Long-term outcome following thrombembolectomy in the upper extremity.

OBJECTIVES: To evaluate short- and long-term mortality and morbidity in patients that were treated for acute upper extremity ischemia. DESIGN: Single center retrospective study. PATIENTS: A consecutive series of 148 patients who were admitted with a diagnosis of acute ischemia of the upper extremity during an 11-year period. METHODS: All charts were reviewed retrospectively and 96% of all survivors participated in clinical follow-up. RESULTS: The median age was 78 years and 64% of patients were females. The 30-day mortality was 8% and the overall 5-year survival 37%. The observed mortality during the follow-up period was significantly higher than expected. Survival was not significantly different in patients who received anticoagulant drugs following discharge from the hospital. The duration of ischemia did not significantly influence long-term arm-function. CONCLUSIONS: Acute embolic episodes in the upper extremity primarily occur in elderly and the peri-operative mortality is high. Mortality following discharge from the hospital remains significantly higher than that of the background population.

Endovascular treatment of severe symptomatic stenosis of the internal carotid artery: early and late outcome.

OBJECTIVES: To report a 6 year experience with carotid percutaneous transluminal angioplasty (CPTA) in a selected group of patients. MATERIAL AND METHOD: We retrospectively reviewed our experience after performing 54 CPTAs, with (n=18) or without (n=36) stent deployment, over a period of 6 years from 1993 to 1999. All patients, except one, suffered from focal hemispheric neurologic symptoms. During the same time period 284 patients underwent carotid endarterectomy. The selection of the 54 patients (16%) for CPTA was based on the carotid angiogram and the sole inclusion criterion for endovascular treatment was a short, concentric, and smooth stenosis of more than 70% without ulceration or severe calcification. All patients who had a patent internal carotid artery after the last control were invited for a clinical duplex examination and all duplex examinations were carried out by a single experienced observer. RESULTS: Early outcome (<30 days): CPTA was judged technically successful in 50 cases (93%). Ten patients (18%) experienced a neurological event in relation to the procedure and one patient (2%) suffered a major stroke. One stent occluded within 30 days. LATE OUTCOME: Forty-six patients (85%) entered the follow-up study after a median of 34 months (range 1-80 months). Six patients (13%) had recurrent symptoms. The colour-duplex examination (n=45) showed internal carotid artery occlusion in 2 patients (5%), and restenosis (>70%) in 10 patients (22%). We found no significant difference in the reoccurrence of neurological symptoms or the rate of restenosis between patients treated with and without stent (Log Rank 0.28, p=0,59). ICA was patent without restenosis in 60% after 48 months in patients treated with CPTA alone, and in 76% after 3 months in patients treated with a stent (N.S.). CONCLUSION: CPTA in a selected group of patients has a mortality and major stroke rate comparable to that of carotid endarterectomy. However, the risk of transient neurological events was high, as well as the incidence of restenosis (>70%) after 3 years. We still consider CPTA an experimental procedure. The indications for this treatment must be clarified if CPTA should be an alternative to surgery with a comparable neurological complication rate.

The giant protein AHNAK is a specific target for the calcium- and zinc-binding S100B protein: potential implications for Ca2+ homeostasis regulation by S100B.

Transformation of rat embryo fibroblast clone 6 cells by ras and temperature-sensitive p53val(135) is reverted by ectopic expression of the calcium- and zinc-binding protein S100B. In an attempt to define the molecular basis of the S100B action, we have identified the giant phosphoprotein AHNAK as the major and most specific Ca(2+)-dependent S100B target protein in rat embryo fibroblast cells. We next characterized AHNAK as a major Ca(2+)-dependent S100B target protein in the rat glial C6 and human U-87MG astrocytoma cell lines. AHNAK binds to S100B-Sepharose beads and is also recovered in anti-S100B immunoprecipitates in a strict Ca(2+)- and Zn(2+)-dependent manner. Using truncated AHNAK fragments, we demonstrated that the domains of AHNAK responsible for interaction with S100B correspond to repeated motifs that characterize the AHNAK molecule. These motifs show no binding to calmodulin or to S100A6 and S100A11. We also provide evidence that the binding of 2 Zn(2+) equivalents/mol S100B enhances Ca(2+)-dependent S100B-AHNAK interaction and that the effect of Zn(2+) relies on Zn(2+)-dependent regulation of S100B affinity for Ca(2+). Taking into consideration that AHNAK is a protein implicated in calcium flux regulation, we propose that the S100B-AHNAK interaction may participate in the S100B-mediated regulation of cellular Ca(2+) homeostasis.

