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Alberto M Martelli

Publications and source records attributed to Alberto M Martelli.

At least 37 records · Page 2Linked to original sources
PubMed2004

Expression of phospholipase C beta family isoenzymes in C2C12 myoblasts during terminal differentiation.

In the present work, we have analyzed the expression and subcellular localization of all the members of inositide-specific phospholipase C (PLCbeta) family in muscle differentiation, given that nuclear PLCbeta1 has been shown to be related to the differentiative process. Cell cultures of C2C12 myoblasts were induced to differentiate towards the phenotype of myotubes, which are also indicated as differentiated C2C12 cells. By means of immunochemical and immunocytochemical analysis, the expression and subcellular localization of PLCbeta1, beta2, beta3, beta4 have been assessed. As further characterization, we investigated the localization of PLCbeta isoenzymes in C2C12 cells by fusing their cDNA to enhanced green fluorescent protein (GFP). In myoblast culture, PLCbeta4 was the most expressed isoform in the cytoplasm, whereas PLCbeta1 and beta3 exhibited a lesser expression in this cell compartment. In nuclei of differentiated myotube culture, PLCbeta1 isoform was expressed at the highest extent. A marked decrease of PLCbeta4 expression in the cytoplasm of differentiated C2C12 cells was detected as compared to myoblasts. No relevant differences were evidenced as regards the expression of PLCbeta3 at both cytoplasmatic and nuclear level, whilst PLCbeta2 expression was almost undetectable. Therefore, we propose that the different subcellular expression of these PLC isoforms, namely the increase of nuclear PLCbeta1 and the decrease of cytoplasmatic PLCbeta4, during the establishment of myotube differentiation, is related to a spatial-temporal signaling event, involved in myogenic differentiation. Once again the subcellular localization appears to be a key step for the diverse signaling activity of PLCbetas.

Animals↗
PubMed2004

Nuclear inositides: facts and perspectives.

Strong evidence has been accumulating over the last 15 years suggesting that phosphoinositides, which are involved in the regulation of a large variety of cellular processes in the cytoplasm and in the plasma membrane, are present within the nucleus. Several advances have resulted in the discovery that nuclear phosphoinositides are involved in cell growth and differentiation. Remarkably, the nuclear inositide metabolism is regulated independently from that present elsewhere in the cell. Although nuclear inositol lipids generate second messengers such as diacylglycerol and inositol 1,4,5-trisphosphate, it is becoming increasingly clear that in the nucleus polyphosphoinositides may act by themselves to influence pre-mRNA splicing and chromatin structure. This review aims at highlighting the most significant and updated findings about inositol lipid metabolism in the nucleus.

Animals↗
PubMed2004

Novel 2'-substituted, 3'-deoxy-phosphatidyl-myo-inositol analogues reduce drug resistance in human leukaemia cell lines with an activated phosphoinositide 3-kinase/Akt pathway.

Activation of the phosphoinositide 3-kinase (PI3-K)/Akt signalling pathway has been linked with resistance to chemotherapeutic drugs, and its down-regulation, by means of pharmacological inhibitors of PI3-K, considerably lowers resistance to various types of therapy in cell lines derived from solid tumours. Recently, a new class of Akt inhibitors, referred to as phosphatidylinositol ether lipids (PIAs), have been synthesized. We tested whether two new PIAs could lower the sensitivity threshold to chemotherapeutic drugs of human leukaemia cell lines with an activated PI3-K/Akt network. We used HL60AR (for apoptosis resistant), K562 and U937 cells. The two pharmacological inhibitors, used at 5 micromol/l, down-regulated Akt kinase activity and phosphorylation. Neither of the two chemicals affected the activity of other signalling proteins in the Akt pathway, such as phosphoinositide-dependent protein kinase-1 or PTEN. When employed at 5 micromol/l, the Akt inhibitors markedly reduced the resistance of the leukaemic cell lines to etoposide or cytarabine. Remarkably, a 5 micromol/l concentration of the inhibitors did not negatively affect the survival rate of human cord blood CD34(+) cells. Overall, our results indicate that new selective Akt pharmacological inhibitors might be used in the future for overcoming Akt-mediated resistance to therapeutic treatments of acute leukaemia cells.

Antineoplastic Agents↗
PubMed2004

Detection of serine 473 phosphorylated Akt in acute myeloid leukaemia blasts by flow cytometry.

