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Inositide signaling pathways can have a role in the Myelodysplastic Syndromes (MDS) progression to acute myeloid leukemia. Erythropoietin (EPO) is currently used in low-risk MDS, where it successfully corrects anemia in 50-70% of patients. However, some MDS patients are refractory to this treatment and little is known about the exact molecular mechanisms underlying the effect of EPO in these subjects. Here, we investigated the role of inositide pathways in low-risk MDS treated with EPO, mainly focusing on the Akt/PI-PLC (Phosphoinositide-Phospholipase C) gamma1 axis, which is activated by the EPO receptor, and PI-PLCbeta1/Cyclin D3 signaling, as Cyclin D3 is associated with hematopoietic proliferation and differentiation. Interestingly, EPO responder patients showed a specific activation of both the Akt/PI-PLCgamma1 pathway and beta-Globin gene expression, while nonresponders displayed an increase in PI-PLCbeta1 signaling. Moreover, in normal CD34+ cells induced to erythroid differentiation, PI-PLCbeta1 overexpression abrogated both EPO-induced Akt phosphorylation and beta-Globin expression. Overall, these findings suggest that PI-PLCbeta1 can act as a negative regulator of erythroid differentiation and confirm the involvement of Akt/PI-PLCgamma1 pathway in EPO signaling, therefore contributing to the comprehension of the effect of EPO in low-risk MDS and possibly paving the way to the identification of MDS patients at higher risk of refractoriness to EPO treatment. [ABSTRACT FROM AUTHOR]

Phosphoinositide-phospholipase C (PI-PLC) beta1 can be considered a specific target for demethylating therapy in high-risk myelodysplastic syndrome (MDS) patients, as azacitidine treatment has been associated with a PI-PLCbeta1-specific promoter demethylation, and induction of PI-PLCbeta1 gene and protein expression. However, little is known about the molecular effect of azacitidine in low-risk MDS or the functional mechanisms linked with azacitidine effect on PI-PLCbeta1 promoter. In the present study, we further investigated the role of epigenetic regulation of PI-PLCbeta1, mainly focusing on the structure of the PI-PLCbeta1 promoter. We first examined the effect of azacitidine on PI-PLCbeta1 promoter methylation and gene expression in low-risk MDS. Moreover, we studied the expression of key molecules associated with the nuclear inositide signaling pathways, such as cyclin D3. By applying a chromatin immunoprecipitation method, we also studied the correlation between the demethylating effect of azacitidine and the degree of recruitment to PI-PLCbeta1 promoter of some transcription factors implicated in hematopoietic stem cell proliferation and differentiation, as well as of the methyl-CpG-binding domain proteins, which specifically interact with methylated DNA. Taken together, our results hint at a specific involvement of PI-PLCbeta1 in epigenetic mechanisms, and are particularly consistent with the hypothesis of a role for PI-PLCbeta1 in azacitidine-induced myeloid differentiation. [ABSTRACT FROM AUTHOR]

Background: An association between oral contraceptive (OC) use and venous thromboembolism (VTE) has long been recognized. However, no summary estimates of the increase in VTE risk associated with OC use have been available since 1995, and no meta-analyses have evaluated the VTE risk of new preparations containing drospirenone.Objective: The aim of the study was to carry out a meta-analysis to summarize existing evidence on the association between venous VTE and OC use, and to investigate how such an association may vary according to the type of OC, OC user characteristics, study characteristics and biases.Methods: Relevant cohort or case-control studies were searched in MEDLINE and other electronic databases up to May 2010, with no language restriction. Data were combined using a generic inverse-variance approach. Meta-regression in addition to stratification was used to explore potential predictors of the summary estimate of risk.Results: Sixteen cohort and 39 case-control studies were included in at least one comparison. Overall, the odds ratio (OR) of OC users versus non-users was 3.41 (95% CI 2.98, 3.92). This estimate was based upon nine cohort studies evaluating approximately 12 000 000 person-years, and 23 case-control studies including approximately 45 000 women. VTE risk for OC users was significantly lower in studies evaluating 'all VTE cases' than in those evaluating 'idiopathic VTE only' (OR 3.09 and 4.94, respectively). Among the carriers of genetic mutations G20210A and Factor V Leiden (FVL), OC users showed a significantly increased VTE risk compared with non-users (OR 1.63; 95% CI 1.01, 2.65, and OR 1.80; 95% CI 1.20, 2.71, respectively). When the newest OCs containing drospirenone were compared with non-drospirenone-containing OCs (except those containing levonorgestrel only), VTE risk did not significantly increase (OR 1.13; 95% CI 0.94, 1.35).Conclusions: This meta-analysis confirms that OC use significantly increases VTE risk. The strength of this association, however, varies according to the generation of OC, type of outcome and presence of a genetic mutation, with ORs ranging from 3 to 5. [ABSTRACT FROM AUTHOR]