S100A6 and S100A11 are specific targets of the calcium- and zinc-binding S100B protein in vivo.

In solution, S100B protein is a noncovalent homodimer composed of two subunits associated in an antiparallel manner. Upon calcium binding, the conformation of S100B changes dramatically, leading to the exposure of hydrophobic residues at the surface of S100B. The residues in the C-terminal domain of S100B encompassing Phe(87) and Phe(88) have been implicated in interaction with target proteins. In this study, we used two-hybrid technology to identify specific S100B target proteins. Using S100B as bait, we identify S100A6 and S100A11 as specific targets for S100B. S100A1, the closest homologue of S100B, is capable of interaction with S100B but does not interact with S100A6 or S100A11. S100B, S100A6, and S100A11 isoforms are co-regulated and co-localized in astrocytoma U373 cells. Furthermore, co-immunoprecipitation experiments demonstrated that Ca(2+)/Zn(2+) stabilizes S100B-S100A6 and S100B-S100A11 heterocomplexes. Deletion of the C-terminal domain or mutation of Phe(87) and Phe(88) residues has no effect on S100B homodimerization and heterodimerization with S100A1 but drastically decreases interaction between S100B and S100A6 or S100A11. Our data suggest that the interaction between S100B and S100A6 or S100A11 should not be viewed as a typical S100 heterodimerization but rather as a model of interaction between S100B and target proteins.

Concerted regulation of wild-type p53 nuclear accumulation and activation by S100B and calcium-dependent protein kinase C.

The calcium ionophore ionomycin cooperates with the S100B protein to rescue a p53-dependent G(1) checkpoint control in S100B-expressing mouse embryo fibroblasts and rat embryo fibroblasts (REF cells) which express the temperature-sensitive p53Val135 mutant (C. Scotto, J. C. Deloulme, D. Rousseau, E. Chambaz, and J. Baudier, Mol. Cell. Biol. 18:4272-4281, 1998). We investigated in this study the contributions of S100B and calcium-dependent PKC (cPKC) signalling pathways to the activation of wild-type p53. We first confirmed that S100B expression in mouse embryo fibroblasts enhanced specific nuclear accumulation of wild-type p53. We next demonstrated that wild-type p53 nuclear translocation and accumulation is dependent on cPKC activity. Mutation of the five putative cPKC phosphorylation sites on murine p53 into alanine or aspartic residues had no significant effect on p53 nuclear localization, suggesting that the cPKC effect on p53 nuclear translocation is indirect. A concerted regulation by S100B and cPKC of wild-type p53 nuclear translocation and activation was confirmed with REF cells expressing S100B (S100B-REF cells) overexpressing the temperature-sensitive p53Val135 mutant. Stimulation of S100B-REF cells with the PKC activator phorbol ester phorbol myristate acetate (PMA) promoted specific nuclear translocation of the wild-type p53Val135 species in cells positioned in early G(1) phase of the cell cycle. PMA also substituted for ionomycin in the mediating of p53-dependent G(1) arrest at the nonpermissive temperature (37.5 degrees C). PMA-dependent growth arrest was linked to the cell apoptosis response to UV irradiation. In contrast, growth arrest mediated by a temperature shift to 32 degrees C protected S100B-REF cells from apoptosis. Our results suggest a model in which calcium signalling, linked with cPKC activation, cooperates with S100B to promote wild-type p53 nuclear translocation in early G(1) phase and activation of a p53-dependent G(1) checkpoint control.

Calcium-dependent interaction of S100B with the C-terminal domain of the tumor suppressor p53.