The phosphoinositide 3-kinase/Akt signalling pathway is a recently recognized important parameter in the prognosis and the response to treatment of acute myeloid leukaemia (AML). Akt kinase is activated by phosphorylation on Thr 308 and Ser 473. Active Akt promotes cell growth and survival to apoptotic insults. Thus, it seems important to evaluate Akt phosphorylation in AML blasts. This work aimed to establish whether it was possible to detect Akt phosphorylation on Ser 473 of AML blasts by means of flow cytometry. High levels of Akt activity and phosphorylation were detected in 13 of 15 cases of AML. Flow cytometric analysis revealed similar patterns of Ser 473 expression as was observed with Akt kinase activity and Western blot analysis of Thr 308 and Ser 473 phosphorylation. Double immunostaining enabled the simultaneous flow cytometric detection of an AML-associated antigen (CD33) and Ser 473 phosphorylated Akt in leukaemic blast populations. Our results indicate that flow cytometry enabled the rapid and quantitative assessment of Ser 473 phosphorylated Akt of AML blasts that, when used in combination with cell surface staining, can provide more accurate phenotyping of AML blasts.

Acute Disease↗
PubMed2004

Caspase-9 is the upstream caspase activated by 8-methoxypsoralen and ultraviolet-A radiation treatment of Jurkat T leukemia cells and normal T lymphocytes.

BACKGROUND AND OBJECTIVES: A combination of 8-methoxypsoralen and ultraviolet-A radiation (PUVA) is used for the treatment of T cell-mediated disorders, including chronic graft-versus-host disease. The mechanisms of action of this therapy, referred to as extracorporeal phototherapy, have not been fully elucidated. PUVA is known to induce apoptosis in T lymphocytes collected by apheresis, however scarce information is available concerning the apoptotic pathways activated by PUVA. DESIGN AND METHODS: We used Jurkat human T leukemia cells and normal T lymphocytes to analyze the PUVA-triggered caspase activation pattern by means of immunoblot analysis, in vitro caspase activity assays, and selective caspase inhibitors coupled to flow cytometric analysis. RESULTS: PUVA treatment induced activation of apical caspases-9 and -8, and of effector caspases-3 and -7 in Jurkat cells and human T lymphocytes. While activation of caspase-9 occurred as early as 1 h after PUVA treatment of Jurkat cells, procaspase-8 cleavage was delayed and was detected 6 h after the exposure. Also in normal T lymphocytes, cleavage of caspase-8 was subsequent to activation of caspase-9. PUVA-dependent proteolytic cleavage of procaspase-8 was blocked by inhibitors selective for either caspase-9 or -3. Moreover, procaspase-8 was cleaved in vitro by activated caspase-3, which gave rise to proteolytic fragments equivalent to those generated in vivo. INTERPRETATION AND CONCLUSIONS: Activation of caspase-8 in PUVA-treated Jurkat cells and normal T lymphocytes is secondary to up-regulation of caspase-9. Overall, our results identify caspase-9 as the critical upstream caspase initiating apoptosis by PUVA in Jurkat T-cells and human T lymphocytes.

Apoptosis↗
PubMed2003

Intranucleolar localization of DNA topoisomerase IIalpha is a distinctive feature of necrotic, but not of apoptotic, Jurkat T-cells.

Two distinct types of cell death have been described: apoptosis and necrosis. However, it is becoming increasingly clear that the differences between these two types are far less numerous than initially thought. Morphological analyses might provide important information to distinguish apoptotic from necrotic samples. We recently reported that in necrotic, but not apoptotic, HL-60 human myeloid leukaemia cells, the nuclear protein topoisomerase IIalpha concentrated in nucleoli. In order to ascertain whether or not this phenomenon was restricted to a peculiar cell type or could be detected also in cells of lymphoid lineage, we performed an investigation aimed at defining the localization of topoisomerase IIalpha in apoptotic and necrotic Jurkat human T lymphoblastoid cells. Immunofluorescence staining demonstrated that topoisomerase IIalpha was excluded from the condensed chromatin of apoptotic cells, whereas in necrotic cells it was localized in discrete nuclear dots. Immuno-electron microscopy analysis showed that topoisomerase IIalpha was undetectable in nucleoli of normal and apoptotic cells, whereas it was present in the nucleolus of necrotic cells irrespectively of the type of inducer used (ethanol, H(2)O(2), HgCl(2)). Taken together, our findings identify topoisomerase IIalpha as a potential morphological marker useful to discriminate between apoptotic and necrotic cells.