Myelodysplastic syndrome (MDS) is an adult hematological disease that evolves into acute myeloid leukemia (AML) in about 30% of the cases. The availability of a highly specific probe moved us to perform in patients affected with MDS/AML, associated with normal karyotype, painting and fluorescence in situ hybridization (FISH) analysis aimed to check the inositide-specific phospholipase C (PI-PLC) ß1 gene, a player in the control of some checkpoints of the cell cycle. Here we present a preliminary observation in which FISH analysis disclosed in a small group of MDS/AML patients with normal karyotype the monoallelic deletion of the PI-PLCß1 gene. On the contrary, PI-PLC ß4, another gene coding for a signaling molecule, located on 20p12.3 at a distance as far as less than 1Mb from PI-PLCß1, is unaffected in MDS patients with the deletion of PI-PLC ß1 gene, hinting at an interstitial deletion. The MDS patients, bearing the deletion, rapidly evolved to AML. The data suggest the possible involvement of PI-PLCß1 in the progression of the disease and pave the way for a larger investigation aimed at identifying a possible high-risk group among MDS patients with a normal karyotype.Leukemia (2004) 18, 1122-1126. doi:10.1038/sj.leu.2403368 Published online 15 April 2004 [ABSTRACT FROM AUTHOR]

Apart from the lipids present in the nuclear envelope, the nucleus also contains lipids which are located further inside and are resistant to treatment with nonionic detergents. Evidence is being accumulated on the importance of internal nuclear lipid metabolism. Nuclear lipid metabolism gives rise to several lipid second messengers that function within the nucleus. Moreover, it is beginning to emerge that nuclear lipids not only act as precursors of bioactive second messengers but may be directly involved in regulation of nuclear structure and gene expression. Over the last 10years, especially the role of the inositol lipid cycle in nuclear signal transduction has been extensively studied. This cycle is activated following a variety of stimuli and is regulated independently from the inositide cycle located at the plasma membrane. However, the nucleus contain other lipids, such as phosphatidylcholine, sphingomyelin, fatty acids and eicosanoids. There are numerous reports which suggest that these classes of nuclear lipids may play roles in the nucleus as important as those of phosphoinositides. This review aims at highlighting the most important aspects regarding the metabolism and signaling activities of nuclear phosphatidylcholine, sphingomyelin, fatty acids and eicosanoids. [ABSTRACT FROM AUTHOR]

It is now well established that the reduced capacity of tumor cells of undergoing cell death through apoptosis plays a key role both in the pathogenesis of cancer and in therapeutic treatment failure. Indeed, tumor cells frequently display multiple alterations in signal transduction pathways leading to either cell survival or apoptosis. In mammals, the pathway based on phosphoinositide 3-kinase (PI3K)/Akt conveys survival signals of extreme importance and its downregulation, by means of pharmacological inhibitors of PI3K, considerably lowers resistance to various types of therapy in solid tumors. We recently described an HL60 leukemia cell clone (HL60AR cells) with a constitutively active PI3K/Akt pathway. These cells were resistant to multiple chemotherapeutic drugs, all-trans-retinoic acid (ATRA), and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Treatment with two pharmacological inhibitors of PI3K, wortmannin and Ly294002, restored sensitivity of HL60AR cells to the aforementioned treatments. However, these inhibitors have some drawbacks that may severely limit or impede their clinical use. Here, we have tested whether or not a new selective Akt inhibitor, 1L-6-hydroxymethyl-chiro-inositol 2(R)-2-O-methyl-3-O-octadecylcarbonate (Akt inhibitor), was as effective as Ly294002 in lowering the sensitivity threshold of HL60 cells to chemotherapeutic drugs, TRAIL, ATRA, and ionizing radiation. Our findings demonstrate that, at a concentration which does not affect PI3K activity, the Akt inhibitor markedly reduced resistance of HL60AR cells to etoposide, cytarabine, TRAIL, ATRA, and ionizing radiation. This effect was likely achieved through downregulation of expression of antiapoptotic proteins such as c-IAP1, c-IAP2, cFLIPL, and of Bad phosphorylation on Ser 136. The Akt inhibitor did not influence PTEN activity. At variance with Ly294002, the Akt inhibitor did not negatively affect phosphorylation of protein kinase C-? and it was less effective in downregulating p70S6 kinase (p70S6K) activity. The Akt inhibitor increased sensitivity to apoptotic inducers of K562 and U937, but not of MOLT-4, leukemia cells. Overall, our results indicate that selective Akt pharmacological inhibitors might be used in the future for enhancing the sensitivity of leukemia cells to therapeutic treatments that induce apoptosis or for overcoming resistance to these treatments.Leukemia (2003) 17, 1794-1805. doi:10.1038/sj.leu.2403044 [ABSTRACT FROM AUTHOR]