In vitro, the S100B protein interacts with baculovirus recombinant p53 protein and protects p53 from thermal denaturation. This effect is isoform-specific and is not observed with S100A1, S100A6, or calmodulin. Using truncated p53 proteins in the N-terminal (p53(1-320)) and C-terminal (p53(73-393)) domains, we localized the S100B-binding region to the C-terminal region of p53. We have confirmed a calcium-dependent interaction of the S100B with a synthetic peptide corresponding to the C-terminal region of p53 (residues 319-393 in human p53) using plasmon resonance experiments on a BIAcore system. In the presence of calcium, the equilibrium affinity of the S100B for the C-terminal region of p53 immobilized on the sensor chip was 24 +/- 10 nM. To narrow down the region within p53 involved in S100B binding, two synthetic peptides, O1(357-381) (residues 357-381 in mouse p53) and YF-O2(320-346) (residues 320-346 in mouse p53), covering the C-terminal region of p53 were compared for their interaction with purified S100B. Only YF-O2 peptide interacts with S100B with high affinity. The YF-O2 motif is a critical determinant for the thermostability of p53 and also corresponds to a domain responsible for cytoplasmic sequestration of p53. Our results may explain the rescue of nuclear wild type p53 activities by S100B in fibroblast cell lines expressing the temperature-sensitive p53val135 mutant at the nonpermissive temperature.

[Endovascular treatment of subclavian artery aneurysm]. / Endovaskulaer behandling af arteria subclavia-aneurisme.

The standard management of subclavian artery aneurysm has until now been surgical repair. The treatment of these aneurysms with transluminal placement of a prosthetic graft stent device is a new approach and seems to be feasible and safe. One case is presented. This new technique warrants further clinical assessment and trial.

Increased bax expression is associated with cell death induced by ganciclovir in a herpes thymidine kinase gene-expressing glioma cell line.

The herpes simplex virus thymidine kinase gene (HSV-tk) was stably transfected into rat C6 glioma cells (C6tk) in order to characterize the mechanisms underlying cell toxicity induced in vitro by the guanosine analog ganciclovir (GCV). The results demonstrate the efficiency of the HSV-tk/GCV system in ablating most of the tumoral cells within 7 to 8 days of treatment with 20 mivroM GCV; however, a few cells still survive. C6tk cells arrest in the S phase of the cell cycle after 2 days of drug treatment before undergoing cell death. Microscopic analysis reveals dying cells with ultrastructural characteristics consistent with apoptosis; we cannot rule out, however, that necrotic cell death may also be occurring. The cytotoxicity induced by GCV is not associated with changes in the expression of p53 protein, suggesting that cell cycle arrest and cell death may occur through a p53-independent pathway. C6tk cells constitutively express Bcl-xL and Bax proteins; when exposed to GCV, Bcl-xL levels do not change but Bax accumulation is rapidly induced. These findings suggest that the balance between Bcl-xL and Bax proteins may be of importance in determining the sensitivity of tumoral cells to GCV.

Calcium and S100B regulation of p53-dependent cell growth arrest and apoptosis.

In glial C6 cells constitutively expressing wild-type p53, synthesis of the calcium-binding protein S100B is associated with cell density-dependent inhibition of growth and apoptosis in response to UV irradiation. A functional interaction between S100B and p53 was first demonstrated in p53-negative mouse embryo fibroblasts (MEF cells) by sequential transfection with the S100B and the temperature-sensitive p53Val135 genes. We show that in MEF cells expressing a low level of p53Val135, S100B cooperates with p53Val135 in triggering calcium-dependent cell growth arrest and cell death in response to UV irradiation at the nonpermissive temperature (37.5 degreesC). Calcium-dependent growth arrest of MEF cells expressing S100B correlates with specific nuclear accumulation of the wild-type p53Val135 conformational species. S100B modulation of wild-type p53Val135 nuclear translocation and functions was confirmed with the rat embryo fibroblast (REF) cell line clone 6, which is transformed by oncogenic Ha-ras and overexpression of p53Val135. Ectopic expression of S100B in clone 6 cells restores contact inhibition of growth at 37.5 degreesC, which also correlates with nuclear accumulation of the wild-type p53Val135 conformational species. Moreover, a calcium ionophore mediates a reversible G1 arrest in S100B-expressing REF (S100B-REF) cells at 37.5 degreesC that is phenotypically indistinguishable from p53-mediated G1 arrest at the permissive temperature (32 degreesC). S100B-REF cells proceeding from G1 underwent apoptosis in response to UV irradiation. Our data support a model in which calcium signaling and S100B cooperate with the p53 pathways of cell growth inhibition and apoptosis.