Antigens, Neoplasm↗
PubMed2003

Diacylglycerol kinase-theta is localized in the speckle domains of the nucleus.

It is well established that the nucleus is endowed with enzymes that are involved in lipid-dependent signal transduction pathways. Diacylglycerol (DAG) is a fundamental lipid second messenger that is produced in the nucleus. Previous reports have shown that the nucleus contains diacylglycerol kinases (DGKs), i.e., the enzymes that, by converting DAG into phosphatidic acid (PA), terminate DAG-dependent events. Here, we show, by immunofluorescence staining and confocal analysis, that DGK-theta localizes mainly to the nucleus of various cell lines, such as MDA-MB-453, MCF-7, PC12, and HeLa. Nuclear DGK-theta co-localizes with phosphatidylinositol 4,5-bisphosphate (PIP(2)) in domains that correspond to nuclear speckles, as revealed by the use of an antibody to the splicing factor SC-35, a well-established marker for these structures. The spatial distribution of nuclear DGK-theta was dynamic in that it was affected by inhibition of mRNA transcription with alpha-amanitin. Immuno-electron microscopy analysis demonstrated that DGK-theta, PIP(2), and phosphoinositide-specific phospholipase Cbeta1 (PLCbeta1) associated with electron-dense particles within the nucleus that correspond to interchromatin granule clusters. Cell fractionation experiments performed in MDA-MB-453, HeLa, and PC12 cells showed a preferential association of DGK-theta with the nucleus. Western blots demonstrated that DGK-theta was enriched in the nuclear matrix fraction prepared from MDA-MB-453 cells. Immunoprecipitation experiments with an antibody to PLCbeta1 revealed in MDA-MB-453 cells an association between this enzyme and both DGK-theta and phosphatidylinositol phosphate kinase Ialpha (PIPKIalpha). Our findings strengthen the contention that speckles represent a crucial site for the nuclear-based inositol lipid cycle. We may speculate that nuclear speckle-located DGK-theta, on cell stimulation with an agonist, converts to PA the DAG derived from PLCbeta1-dependent PIP(2) hydrolysis.

Amanitins↗
PubMed2003

Nuclear lipids: new functions for old molecules?

It is becoming increasingly evident that stimulation of nuclear lipid metabolism plays a central role in many signal transduction pathways that ultimately result in various cell responses including proliferation and differentiation. Nuclear lipid metabolism seems to be at least as complex as that existing at the plasma membrane. However, a distinctive feature of nuclear lipid biochemical pathways is their operational independence from their cell periphery counterparts. Although initially it was thought that nuclear lipids would serve as a source for second messengers, recent evidence points to the likelihood that lipids present in the nucleus also fulfil other roles. The aim of this review is to highlight the most intriguing advances made in the field over the last year, such as the production of new probes for the in situ mapping of nuclear phosphoinositides, the identification of two sources for nuclear diacylglycerol production, the emerging details about the peculiar regulation of nuclear phosphoinositide synthesizing enzymes, and the distinct possibility that nuclear lipids are involved in processes such as chromatin organization and pre-mRNA splicing.

Cell Nucleus↗
PubMed2003

Up-regulation of nuclear PLCbeta1 in myogenic differentiation.

Phospholipase C beta(1) (PLCbeta(1)) signaling in both cell proliferation and differentiation has been largely investigated, but its role in myoblast differentiation is still unclear. The C2C12 myogenic cell line has been used in this study in order to find out the role of the two subtypes of PLCbeta(1), i.e., a and b in this process. C2C12 myoblast proliferate in response to mitogens and upon mitogen withdrawal differentiates into multinucleated myotubes. We found that differentiation of C2C12 skeletal muscle cells is characterized by a marked increase in the amount of nuclear PLCbeta(1)a and PLCbeta(1)b. Indeed, treatment with insulin induces a dramatic rise of both PLCbeta(1) subtypes expression and activity, as determined by immunochemical and enzymatic assays. Immunofluorescence experiments with anti-PLCbeta(1) specific monoclonal antibody showed a low level of cytoplasmatic and nuclear staining during the initial 12 h of differentiation whilst a massive nuclear staining is appreciable in differentiating cells. The time course of PLCbeta(1) expression versus Troponin T expression clearly indicates that the increase in the amount of PLCbeta(1) takes place 24 h earlier than that of Troponin T. Moreover, the overexpression of the PLCbeta(1)M2b mutant, lacking the nuclear localization signal and entirely located in the cytoplasm, represses the formation of mature multinucleated myotube. Taken together these results suggest that nuclear PLCbeta(1) is a key player in myoblast differentiation, functioning as a positive regulator of this process.

