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Pulmonary toxicity induced by asbestos is thought to be mediated through redox-cycling of fiber-bound and bioavailable iron (Fe).This impacts an oxidative stress which potentially mediates a cascade of events culminating in inflammation, fibrosis and cancer (Ghio et al, 2008 and 2009). The present study aimed to elucidate in a murine model of asbestos exposure the distribution of ferric and ferrous iron and its relation with the development of fibrosis. C57/Bl/6 mice were exposed to saline or crocidolite asbestos in saline (55 and 110 µg intra-tracheally) and examined at 1 and 6 months for lung pathology. The left lung was embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E) and Masson’s trichrome. A modified Perl’s or Turnbull’s blue histochemical stain was used to demonstrate Fe3+ and Fe2+ respectively (Wang et al, 2002). One month following exposure, mice instilled with crocidolite displayed a significant inflammatory injury on staining with H&E (relative to animal exposed to saline). Crocidolite fibers could be observed most frequently in the region of distal bronchioles. The retention of these fibers was associated with an accumulation of inflammatory cells, predominantly macrophages. This response persisted 6 months following asbestos instillation. Increased collagen was observed at one and 6 months after asbestos exposure. One important observation made in 1-month H&E-stained slides of asbestos-exposed mice was the presence of focal atypical hyperplastic lesions. These lesions did not progress in severity from 1 to 6 months. Histological lung sections from saline exposed mice did not stain for both ferric and ferrous Fe. However, one month following crocidolite instillation, a number of Fe3+-positive (but no Fe2+-positive) cells (predominantly macrophages) were present, in the lower respiratory tract. At 6 months postinjection, an increase number of Fe3+-positive cells was observed and also some Fe2+ positive cells were present. The observation of some Fe2+ positive cells in the lung of asbestos exposed mice at 6 months postinjection suggests a shift in the oxidation state of the accumulated iron, with more cells becoming Fe2+-positive at a late stage. These growing amounts of Fe2+ could support a continuated generation of oxidative stress resulting in clinical manifestations, which culminate in inflammarory, fibrotic and hyperplastic lesions., Italian Journal of Anatomy and Embryology, Vol 118, No 2 (Supplement) 2013

There exists an active lipid metabolism in the nucleus, which is regulated differentially from the lipid metabolism taking place elsewhere in the cell. Evidence has been accumulated that nuclear lipid metabolism is closely involved in a variety of cell responses, including proliferation, differentiation, and apoptosis. A fundamental lipid second messenger which is generated in the nucleus is diacylglycerol, that is mainly known for its role as an activator of some protein kinase C isoforms. Diacylglycerol kinases attenuate diacylglycerol signaling by converting this lipid to phosphatidic acid, which also has signaling functions. Ten mammalian diacylglycerol kinase isoforms have been cloned so far, and some of them are found also in the nucleus, either as resident proteins or after migration from cytoplasm in response to various agonists. Experiments using cultured cells have demonstrated that nuclear diacylglycerol kinases have prominent roles in cell cycle regulation and differentiation. In this review, the emerging roles played by diacylglycerol kinases in the nucleus, such as the control of G1/S phase transition, are discussed.

The existence of intranuclear lipid-dependent signal transduction systems has been demonstrated by several independent groups. Remarkably, intranuclear lipid-dependent signal transduction pathways are regulated independently from their membrane/cytosolic counterparts. A sizable body of evidence suggests that nuclear lipid signaling controls critical biological functions such as cell proliferation, differentiation, and apoptosis. Diacylglycerol (DG) is a fundamental lipid second messenger which is produced in the nucleus. Since the levels of nuclear DG fluctuate during the cell cycle progression, it has been suggested that this lipid second messenger has important regulatory roles. Most likely, nuclear DG 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 (DGKs), i.e. the enzymes that, by converting DG into phosphatidic acid (PA), terminate DG-dependent events. This review aims at highlighting the different isozymes of DGKs present within the nucleus as well as at discussing their potential functions with particular emphasis placed on DNA replication.

The nuclear matrix is defined as the residual framework after the removal of the nuclear envelope, chromatin, and soluble components by sequential extractions. According to several investigators the nuclear matrix provides the structural basis for intranuclear order. However, the existence itself and the nature of this structure is still uncertain. Although the techniques used for the visualization of the nuclear matrix have improved over the years, it is still unclear to what extent the isolated nuclear matrix corresponds to an in vivo existing structure. Therefore, considerable skepticism continues to surround the nuclear matrix fraction as an accurate representation of the situation in living cells. Here, we summarize the experimental evidence in favor of, or against, the presence of a diffuse nucleoskeleton as a facilitating organizational nonchromatin structure of the nucleus.

Several studies have demonstrated the existence of an autonomous intranuclear phosphoinositide cycle that involves the activation of nuclear PIPLC and the generation of diacylglycerol (DG) within the nucleus. Although several distinct isozymes of PIPLC have been detected in the nucleus, the isoform that has been most consistently highlighted as being nuclear is PI-PLC-ß1. Nuclear PI-PLC-ß1 has been linked with either cell proliferation or differentiation. Remarkably, the activation mechanism of nuclear PI-PLC-ß1 has been shown to be different from its plasma membrane counterpart, being dependent on phosphorylation effected by p44/42 mitogen activated protein (MAP) kinase. In this review, we report the most up-dated findings about nuclear PI-PLC-ß1, such as the localization in nuclear speckles, the activity changes during the cell cycle phases, and the possible involvement in the progression of myelodisplastic syndrome to acute myeloid leukemia.