Cysteine oxidation in the mitogenic S100B protein leads to changes in phosphorylation by catalytic CKII-alpha subunit.

The glial-derived calcium-binding protein S100B can be secreted to act as a neurotrophic factor or a mitogen, stimulating proliferation of glial cells. The extracellular S100B activities rely on the oxidation of the protein cysteine residues (Kligman, D., and Marshak, D. R. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 7136-7139; Winningham-Major, F., Staecker, J. L., Barger, S. W., Coats, S., and Van Eldik, L. J. (1989) J. Cell Biol. 109, 3063-3071). Here we show that oxidation of the S100B cysteine residues, Cys-68 and Cys-84, induces a conformational change in the protein structure, unmasking a canonical CKII phosphorylation site located within the typical EF-hand calcium-binding site IIbeta. Intrasubunit disulfide-bridged S100B monomer and disulfide-bonded S100B dimer are phosphorylated by the catalytic CKII-alpha subunit on Ser-62 with a Km of 0.5 microM and a Vmax of 10 pmol/min/100 pmol of S100B. Oxidized S100B is the best in vitro CKII-alpha substrate identified so far. Next we show that intrasubunit disulfide-bridged S100B monomer is the most potent S100B species to stimulate [3H]thymidine uptake by C6 glial cells in culture. In addition, the phosphorylated intrasubunit disulfide-bridged S100B monomer retains apparent mitogenic activity toward C6 glial cells, and hence, 32P-labeled S100B should be a useful probe for characterizing the mechanisms by which extracellular oxidized S100B functions. Finally, we show that formation of intrasubunit disulfide-bridged S100B monomer is stimulated by peroxynitrite anion, suggesting that production of mitogenic S100B species could be enhanced in neuropathology associated with peroxynitrite anion production.

The in vitro phosphorylation of p53 by calcium-dependent protein kinase C--characterization of a protein-kinase-C-binding site on p53.

We show that, in vitro, Ca2+-dependent protein kinase C (PKC) phosphorylates recombinant murine p53 protein on several residues contained within a conserved basic region of 25 amino acids, located in the C-terminal part of the protein. Accordingly, synthetic p53-(357-381)-peptide is phosphorylated by PKC at multiple Ser and Thr residues, including Ser360, Thr365, Ser370 and Thr377. We also establish that p53-(357-381)-peptide at micromolar concentrations has the ability to stimulate sequence-specific DNA binding by p53. That stimulation is lost upon phosphorylation by PKC. To further characterise the mechanisms that regulate PKC-dependent phosphorylation of p53-(357-381)-peptide, the phosphorylation of recombinant p53 and p53-(357-381)-peptide by PKC were compared. The results suggest that phosphorylation of full-length p53 on the C-terminal PKC sites is highly dependent on the accessibility of the phosphorylation sites and that a domain on p53 distinct from p53-(357-381)-peptide is involved in binding PKC. Accordingly, we have identified a conserved 27-amino-acid peptide, p53-(320-346)-peptide, within the C-terminal region of p53 and adjacent to residues 357-381 that interacts with PKC in vitro. The interaction between p53-(320-346)-peptide and PKC inhibits PKC autophosphorylation and the phosphorylation of substrates, including p53-(357-381)-peptide, neurogranin and histone H1. Conventional Ca2+-dependent PKC alpha, beta and gamma and the catalytic fragment of PKC (PKM) were nearly equally susceptible to inhibition by p53-(320-346)-peptide. The Ca2+-independent PKC delta was much less sensitive to inhibition. The significance of these findings for understanding the in vivo phosphorylation of p53 by PKC are discussed.

Casein kinase 2 inhibits the renaturation of complementary DNA strands mediated by p53 protein.

