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Biomedical subjects

S Alemà

Publications and source records attributed to S Alemà.

At least 19 recordsLinked to original sources
PubMed2000

Inhibition of ErbB-2 mitogenic and transforming activity by RALT, a mitogen-induced signal transducer which binds to the ErbB-2 kinase domain.

The product of rat gene 33 was identified as an ErbB-2-interacting protein in a two-hybrid screen employing the ErbB-2 juxtamembrane and kinase domains as bait. This interaction was reproduced in vitro with a glutathione S-transferase fusion protein spanning positions 282 to 395 of the 459-residue gene 33 protein. Activation of ErbB-2 catalytic function was required for ErbB-2-gene 33 physical interaction in living cells, whereas ErbB-2 autophosphorylation was dispensable. Expression of gene 33 protein was absent in growth-arrested NIH 3T3 fibroblasts but was induced within 60 to 90 min of serum stimulation or activation of the ErbB-2 kinase and decreased sharply upon entry into S phase. New differentiation factor stimulation of mitogen-deprived mammary epithelial cells also caused accumulation of gene 33 protein, which could be found in a complex with ErbB-2. Overexpression of gene 33 protein in mouse fibroblasts inhibited (i) cell proliferation driven by ErbB-2 but not by serum, (ii) cell transformation induced by ErbB-2 but not by Ras or Src, and (iii) sustained activation of ERK 1 and 2 by ErbB-2 but not by serum. The gene 33 protein may convey inhibitory signals downstream to ErbB-2 by virtue of its association with SH3-containing proteins, including GRB-2, which was found to associate with gene 33 protein in living cells. These data indicate that the gene 33 protein is a feedback inhibitor of ErbB-2 mitogenic function and a suppressor of ErbB-2 oncogenic activity. We propose that the gene 33 protein be renamed with the acronym RALT (receptor-associated late transducer).

3T3 Cells↗
PubMed2000

Activation of TrkA tyrosine kinase in embryonal carcinoma cells promotes cell compaction, independently of tyrosine phosphorylation of catenins.

Cadherins are transmembrane receptors whose extracellular domain mediates homophilic cell-cell interactions, while their cytoplasmic domain associates with a family of proteins known as catenins. Although the mechanisms that regulate the assembly and functional state of cadherin-catenin complexes are poorly understood, current evidence supports a role for protein tyrosine kinase activity in regulating cell adhesion and migration. Tyrosine phosphorylation of catenins is thought to mediate loss of intercellular adhesion promoted by activation of receptor tyrosine kinases in epithelial cells. Here, we show that activation of ectopically expressed TrkA, the tyrosine kinase receptor for nerve growth factor (NGF), stimulates embryonal carcinoma P19 cells to develop extensive intercellular contacts and to assemble into closely packed clusters. Thus, activation of receptor tyrosine kinases can differentially regulate adhesiveness by cell-type-specific mechanisms. Furthermore, activation of TrkA in P19 and epithelial MDCK cells induces tyrosine phosphorylation of p120(ctn) and of beta-catenin, irrespective of the elicited cellular response. The selective Src tyrosine kinase inhibitor PP2, however, suppresses NGF- or HGF-induced tyrosine phosphorylation of catenins in both P19 and MDCK cells without interfering with the acquisition of a compacted or scattered phenotype. These findings provide a cogent argument for considering that tyrosine phosphorylation of catenins is dispensable for their interaction with cadherins and, ultimately, for the modulation of cadherin-based cell adhesion by receptor tyrosine kinases.

Amino Acid Sequence↗
PubMed1999

Induction of telomerase activity in v-myc-transformed avian cells.