Animals↗
PubMed2003

Threonine 308 phosphorylated form of Akt translocates to the nucleus of PC12 cells under nerve growth factor stimulation and associates with the nuclear matrix protein nucleolin.

We have examined the issue of whether or not in PC12 cells it may be observed a nerve growth factor (NGF) nuclear translocation of an active (phosphorylated) Akt. Western blot analysis with antibodies to either total or phosphorylated Akt showed a maximal nuclear translocation after 15 min of NGF stimulation. NGF increased rapidly and transiently the enzymatic activity of immunoprecipitable nuclear Akt and after 45 min the values returned to a level close to the basal one. Enzyme translocation was blocked by the selective phosphoinositide 3-kinase inhibitor, LY294002. Confocal microscopy of samples stained with antibody to Akt showed an evident increase in immunostaining intensity in the nuclear interior after NGF treatment. Treatment of cells with inhibitors of protein phosphatase PP2A, calyculin A, or okadaic acid, maintained the phosphorylation levels of nuclear Akt. Immunoprecipitation experiments revealed an association between Akt and PP2A that was maximal when nuclear Akt activity was decreased. Both total and active Akt associated with the nuclear matrix and, in particular, with the protein nucleolin, with which Akt co-immunoprecipitated. These findings strongly suggest that the intranuclear translocation of active Akt is an important step in the signaling pathways elicited by the neurotrophin NGF and that the intranuclear control of Akt is achieved through the action of PP2A.

Animals↗
PubMed2003

The phosphoinositide 3-kinase/AKT1 pathway involvement in drug and all-trans-retinoic acid resistance of leukemia cells.

Disruption of the apoptotic pathways may account for resistance to chemotherapy and treatment failures in human neoplastic disease. To further evaluate this issue, we isolated a HL-60 cell clone highly resistant to several drugs inducing apoptosis and to the differentiating chemical all-trans-retinoic acid (ATRA). The resistant clone displayed an activated phosphoinositide 3-kinase (PI3K)/AKT1 pathway, with levels of phosphatidylinositol (3,4,5) trisphosphate higher than the parental cells and increased levels of both Thr 308 and Ser 473 phosphorylated AKT1. In vitro AKT1 activity was elevated in resistant cells, whereas treatment of the resistant cell clone with two inhibitors of PI3K, wortmannin or Ly294002, strongly reduced phosphatidylinositol (3,4,5) trisphosphate levels and AKT1 activity. The inhibitors reversed resistance to drugs. Resistant cells overexpressing either dominant negative PI3K or dominant negative AKT1 became sensitive to drugs and ATRA. Conversely, if parental HL-60 cells were forced to overexpress an activated AKT1, they became resistant to apoptotic inducers and ATRA. There was a tight relationship between the activation of the PI3K/AKT1 axis and the expression of c-IAP1 and c-IAP2 proteins. Activation of the PI3K/AKT1 axis in resistant cells was dependent on enhanced tyrosine phosphorylation of the p85 regulatory subunit of PI3K, conceivably due to an autocrine insulin-like growth factor-I production. Our findings suggest that an up-regulation of the PI3K/AKT1 pathway might be one of the survival mechanisms responsible for the onset of resistance to chemotherapeutic and differentiating therapy in patients with acute leukemia.

Antineoplastic Agents↗
PubMed2002

The nuclear phosphoinositide 3-kinase/AKT pathway: a new second messenger system.