Considerable effort is currently being devoted to understand the functions of protein p53, a major regulator of cell proliferation. The protein p53 has been reported to catalyse the annealing of complementary DNA or RNA strands. We report that this activity is inhibited in the presence of the serine/threonine protein kinase CK2. It is shown that this inhibition can be explained by the occurrence of a high-affinity molecular association between p53 and CK2. The molecular complex involves an interaction between the C-terminal domain of p53 and the beta subunit of the oligomeric kinase. Accordingly, the isolated alpha subunit of the kinase was without effect. In addition, after phosphorylation by CK2, phosphorylated p53 lost its DNA annealing activity. Because the C-terminal domain of p53 is both involved in the association with CK2 and phosphorylated by it, our results suggest that either protein-protein interaction or phosphorylation of this domain might control the base pairing of complementary sequences promoted by p53 in processes related to DNA replication and repair.

Interactions of myogenic bHLH transcription factors with calcium-binding calmodulin and S100a (alpha alpha) proteins.

MyoD belongs to a family of myogenic basic helix-loop-helix (bHLH) transcription factors that activate muscle-specific genes. The basic helix I sequence of the bHLH motif contains a consensus sequence for protein kinase C (PKC) substrates. We show here that MyoD is indeed phosphorylated by PKC in vitro on Thr 115 within the basic part of the bHLH motif. By analogy with calmodulin-target peptide models, we also identified within the consensus basic helix I motif of myogenic proteins a conserved putative calmodulin/S100-binding domain. Calcium-dependent interaction between MyoD with calmodulin and the abundant muscle S100a(alpha alpha) proteins was demonstrated by affinity chromatography and cross-linking experiments. The binding of calmodulin and S100a inhibited MyoD phosphorylation by PKC as well as MyoD DNA binding activity. S100a was found to be more efficient than calmodulin in antagonizing DNA binding to MyoD. We next developed a rapid purification method for bacterial recombinant MyoD-bHLH domain by affinity chromatography using a calmodulin-Sepharose column and investigated the phosphorylation of that peptide by PKC and its interactions with calmodulin and S100a. We confirmed the phosphorylation of the threonine residue 115 in the MyoD-bHLH by PKC with a Km of 0.8 microM. Calmodulin and S100a binding inhibited MyoD-bHLH phosphorylation by PKC. A strict calcium-dependent interaction between calcium binding proteins and the MyoD-bHLH was identified by native gel electrophoresis and fluorescence spectroscopy with 5-(dimethylamino)naphthalene-1-sulfonylcalmodulin. The MyoD-bHLH bound to fluorescently labeled 5-(dimethylamino)naphthalene-1-sulfonylcalmodulin with a dissociation constant around 20 nM. S100a inhibited stoichiometrically the binding of the bHLH peptide for labeled calmodulin, suggesting an affinity of S100a for the bHLH peptide at least 1 order of magnitude higher than calmodulin. In favor of an in vivo interaction between S100a and MyoD, we report that S100a- and MyoD-like immunoreactivities colocalize in H9c2 cells, and that a significant amount of MyoD-like immunoreactivity is recovered in the S100a immunoprecipitate from crude H9c2 cell extract in the presence of calcium. We propose that myogenic proteins represent a new family of calmodulin/S100-binding PKC substrates and that calmodulin/S100a could participate in the regulation of the bHLH myogenic protein activities.

The protein kinase C activator, phorbol ester, cooperates with the wild-type p53 species of Ras-transformed embryo fibroblasts growth arrest.