Telomerase activity is detectable in the majority of tumors or immortalized cell lines, but is repressed in most normal human somatic cells. It is generally assumed that reactivation of telomerase prevents the erosion of chromosome ends which occurs in cycling cells and, hence, hinders cellular replicative senescence. Here, we show that the expression of v-Myc oncoprotein by retroviral infection of telomerase-negative embryonal quail myoblasts and chicken neuroretina cells is sufficient for reactivating telomerase activity, earlier than telomere shortening could occur. Furthermore, the use of a conditional v-Myc-estrogen receptor protein (v-MycER) causes estrogen-dependent expression of detectable levels of telomerase activity in recently infected chick embryo fibroblasts and neuroretina cells. We conclude that the high levels of telomerase activity in v-Myc-expressing avian cells are not the mere consequence of transformation or of a differentiative block, since v-Src tyrosine kinase, which prevents terminal differentiation and promotes cell transformation, fails to induce telomerase activity.

Animals↗
PubMed1999

Distinct effects of Rac1 on differentiation of primary avian myoblasts.

Rho family GTPases have been implicated in the regulation of the actin cytoskeleton in response to extracellular cues and in the transduction of signals from the membrane to the nucleus. Their role in development and cell differentiation, however, is little understood. Here we show that the transient expression of constitutively active Rac1 and Cdc42 in unestablished avian myoblasts is sufficient to cause inhibition of myogenin expression and block of the transition to the myocyte compartment, whereas activated RhoA affects myogenic differentiation only marginally. Activation of c-Jun N-terminal kinase (JNK) appears not to be essential for block of differentiation because, although Rac1 and Cdc42 GTPases modestly activate JNK in quail myoblasts, a Rac1 mutant defective for JNK activation can still inhibit myogenic differentiation. Stable expression of active Rac1, attained by infection with a recombinant retrovirus, is permissive for terminal differentiation, but the resulting myotubes accumulate severely reduced levels of muscle-specific proteins. This inhibition is the consequence of posttranscriptional events and suggests the presence of a novel level of regulation of myogenesis. We also show that myotubes expressing constitutively active Rac1 fail to assemble ordered sarcomeres. Conversely, a dominant-negative Rac1 variant accelerates sarcomere maturation and inhibits v-Src-induced selective disassembly of I-Z-I complexes. Collectively, our findings provide a role for Rac1 during skeletal muscle differentiation and strongly suggest that Rac1 is required downstream of v-Src in the signaling pathways responsible for the dismantling of tissue-specific supramolecular structures.

Animals↗
PubMed1998

Eps8, a tyrosine kinase substrate, is recruited to the cell cortex and dynamic F-actin upon cytoskeleton remodeling.

Eps8 is a recently identified substrate of receptor and nonreceptor tyrosine kinases implicated in the control of cell proliferation. To investigate potential functions of Eps8, its intracellular localization has been examined in several cell types. In cycling fibroblasts immunolabeling with antibodies to Eps8 reveals a punctate pattern within the perinuclear region and staining of motile peripheral cell extensions and cell-cell contact regions. Stimulation of quiescent Swiss 3T3 fibroblasts with serum induces a striking reorganization of the actin cytoskeleton which is accompanied by the enrichment of Eps8 and cortactin in membrane ruffles and lamellipodia. A similar accumulation of Eps8 to membrane ruffles is observed in cells treated with phorbol esters, which also induce marked changes of the F-actin cytoskeleton. The localization of Eps8 at the cell cortex is largely independent from the binding of Eps8 to an EGFR/ErbB-2 chimeric receptor. Moreover, fractionation studies reveal that a portion of the Eps8 molecules present in the cell periphery, unlike cortactin and the receptor, is resistant to mild extraction with detergent. Upon cellular transformation by the tyrosine kinase v-Src, a pool of Eps8 is recruited to newly formed specialized regions of the cytoskeleton, such as actin bodies in terminally differentiated myotubes and podosomes in fibroblasts, where cortactin and a variety of cytoskeletal proteins are also found. Extraction with Triton X-100 preserves the association of Eps8 to podosomes and leaves the majority of the v-Src tyrosine-phosphorylated Eps8 in the detergent-resistant fraction. The observed recruitment of Eps8 to highly dynamic cytoskeletal structures of normal and transformed cells suggests that Eps8 may play a role in the reorganization of the cytoskeleton, perhaps acting as a docking site for other signaling molecules.