TRAIL is a member of the tumor necrosis factor superfamily which induces apoptosis in cancer but not in normal cells. Akt1 promotes cell survival and blocks apoptosis. The scope of this paper was to investigate whether a HL60 human leukemia cell clone (named AR) with constitutively active Akt1 was resistant to TRAIL. We found that parental (PT) HL60 cells were very sensitive to a 6 h incubation in the presence of TRAIL and died by apoptosis. In contrast, AR cells were resistant to TRAIL concentrations as high as 2 microg/ml for 24 h. Two pharmacological inhibitors of PI3K, Ly294002 and wortmannin, restored TRAIL sensitivity of AR cells. AR cells stably overexpressing PTEN had lower Akt1 activity and were sensitive to TRAIL. Conversely, PT cells stably overexpressing a constitutive active form of Akt1 became TRAIL resistant. TRAIL activated caspase-8 but not caspase-9 or -10 in HL60 cells. We did not observe a protective effect of Bcl-X(L) or Bcl-2 against the cytotoxic activity of TRAIL, even though TRAIL induced cleavage of BID. There was a close correlation between TRAIL sensitivity and intranuclear presence of the p50 subunit of NF-kappaB. Higher levels of the FLICE inhibitory protein, cFLIP(L), were observed in TRAIL-resistant cells. Both the cell permeable NF-kappaB inhibitor SN50 and cycloheximide lowered cFLIP(L)expression and restored sentivity of AR cells to TRAIL. Our results suggest that Akt1 may be an important regulator of TRAIL sensitivity in HL60 cells through the activation of NF-kappaB and up-regulation of cFLIP(L) synthesis. [ABSTRACT FROM AUTHOR]

Several independent groups have shown that lipid-dependent signal transduction systems operate in the nucleus and that they are regulated independently from their membrane and cytosolic counterparts. A sizable body of evidence suggests that nuclear lipid signaling controls critical biological functions such as cell proliferation and differentiation. Diacylglycerol is a fundamental lipid second messenger which is produced in the nucleus. The levels of nuclear diacylglycerol fluctuate during the cell cycle progression, suggesting that such a molecule has important regulatory roles. Most likely, nuclear diacylglycerol serves as a chemoattractant for some isoforms of protein kinase C that migrate to the nucleus in response to a variety of agonists. The nucleus also contains diacylglycerol kinases, i.e. the enzymes that, by converting diacylglycerol into phosphatidic acid, terminate diacylglycerol-dependent events. A number of diacylglycerol kinases encoded by separate genes are present in the mammalian genome. This review aims at highlighting the different isotypes of diacylglycerol kinases identified at the nuclear level as well as at discussing their potential function and regulation. [ABSTRACT FROM AUTHOR]

HeLa metaphase chromosomes were exammed by means of 'in lens' field emission scanning electron microscopy, which permits high resolution detection of uncoated biological samples. By using uncoated chromosomes as a model for comparison we report evidence of how traditional scanning electron microscopy techniques such as metal coating and conductive methods can generate errors in chromosome structure evaluation, since both give rise to morphological artifacts. By comparing the morphology of uncoated chromosomes obtained by two different isolation procedures, such as that utilized in standard cytogenetics and the polyamine method, we have drawn the following conclusions: (a) the standard cytogenetic method gives rise to a chromosome structure consisting of a flattened network of 10 nm fibers, in which higher order chromatin organization is absent. (b) Chromosomes obtained by the polyamine method show both three-dimensional profile and higher level folding of chromatin fibers, supporting the loop chromosome organization previously suggested by scanning electron microscopy observation of hexylene glycol isolated chromosomes. [ABSTRACT FROM AUTHOR]

In order to investigate at ultrastructural level the mechanism of DNA synthesis progression during the different moments of S-phase a bromodeoxyuridine-anti bromodeoxyuridine (BrdU-anti BrdU) method has been applied to synchronized 3T3 fibroblasts. After 30 min BrdU incorporation, five different labelling patterns can be identified and should be related to early, middle and late S-phase. These patterns are represented mainly by diffuse labelling localized in different nuclear domains and by quite rare cases in which the labelling is limited to isolated clusters of gold particles. After a 5-min pulse with BrdU it is possible to observe isolated clusters of gold particles at each moment of S-phase, which, however, exhibit the same distribution of the five principal labelling patterns observed after 30 min incorporation. In both cases labelling can be detected in the interchromatin regions during early S-phase, at the boundary between interchromatin and heterochromatin during middle S-phase and in the heterochromatin domains during late S-phase. Considering their size, the isolated spots of labelling could be interpreted as single replication units which are subsequently activated throughout the different moments of the S-phase. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented about high risk myelodysplastic syndromes that is published within the issue.