Clone 112 cells, a rat embryo fibroblast cell line cotransfected by an activated ras gene and a temperature-sensitive mutant p53 gene (p53val135) grow well at 37 degrees C but cease DNA synthesis and cell division when shifted to 32 degrees C (Michalovitz, D., Halevy, O., and Oren, M. (1990) Cell 62, 671-680). Characterization of the p53 protein in exponentially growing clone 112 cells at 37 degrees C revealed that both wild-type (reactive with the monoclonal antibody PAb 246) and mutant (reactive with PAb 240) p53 conformational forms are co-expressed. These results indicate that in clone 112 cells the growth suppressor activity of the wild-type p53 species is inactivated at 37 degrees C. We show that clone 112 cells grown at 37 degrees C elicits specific growth inhibition response to stimulation by the tumor promoter phorbol ester, phorbol 12-myristate 13-acetate (PMA). At 37 degrees C, PMA induced nuclear accumulation of the p53 protein, a behavior that is also observed in growth-arrested cells at 32 degrees C. Furthermore, when cells are growth arrested at 32 degrees C, PMA prevented the cells from re-entering the cell cycle when they are shifted back to 37 degrees C. All these observations suggest that PMA can cooperate with the wild-type p53 in cell growth arrest. At 37 and 32 degrees C, PMA stimulation of clone 112 cells resulted in specific enhancement of phosphorylation of the wild-type p53 species but not of the mutant form. We also demonstrate that the growth arrest of clone 112 cells at 37 degrees C is correlated with stimulation of the nuclear wild-type p53-DNA binding activities. The PMA-mediated increase in p53 DNA binding activity coincides with the loss of the PAb 421 epitope on the p53.DNA complex. PAb 421 non-reactivity with p53 has been shown by others to occur in growth-arrested cells and upon phosphorylation of p53 by protein kinase C. We also provide evidence that, in vitro, the protein kinase C mode of phosphorylation stimulates DNA binding activities of purified recombinant wild-type p53 and that in a mutant conformation p53 is not a substrate for protein kinase C. We propose that wild-type p53 and protein kinase C, the cellular receptor of phorbol ester, could participate in the negative feedback controls associated with the phosphoinositide-derived signals common to a number of mitogenic stimulations.

Characterization of baculovirus recombinant wild-type p53. Dimerization of p53 is required for high-affinity DNA binding and cysteine oxidation inhibits p53 DNA binding.

A high-yield, rapid and non-denaturing purification protocol for baculovirus recombinant wild-type p53 is described. Gel-filtration chromatography and chemical cross-linking experiments indicated that purified p53 assembles into multimeric forms ranging from tetramer to higher oligomers. A gel-mobility-shift assay and protein-DNA cross-linking studies demonstrated that purified baculovirus recombinant p53 binds to consensus DNA target as a dimer but that additional p53 molecules may then associate with the preformed p53-dimer-DNA complexes to form larger p53 DNA complexes. These observations suggest that the p53 tetramers and higher oligomers that form the minimal p53 association in solution dissociate upon DNA binding to form p53 dimer-DNA complexes. Binding of the mAB PAb 421 to the oligomerization-promoting domain on p53 stimulated sequentially formation of both p53-dimer-DNA and larger p53-DNA complexes. This observation suggests that factors may exist in vivo that could participate in the formation and the stabilization of the various p53-DNA complexes. Further characterization of the purified p53 revealed that the protein possesses highly reactive cysteine residues. We show that intrachain disulfide bonds form within the purified p53 molecules during storage in the absence of reducing agent. Zn2+ binding to p53 protect sulfhydryl groups from oxidation. Cysteine oxidation by intramolecular disulfide-bond formation did not modify the wild-type immunoreactive phenotype of the p53 protein but totally inhibited its DNA-binding activities. The oxidation of the p53 cysteine residues was also observed for nuclear p53 in baculovirus-infected insect cells. The redox status of the nuclear p53 regulates its DNA-binding activity in vitro confirming the essential role of the reduced state of cysteine residues in p53 for detectable DNA-binding activity.

De novo synthesis of GAP-43: in situ hybridization histochemistry and light and electron microscopy immunocytochemical studies in regenerating motor neurons of cranial nerve nuclei in the rat brain.