3T3 Cells↗
PubMed1998

The neuronal alpha6 subunit forms functional heteromeric acetylcholine receptors in human transfected cells.

We examine some of the biological and physiological properties of the avian alpha6 neuronal nicotinic acetylcholine receptor (nAChR) subunit. We show here that, beginning at embryonic day 5, alpha6 mRNA is abundantly expressed in the developing chick neuroretina, where it coexists with other nicotinic receptor subunit mRNAs such as alpha3, beta2 and beta4. In contrast, alpha6 mRNA is absent from the optic tectum and from the peripheral ganglia. Despite numerous efforts, the alpha6 subunit has long failed the critical test of functional reconstitution. Here we use patch-clamp techniques and confocal laser microscopy to measure ACh-activated currents and nicotine-elicited Ca2+ transients in human BOSC 23 cells transfected with chick alpha6 in combination with other chick nAChR neuronal subunits. Heterologously expressed alpha6 and beta4 subunits form functional heteromeric nAChRs, which are permeable to Ca2+ ions and blocked by the nicotinic antagonist methyllycaconitine (10 microM). Likewise, ACh elicits measurable currents in cells transfected with alpha6 and beta2. Hill analysis of the dose-response curves in cells transfected with alpha3, beta4 and alpha6 cDNAs, suggests the assembly of functional alpha3beta4alpha6 receptor, with an apparent affinity for ACh threefold lower than alpha3beta4. Our results indicate that alpha6-containing nAChRs assemble in heterologous expression systems and are probably present in retinal cells.

Acetylcholine↗
PubMed1997

Regulation of the tyrosine kinase substrate Eps8 expression by growth factors, v-Src and terminal differentiation.

SH3-containing proteins are involved in signal transduction by a number of growth factor receptors and in the organization of the cytoskeleton. The recently identified Eps8 protein, which contains an SH3 domain, is coupled functionally and physically to the EGFR and is tyrosine phosphorylated by this receptor and other receptors as well. Here, we examined the regulation of eps8 expression in response to mitogenic or differentiative signals. We show that Eps8 is expressed at low levels in resting fibroblasts, but its expression is strongly induced during activation by serum, phorbol esters and the v-src oncogene. Conversely, expression of Eps8, but not of other EGFR substrates such as Shc or Eps15, is virtually extinguished in non-proliferating, terminally differentiated murine myogenic cells. The putative role of Eps8 protein as a v-Src substrate was analysed in murine fibroblasts and in quail myogenic cells expressing a temperature-sensitive variant of the tyrosine kinase. Tyrosine phosphorylation of Eps8 was detected only at the permissive temperature. A non-myristylated, transformation-defective mutant of v-Src did not phosphorylate Eps8, whereas it phosphorylated Shc. Together, these findings indicate that Eps8 may be a critical substrate of v-Src. They further establish Eps8 as an example of a signal transducer whose expression senses the balance between growth and differentiation and might, therefore, be involved in the determination of the phenotype.

3T3 Cells↗
PubMed1997

Alpha 5 subunit forms functional alpha 3 beta 4 alpha 5 nAChRs in transfected human cells.

nAChRs heterologously expressed in human cells after transient transfection with alpha 3 beta 4 alpha 5 or alpha 3 beta 4 subunit cDNAs exhibited similar sensitivities to antagonists and comparable functional channel profiles. However, the sum of two Hill equations was required for best fitting the ACh dose-current response curves after co-expression of alpha 5, alpha 3 and beta 4 subunits. One component was comparable to that obtained in alpha 3 beta 4-transfected cells, while the additional component, putatively attributed to an alpha 3 beta 4 alpha 5 nAChR population, showed a Hill coefficient > 2 and a nine-fold greater half-maximal ACh concentration (EC50). These results suggest that the alpha 5 subunit participates in the assembly of alpha 3 beta 4 alpha 5 nAChRs complexes in human cells, adding a new member to the family of neuronal nicotinic receptors.