Lipid second messengers, particularly those derived from the polyphosphoinositide cycle, play a pivotal role in several cell signaling networks. Phosphoinositide 3-kinases (PI3Ks) generate specific inositol lipids that have been implicated in a plethora of cell functions. One of the best-characterized targets of PI3K lipid products is the serine/threonine protein kinase Akt. Recent findings have implicated Akt in cancer progression because it stimulates cell proliferation and suppresses apoptosis. Evidence accumulated over the past 15 years has highlighted the presence of an autonomous nuclear inositol lipid metabolism, and suggests that lipid molecules are important components of signaling pathways operating within the nucleus. PI3Ks, their lipid products, and Akt have also been identified at the nuclear level. In this review, we shall summarize the most updated findings about these molecules in relationship with the nuclear compartment and provide an overview of the possible mechanisms by which they regulate important cell functions.

Animals↗
PubMed2002

Nuclear inositol lipid signaling and its potential involvement in malignant transformation.

Growth, differentiation, and apoptosis of eukaryotic cells are mediated by extremely complex signaling pathways and a high degree of coordination is required for regulating cell proliferation. In multicellular organisms homeostasis is achieved through signal transduction events. If these homeostatic mechanisms are interrupted, a disease, such as cancer, may ensue. Lipid second messengers, particularly those derived from polyphosphoinositide cycle, play a pivotal role in several cell signaling networks. Evidence accumulated over the past 15 years has highlighted the presence of an autonomous nuclear inositol lipid metabolism, and suggests that lipid signaling molecules are important components of signaling pathways operating within the nucleus. Recent findings are starting to elucidate how the nuclear phosphoinositide cycle is regulated and what down-stream molecules are targeted through this cycle. In this review, we shall summarize the most updated data about inositol lipid-dependent nuclear signaling pathways that might have a relevance for the development of cancer. In the near future, this knowledge might also prove to have relevance for the diagnosis and treatment of the neoplastic disease.

Animals↗
PubMed2002

Molecular characterization of the human PLC beta1 gene.

Inositide-specific phospholipase C (PLC) signaling constitutes a central intermediate in a number of cellular functions among which the control of cell growth raises a particular interest. Indeed, we have previously shown that nuclear phospholipase C beta1 (PLC beta1) is central for the regulation of mitogen-induced cell growth. We have also assigned by fluorescence in situ hybridization (FISH) analysis the PLC beta1 to human chromosome 20p12. In this study, we have carried out a detailed analysis of the human gene, showing the existence of alternative splicing, which gives rise, besides the two forms (1a and 1b) already shown in rodents, to a new 600 bp smaller form coding for a 110 kDa protein. We have also identified a new exon at the 5', showing no homology with the rodent sequence. Here we provide the complete determination of the exon/intron structure of the gene spanning 250 kb of DNA. We found that the exons are quite small, ranging from 49 to 222 bp, while the introns vary between 108 bp and 34,400 bp. The availability of the understanding of the genome organization of this inositide-specific PLC, which represents a key step of the cell cycle related signaling, could actually pave the way for further genetic analysis of p12 region of human chromosome 20 in diseases involving alterations of the control of cell growth such as malignancies.

3' Untranslated Regions↗
PubMed2002

Nuclear PLCbeta(1) acts as a negative regulator of p45/NF-E2 expression levels in Friend erythroleukemia cells.

It is well established that phospholipase C (PLC) beta(1) plays a role in the nuclear compartment and is involved in the signalling pathway that controls the switching of the erythroleukemia cells programming from an undifferentiated to a differentiated state. Constitutive overexpression of nuclear PLCbeta(1) has been previously shown to inhibit Friend cells differentiation. For further characterization, we investigated the localization of PLCbeta(1)a and PLCbeta(1)b in Friend cells by fusing their cDNA to enhanced green fluorescent protein (GFP). To investigate the potential target of nuclear PLCbeta(1) in Friend differentiation, we studied the expression of p45/NF-E2 transcription factor, which is an enhancer binding protein for expression of the beta-globin gene and the expression of GATA proteins that are important for the survival and differentiation of erythroid cells. Our data suggest that the overexpression of PLCbeta(1) (both 1a and 1b) only in the nuclear compartment significantly reduces the expression of p45/NF-E2. The effect observed is attributable to the specific action of nuclear PLCbeta(1) signalling given that GATA-1 and GATA-3 are not affected at all. Here we show the existence of a unique target, i.e. the transcription factor p45/NF-E2, whose expression is specifically inhibited by the nuclear signalling evoked by PLCbeta(1) forms.

Animals↗
PubMed2002

Molecular characterization of protein kinase C-alpha binding to lamin A.

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.

Amino Acid Sequence↗