group (P<0.0001). The median age at CML diagnosis was significantly higher in the nTr-CML than in the Tr-CML group (P<0.0001). The proportion of hematological malignancies as first tumor type was not different in the two groups (44% in nTr-CML versus 56% in Tr-CML). Our study underlines that nTr-CML as a second malignancy is a rare entity associated with elderly age.

Concomitant primary polycythemia vera and follicle center cell non-Hodgkin lymphoma: a case report and review of the literature.

The association of myeloproliferative and lymphoproliferative disorders is well known after cytotoxic drug or radiation exposure, while it is remarkably rare prior to therapy. We report on a patient simultaneously diagnosed as having polycythemia vera and II3A follicle center cell non-Hodgkin lymphoma (grade 1). At this timepoint, he is on 12-year follow-up, characterized by post-polycythemia myeloid metaplasia with myelofibrosis and persistent complete remission of lymphoma. The conventional marrow cytogenetic analysis performed during the course of the disease demonstrated an abnormal karyotype with deletion of the long arm of chromosome 20 and trisomy 8, while molecular analysis failed to detect BCR-ABL rearrangement in peripheral blood cells. To the best of our knowledge based on a computer-aided review of the literature (MED-LINE 1966-2002), this is the sixth case of concomitant primary polycythemia vera and lymphoma of non-Hodgkin type. Besides, there is a single literature report on polycythemia vera coexisting with the Hodgkin's lymphoma. In our case as well as in the recorded ones, two independent malignant clones of myeloid and lymphoid origin, respectively, seem to have arisen. Further reports, supported by chromosomal and molecular studies, could improve our knowledge on this extremely infrequent disease association.