Acetylcholine↗
PubMed1997

Immunohistochemical detection of p140trkA and p75LNGFR neurotrophin receptors in neuroblastoma.

In neuroblastoma, high levels of mRNA for p140trkA and p75LNGFR neurotrophin receptors are predictive of favorable outcome. Their evaluation by Northern blot, however, requires substantial amounts of tissue and this prevents their routine evaluation as well as the possibility for multicenter studies to be easily carried out. In an attempt to overcome these limitations, the feasibility and reliability of determining both neurotrophin receptors on cryostat sections by immunohistochemistry were assessed, and these findings were compared to those obtained from Northern blot analysis. Primary tumor samples from 28 untreated patients at all stages were evaluated by using H10 anti-p140trkA and ME20.4 anti-p75LNGFR mAbs. Although weak, positive immunostaining was found in 9 of 28 tumors for p140trkA and in 5 of 28 tumors for p75LNGFR. As compared to Northern blot, the concordance rate was 79% (22 of 28 cases) for p140trkA (p < 0.05) and 71% (20 of 28 cases) for p75LNGFR (p < 0.05). No case negative for Northern blot was found to be positive with immunohistochemistry. Since only high mRNA levels for both receptors have been shown to be clinically relevant, their immunohistochemical detection, although less sensitive than Northern blot, can be just as sufficient and reliable as a prognostic tool, and possibly with a better cost-benefit ratio.

Antibodies, Monoclonal↗
PubMed1997

Functional properties of neuronal nicotinic acetylcholine receptor channels expressed in transfected human cells.

To study how subunit composition affects the functional properties of neuronal nicotinic acetylcholine receptors (nAChRs), we examined the behaviour of acetylcholine (ACh)-induced single-channel currents in human BOSC 23 cells transiently transfected with various subunit cDNA combinations. For all nAChRs examined (chick and rat alpha 3 beta 4, chick alpha 3 beta 2, alpha 4 beta 2, alpha 7 and alpha 8), expression levels were high enough to allow measurements of acetylcholine-evoked whole-cell currents and nicotine-elicited Ca2+ transients as well as the functional characterization of nAChR channels. Unitary acetylcholine-evoked events of alpha 8 nAChR had a slope conductance of 23 pS, whereas two conductance classes (19-23 and 32-45 pS) were identified for all other nAChR channels. The mean channel open times were significantly longer for homomeric alpha 7 and alpha 8 nAChRs (6-7 ms) than for heteromeric nAChRs (1-3 ms), with the exception of alpha 3 beta 4 nAChRs (8.4 ms for rat, 7 ms for chick). At least two species of heterologously expressed nAChRs (alpha 3 beta 4 and alpha 3 beta 2) exhibited single-channel characteristics similar to those reported for native receptors. The variety of nAChR channel conductance and kinetic properties encountered in human cells transfected with nAChR subunits contributes to the functional diversity of nAChRs in nerve cells.

Acetylcholine↗
PubMed1996

Identification of a determinant of acetylcholine receptor gating kinetics in the extracellular portion of the gamma subunit.

A large body of structure-function studies has identified many of the functional motifs underlying ion permeation through acetylcholine receptor (AChR) channels. The structural basis of channel gating kinetics is, however, incompletely understood. We have previously identified a novel shorter form of the AChR gamma subunit, which lacks the 52 amino acids within the extracellular amino-terminal half, encoded by exon 5. To define the contribution of the missing domain to AChR channel function, we have transiently coexpressed the mouse short gamma subunit [gamma(s)] with alpha, beta and delta subunits in human cells and recorded single-channel currents from the resulting AChRs. Our findings show that replacement of the gamma by the gamma(s) subunit confers a long duration characteristic to AChR channel openings without altering unitary conductance sizes or receptor affinity for the transmitter. We also show that alpha beta gamma(s) delta AChR channels exhibit a peculiar voltage sensitivity characterized by a short opening duration when the membrane potential is hyperpolarized. Together, these findings indicate that the domain in the extracellular amino-terminal half of the gamma subunit that encompasses a conserved disulphide loop and a critical tyrosine residue implicated in receptor oligomerization and insertion at the cell surface is a functional motif that also modulates AChR channel gating kinetics. The results also provide a molecular explanation of the functional diversity exhibited by skeletal muscle AChRs during development.