A major factor undermining successful cancer treatment is the occurrence of resistance to conventional treatments such as chemotherapy and ionizing radiation. Evidence accumulated over the recent years has indicated the phosphoinositide 3-kinase/Akt signal transduction pathway as one of the major factors implicated in cancer resistance to conventional therapies. Indeed, the phosphoinositide 3-kinase/Akt axis regulates the expression and/or function of many anti-apoptotic proteins which strongly contributes to cancer cell survival. As a result, small molecules designed to specifically target key components of this signaling network are now being developed for clinical use as single therapeutic agents and/or in combination with other forms of therapy to overcome resistance. Initially, the phosphoinositide 3-kinase/Akt signal transduction pathway has been mainly investigated in solid tumors. Recently, however, this network has also been recognized as an important therapeutic target in human leukemias. Specific inhibition of this signalling pathway may be a valid approach to treat these diseases and increase the efficacy of standard types of therapy.

Previous results from our laboratory have identified lamin A as a protein kinase C (PKC)-binding protein. Here, we have identified the regions of PKC-alpha that are crucial for this binding. By means of overlay assays and fusion proteins made of glutathione-S-transferase (GST) fused to elements of rat PKC-alpha, we have established that binding occurs through both the V5 region and a portion of the C2 region (i.e., the calcium- dependent lipid binding (CaLB) domain) of the kinase. In particular, we have found that amino acid 200-217 of the CaLB domain are essential for binding lamin A, as a synthetic peptide corresponding to this stretch of amino acids prevented the interaction between the CaLB domain and lamin A. We also show that the presence of four lysine residues of the CaLB domain (K205, K209, K211, and K213) was essential for the binding. We have determined that binding of elements of PKC-alpha to lamin A does not require the presence of cofactors such as phosphatidylserine (PS) and Ca (2+). We have also found that the binding site of lamin A for the CaLB domain of PKC-alpha is localized in the carboxyl-terminus of the lamin, downstream of amino acid 499. Our findings may prove to be important to clarify the mechanisms regulating PKC function within the nucleus and may also lead to the synthesis of isozyme-specific drugs to attenuate or reverse PKC-dependent nuclear signaling pathways important for the pathogenesis of cancer.

The nuclear matrix is defined as the residual framework after the removal of the nuclear envelope, chromatin, and soluble components by sequential extractions. According to several investigators the nuclear matrix provides the structural basis for intranuclear order. However, the existence itself and the nature of this structure is still uncertain. Although the techniques used for the visualization of the nuclear matrix have improved over the years, it is still unclear to what extent the isolated nuclear matrix corresponds to an in vivo existing structure. Therefore, considerable scepticism continues to surround the nuclear matrix fraction as an accurate representation of the situation in living cells. Here, we summarize the experimental evidence in favor of, or against, the presence of a diffuse nucleoskeleton as a facilitating organizational nonchromatin structure of the nucleus.

The evaluation of regulatory proteins is important for biological studies and is also established as a prognostic marker for cancer diagnosis. Very recently, it has been highlighted that the serine/threonine kinase Akt plays a fundamental role in survival pathways and is also involved in the onset of resistance to anti-neoplastic drugs and ionizing radiation in cell lines derived from solid tumors. For its full activation Akt needs to be phosphorylated on Serine 473 residue. Molecules that are fundamental in determining resistance to therapeutic treatments might serve in the future as clinical markers to tailor therapy and/or predict treatment response. The aim of this study was to ascertain whether or not flow cytometric analysis of total Akt and of its form phosphorylated on Serine 473 could be related to standard techniques such as Western blotting with phosphospecific antibodies and in vitro kinase assay. To this end, we employed as experimental models HL-60 and PC-12 lines in which there is an enhancement of Akt activity. Our results showed that flow cytometry analysis, performed on fixed and permeabilized cells, correlated well with the results provided by in vitro activity assays and Western blots. Therefore, our findings might indicate that flow cytometric study of Akt (both total and phosphorylated) content may be applied in routine work for phenotyping of hematological and non-hematological neoplasias, and allow for its use as a useful marker for the classification and the prognosis of neoplastic diseases.

D-3 phosphorylated inositides are a peculiar class of lipids, synthesized by phosphatidylinositol 3-kinase (PtdIns 3-K), which are also present in the nucleus. In order to clarify a possible role for nuclear D-3 phosphorylated inositides during human erythroid differentiation, we have examined the issue of whether or not, in K562 human erythroleukemia cells, erythropoietin (EPO) may generate nuclear translocation of an active PtdIns 3-K. Immunoprecipitation with an anti-p85 regulatory subunit of PtdIns 3-K, revealed that both the intranuclear amount and the activity of the kinase increased rapidly and transiently in response to EPO. Enzyme translocation was blocked by the specific PtdIns 3-K pharmacological inhibitor, LY294002, which also inhibited erythroid differentiation. In vivo, intranuclear synthesis of phosphatidylinositol (3,4,5) trisphosphate (PtdIns (3,4,5)P(3)) was stimulated by EPO. Almost all PtdIns 3-K that translocated to the nucleus was highly phosphorylated on tyrosine residues of the p85 regulatory subunit. These findings strongly suggest that an important step in the signaling pathways that mediate EPO-induced erythroid differentiation may be represented by the intranuclear translocation of an active PtdIns 3-K.

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.