In order to investigate the modulation of the synthesis and the subcellular localization of the growth associated protein GAP-43 in neuronal cell bodies we have taken advantage of the well known regenerative properties of axotomized motor neurons of the facial and hypoglossal nuclei. Alterations in the levels of GAP-43 mRNA containing cells were studied by in situ hybridization histochemistry. The protein localization was examined using immunohistochemistry at the light and electron microscopic levels. Neurons from the control side showed undetectable levels of both GAP-43-like immunoreactivity and GAP-43 mRNA levels. Whereas axotomized neurons exhibited a marked increase in GAP-43 mRNA levels and in GAP-43-like immunoreactivity. Three to 50 days after axotomy, motor neurons ipsilateral to the lesion displayed a dense reticular or filamentous perinuclear distribution of the immunoreactivity in somata and proximal dendritic processes, corresponding to the location of the Golgi apparatus in these neurons. At the electron microscopic level the immunoreactivity was located in the cisternae of the Golgi complex and found to be associated with trans-side vesicles of these complexes. The myelinated fibers of the transectomized facial nerve also presented an intense GAP-43-like immunoreactivity. Twenty-one days after the axotomy a decay in the number of immunostained neurons and in the intensity of immunolabeled somata was observed. Our study reveals a rapid induction of GAP-43 mRNA and protein after axotomy. The localization of the newly synthesized GAP-43-like immunoreactivity to the Golgi apparatus seen in the present work suggests an early association of this protein with newly formed membranes prior to transport toward the terminals through the axons.

Expression of neuromodulin (GAP-43) and its regulation by basic fibroblast growth factor during the differentiation of O-2A progenitor cells.

In a recent work we have shown that neuromodulin (Nm, also known as GAP-43), a protein kinase C substrate, previously believed to be expressed exclusively in neurons, is also present in glial cells. Here we investigated the expression of Nm and its mRNA in O-2A glial progenitor cells (common precursor for oligodendrocytes and type-2 astrocytes) during their development in secondary culture and under the influence of basic fibroblast growth factor (bFGF). The different stages of oligodendrocyte development were characterized by the expression of surface markers: A2B5, which identifies O-2A glial precursor cells, and O4 and galactocerebroside (GC), which characterize later developmental stages. The number of cells expressing Nm (about 90% at culture initiation) decreased rapidly during the first 2 days and reached a plateau at around 30-40%. The level of Nm mRNA followed a similar kinetic. Immunocytochemistry demonstrated that at 4 days in vitro about 25-30% cells were A2B5+, 30-40% Nm+, a high percentage (60-70%) O4+, and 35-40% GC+. Nearly all of the morphologically immature A2B5+ cells expressed also the Nm antigen, very few of the O4+ cells still expressed Nm and almost no cells expressed both GC and Nm. Most O4+ cells developed a typical oligodendrocyte morphology and were essentially GC+. This study also showed that in the presence of serum, the A2B5+ Nm+ and O4+ Nm+ (GC-) cells retained their bipotentiality and differentiated into GFAP+ (glial fibrillary acidic protein) Nm+ type-2 astrocytes. The bFGF was found to stimulate the proliferation of Nm+ 0-2A precursor cells and to increase the level of Nm mRNA. At 4 days under this culture condition, the predominant cell type was A2B5+ and Nm+. Only 25-35% of the cells were O4+, but 90-95% of them were Nm+. Very few GC+ cells were visible in the presence of bFGF, but 20-40% of them were Nm+. These data indicate that Nm is essentially associated to glial O-2A precursor cells and further confirm that bFGF blocks the differentiation of these cells. It is suggested that Nm plays a role in the plasticity (developmental potential) of the bipotential 0-2A progenitor cells.

Characterization of the tumor suppressor protein p53 as a protein kinase C substrate and a S100b-binding protein.

We report here that the negative cell cycle regulator protein p53 is an in vivo and in vitro substrate for protein kinase C, a cellular receptor for the tumor-promoter phorbol esters. We also demonstrate that p53 interacts in a calcium-dependent manner with S100b, a member of the S100 protein family involved in cell cycle progression and cell differentiation, and that such an interaction inhibits in vitro p53 phosphorylation by protein kinase C. The interaction between p53 and S100b was utilized for the purification of cellular and recombinant murine p53 by affinity chromatography with S100b-Sepharose. Furthermore, and of particular interest, we have shown that purified p53 undergoes temperature-dependent oligomerization and that the interaction between S100b and p53 not only induces total inhibition of p53 oligomerization but also promotes disassembly of the p53 oligomers. We suggest that these effects result from the binding of S100b to the multifunctional basic C-terminal domain of p53 and propose that p53 may be a cellular target for the S100 protein family members involved in the control of the cell cycle at the G0-G1/S boundary.