In order to expand the present knowledge of the pathogenic potential ofBlastoschizomyces capitatusin central venous catheter (CVC)-related bloodstream infections, six strains of the organism recovered from three leukemic patients with CVC-related fungemia in different years were investigated. Isolates and control strains were tested for their genetic relatedness and for their ability to produce slime in glucose-containing solutions. DNA restriction enzyme analysis revealed that all strains ofB. capitatuswere identical, whereas slime production assays and examination of ex vivo material showed that they were able to produce large amounts of slime. Slime production may therefore play a relevant pathogenic role in cases of CVC-related fungemia caused byB. capitatus. [ABSTRACT FROM AUTHOR]

Dynamic cell cycle analysis is based on the incorporation of labelled precursors into DNA. Although antibodies to BrdU are very useful for analysing in flow cells which synthesize DNA, this approach has two main limitations. First, the detection of low incorporating cells is often difficult; second, four parameter flow cytometry is not able to correlate cell cycle to any other cellular marker. We have developed a methodology that, employing an IgG as a second antibody and side scatter instead of propidium iodide fluorescence, allows a better discrimination of BudR cells. This approach allows the collection of an extra-fluorescent signal, and the analysis of specific cellular markers within the cell cycle. [ABSTRACT FROM AUTHOR]

Background: Data on the potential efficacy of acupuncture (AC) in controlling intense or very intense pain in patients with Herpes Zoster (HZ) has not been so far adequately assessed in comparison with standard pharmacological treatment (ST) by a controlled trial design. Methods: Within the VZV Pescara study, pain was assessed in HZ patients on a Visual Analogue Scale (VAS) and by the McGill Pain Questionnaire (MPQ) both at the beginning and at the end of treatment. Response rates, mean changes in pain intensity, differences in total pain burden with an area-under-the-curve (AUC) method over a 1-year follow-up and differences in the incidence of Post-Herpetic Neuralgia (PHN) were evaluated. Results: One hundred and two patients were randomized to receive either AC (n = 52) or ST (n = 50) for 4 weeks. Groups were comparable regarding age, sex, pain intensity at presentation and missed antiviral prescription. Both interventions were largely effective. No significant differences were observed in response rates (81.6 % vs 89.2 %, p = 0.8), mean reduction of VAS (4.1 +/−2.3 vs 4.9 +/−1.9, p = 0.12) and MPQ scores (1.3 +/-0.9 vs 1.3 +/-0.9, p = 0.9), incidence of PHN after 3 months (48.4 % vs 46.8 %, p = 0.5), and mean AUC during follow-up (199 +/-136 vs 173 +/-141, p = 0.4). No serious treatment-related adverse event was observed in both groups. Conclusions: This controlled and randomized trial provides the first evidence of a potential role of AC for the treatment of acute herpetic pain Beschreibung: Es handelt sich bei der vorliegenden Arbeit um eine nicht-verblindete, randomisierte klinische Studie, welche den Einfluss der Akupunktur auf die Schmerzentwicklung bei einer Herpes-Zoster(HZ)-Infektion im Vergleich zu einer medikamentösen Standardtherapie (ST) untersucht []. Sie wurde als Teil einer Multicenterstudie zur Identifizierung von Risikofaktoren für eine post-herpetische Neuralgie durchgeführt []. Als Hauptzielparameter dieser Untersuchung wurde die Reduktion der Schmerzintensität (VAS) nach einer vierwöchigen Behandlungsphase definiert. Sekundäre Zielparameter waren die Responderrate (VAS-Score Reduktion während der Behandlung um mindestens zwei Punkte), die Reduktion der Schmerzintensität nach Behandlung gemessen anhand des McGill Schmerzfragebogens, die Inzidenz einer post-herpetischen Neuralgie (Schmerz im betroffenen Areal drei Monate nach Studieneinschluss), die gesamte Schmerzbelastung während eines einjährigen Follow-ups (Flächeninhalt unter der Kurve bei einer Auftragung der VAS-Scores über die Zeit) sowie das Auftreten schwerer unerwünschter Ereignisse während der Behandlung. Für keine dieser Größen wurde ein signifikanter Unterschied zwischen den Studiengruppen festgestellt. Die Schmerzstärke verringerte sich sowohl nach Akupunktur als auch nach ST in hoch signifikantem Maße. [Copyright &y& Elsevier]