Acetylcholine↗
PubMed1996

Remodeling of cytoskeleton and triads following activation of v-Src tyrosine kinase in quail myotubes.

To study the cellular signals underlying the regulatory mechanisms involved in maintenance of sarcomeric integrity, we have used quail skeletal muscle cells that reach a high degree of structural maturation in vitro, and also express a temperature-sensitive mutant of the v-Src tyrosine kinase that allows the control of differentiation in a reversible manner. By immunofluorescence and electron microscopy we show that v-Src activity in myotubes leads to an extensive cellular remodeling which affects components of the sarcomeres, the cytoskeleton network and the triad junctions. We have previously shown that activation of v-Src causes a selective dismantling of the I-Z-I segments coupled to the formation of aggregates of sarcomeric actin, alpha-actinin and vinculin, called actin bodies. We now show that intermediate filaments do not participate in the formation of actin bodies, while talin, a component of costameres, does. The I-Z-I segments are completely dismantled within 24 hours of v-Src activity, but the A-bands persist for a longer time, implying distinct pathways for the turnover of sarcomeric subdomains. Immunofluorescence labeling of markers of the triad junctions demonstrates that the localization of the alpha 1 subunit of the dihydropyridine receptor is disrupted earlier than that of the ryanodine receptor after tyrosine kinase activation. Furthermore, the location of junctional sarcoplasmic reticulum and transverse tubule membranes is maintained in myotubes in which the I-Z-I have been removed and the regular disposition of the intermediate filaments is disrupted, supporting a role for sarcoplasmic reticulum in the proper positioning of triad junctions. Altogether these results point to a tyrosine kinase signaling cascade as a mechanism for selectively destabilizing sarcomere subdomains and their tethering to the cytoskeleton and the sarcolemma.

Actins↗
PubMed1995

Neuronal differentiation of P19 embryonal cells exhibits cell-specific regulation of neurotrophin receptors.

During development, the regulation of expression of the Trk family of tyrosine kinase receptors plays an important role in defining the cellular responses to neurotrophin action. We report here that neurotrophin receptors are differentially expressed in distinct populations of retinoic acid-differentiated P19 cells. TrkB, the tyrosine kinase receptor for brain-derived neurotrophic factor, and LNGFR, the low-affinity receptor for all neutrophins, are preferentially expressed in P19-derived neurones. In contrast, retinoic acid induces the expression of TrkA, the high-affinity receptor for nerve growth factor, and of a non-catalytic form of TrkB, in non-neural subsets of differentiated cells. We propose P19 cells as a model system to study the mechanisms controlling the expression of neurotrophin receptors and the responsiveness of developing neurones to a specific neurotrophin.

Animals↗
PubMed1995

Immunohistochemical detection of high-affinity nerve growth factor receptor in neuroblastoma.

High levels of mRNA (as assessed by northern blot) for the high-affinity nerve growth factor receptor (p140TRK) are predictive of favourable outcome in neuroblastoma. The feasibility of determining p140trk on frozen sections using a recently developed monoclonal antibody was evaluated, and immunohistochemical findings were compared to those obtained from northern blot analysis. Primary tumour samples from 28 untreated patients were quick frozen and an indirect immunofluorescence assay was performed on 4-microns acetone-fixed cryostat sections. 9 cases were positive with immunohistochemistry, and these were among the 15 cases also positive by northern blot. None of the cases negative by northern blot were positive with immunohistochemistry. The concordance rate was 79% (P < 0.03), with a sensitivity of 60% and a specificity of 100%. Immunohistochemistry can thus be rather reliable for assessing p140trk expression, even when only very small amounts of tissue are available, such as with needle biopsy.

Biomarkers, Tumor↗
PubMed1995

Maintenance of the differentiated state in skeletal muscle: activation of v-Src disrupts sarcomeres in quail myotubes.

We have used quail skeletal myotubes expressing a temperature-sensitive allele of the v-src oncogene to address the issue of the homeostasis of sarcomeric myofibrils in differentiated muscle cells. Reactivation of the v-Src tyrosine kinase by shifting the cultures to the permissive temperature leads within minutes to the formation of F-actin-containing bodies (ABs), that originate in the ventral region of the myotubes and increase in number concomitantly with the dismantling of the I-Z-I complex of the sarcomeres. This process is detailed by confocal and electron microscopy. Indirect immunofluorescence reveals that ABs contain muscle-specific protein isoforms associated with the I-Z-I complexes and vinculin, a component of the cytoskeletal network. Anti-phosphotyrosine antibodies label proteins in ABs and Z-discs. Evidence is presented indicating that this phenomenon specifically depends on the persistent activation of v-Src, rather than on a general increase in phosphotyrosine content such as that induced by vanadate. AB formation is prevented by activation of protein kinase C by phorbol ester or by treatment with the kinase inhibitor 2-aminopurine, without any detectable effect on tyrosine phosphorylation. Taken together these findings indicate that phosphorylation of specific target proteins by v-Src, although necessary, is not sufficient per se to induce AB formation. In addition, the signal transduction cascade that culminates in MAP kinase activation and its nuclear translocation is activated both by v-Src and phorbol ester, and is relatively unaffected by 2-aminopurine. These findings imply that both phorbol esters and 2-aminopurine operate, at least in part, at the level of alternative pathways that may diverge upstream of the MAP kinase and are presumably mediating the early effects of v-Src on the differentiated phenotype.

Actins↗
PubMed1994

Oncogenes and muscle differentiation: multiple mechanisms of interference.

In vitro transformation of replicating myogenic cells by a variety of oncogenes is always accompanied by inhibition of the transition to the terminally differentiated state. Three main levels of interference by oncogenes may ultimately prevent transcription of skeletal muscle-specific genes. First, the transcription of muscle-regulatory factors (MRF) of the MyoD family can be extinguished in transformed myoblasts. Second, several oncoproteins are capable of silencing the transcriptional capacity of MRF. Third, the DNA binding activity of MRF can be negatively modulated by phosphorylation or competition by dominant negative partners. Oncogenes can function at multiple potential levels of regulation, depending on the myogenic cell context.

Animals↗
PubMed1992

Transformation of NIH3T3 cells by Rous sarcoma virus occurs with high efficiency in the absence of proviral rearrangements or amplification.

NIH3T3 cells could be transformed by a mammaltropic strain of Rous sarcoma virus (RSV) with an efficiency 10(3) times greater than that observed in Balb/c 3T3 cells or other mammalian cell lines and almost identical to that of chick embryo fibroblasts. In infected NIH3T3 cells a single, properly integrated, provirus was sufficient to induce focus formation; moreover, kinase activity of pp60v-src and tyrosine phosphorylation of cellular proteins could be detected very soon after infection in the majority of cells. On the other hand, in transformed foci from RSV-infected Balb/c 3T3 cells both rearrangements and amplification of proviral sequences were frequently detected. Accordingly, expression of pp60v-src and ensuing tyrosine phosphorylation of cellular proteins occurred, at high levels, only in a minority of the infected cells. Furthermore, by using a murine retrovirus carrying the v-src oncogene and an independent selectable marker, we found that Balb/c 3T3 cells were transformed with a 100-fold lower efficiency than NIH3T3 cells, yet the majority of infected untransformed Balb/c 3T3 cells expressed active pp60v-src. These findings are consistent with the existence in most mammalian cell lines of a major restriction to v-src-induced transformation, operating at the level of proviral expression, that is apparently absent in NIH3T3 cells.

3T3 Cells↗