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M Passalacqua

Publications and source records attributed to M Passalacqua.

At least 37 records · Page 2Linked to original sources
PubMed1996

Extracellular high-mobility group 1 protein is essential for murine erythroleukaemia cell differentiation.

A high-mobility group 1 (HMG1) protein type isolated from murine erythroleukaemia (MEL) cells promotes acceleration of the differentiation process when added to a MEL cell culture together with the inducer hexamethylene bisacetamide. We now provide direct evidence that the presence of HMG1 protein in the extracellular medium is essential for terminal erythroid differentiation. An extracellular function for HMG1 protein in MEL cell is further supported by a demonstration that this protein is released from MEL cells exposed to the chemical inducer and that the addition of an anti-(HMG1 protein) monoclonal antibody to the cell culture inhibits the differentiation process almost completely. The release of HMG1 protein from MEL cells is modulated by compounds affecting cell calcium homoeostasis, such as a calcium ionophore or verapamil. In fact, in the presence of the ionophore an increased rate of differentiation is accompanied by an enhanced extracellular release of HMG1 protein, whereas in the presence of verapamil both phenomena are significantly decreased.

Acetamides↗
PubMed1996

A 6 kDa protein homologous to the N-terminus of the HMG1 protein promoting stimulation of murine erythroleukemia cell differentiation.

Murine erythroleukemia (MEL) cells, in addition to an mRNA coding for a 30 kDa high mobility group (HMG)-1 protein, contain an mRNA coding for a 6 kDa HMG1 protein having the following structural properties: (1) its primary structure has 90% homology with the N-terminal sequence of the 30 kDa HMG1 protein; (2) it contains a consensus region of the HMG1 protein family; (3) it is deprived of the cluster of acidic amino acids that characterizes the C-terminal region of the 30 kDa HMG1 protein. This novel small Mr HMG1 protein has been expressed in prokaryotic cells and tested to establish similarities and differences in activity compared to the homologous higher Mr HMG1 protein. It has been found that the low Mr HMG1 form is not released from MEL cells following induction to erythroid differentiation, but is still effective, although with much less efficiency, when added to the external medium, in promoting acceleration in the rate of MEL cell differentiation as well as in activation of alpha-protein kinase C. Altogether these results provide evidence for the presence in MEL cells of a multigene family that encodes at least two different HMG1-type sequences most presumably involved, at distinct cellular sites, in different functions although commonly related to the promotion of cell differentiation. Additional information can be considered concerning the relationship between the characteristic N-terminal sequence of HMG1 protein and the extracellular activity on MEL cell differentiation.

Animals↗
PubMed1996

Correlation between levels of delta protein kinase C and resistance to differentiation in murine erythroleukemia cells.

It has been demonstrated that the level of delta protein kinase C is inversely correlated to the responsiveness of murine erythroleukemia cells to chemical induction to terminal erythroid differentiation. In these cells, deltaPKC is largely present in a membrane associated form, and thus in a constitutively active state, a condition which characterizes the undifferentiated phenotype. Accordingly, commitment to cell differentiation has been shown to be preceded by down regulation of deltaPKC, a process significantly accelerated and induced to almost completion by the differentiation enhancing factor (DEF) in a dose dependent manner. The present results provide a better understanding of the role of deltaPKC in characterizing the undifferentiated MEL cell phenotype and suggest a relationship between the acceleration in the rate of differentiation induced by DEF and the down regulation of this kinase form.

Animals↗
PubMed1995

Antisense oligodeoxynucleotide inhibition of delta protein kinase C expression accelerates induced differentiation of murine erythroleukaemia cells.

The potential regulatory role of delta protein kinase C (delta PKC) in murine erythroleukaemia cell differentiation was studied by using antisense oligodeoxynucleotides targeting the translation initiation region of mouse delta PKC mRNA. Cell treatment with antisense oligonucleotides, at a concentration of 20 microM, followed by hexamethylenebisacetamide induction, produced a specific 2-fold increase in the differentiation rate of both slowly and rapidly differentiating murine erythroleukaemia cell clones. Cell permeabilization by a cationic lipid resulted in a decrease of one order of magnitude in the amounts of antisense oligonucleotides necessary to elicit the maximal response, and accelerated the kinetics of the stimulatory effect. These changes in murine erythroleukaemia cell differentiation rates, observed in both cell clones, were associated with 60% and 50% decreases, respectively, in delta PKC immunoreactive protein in slowly and rapidly differentiating cells. The present results indicate strongly that basal levels of delta PKC in murine erythroleukaemia cells are essential in regulating the initial differentiation rate of these cells in response to chemical induction, and provide further evidence that this PKC isoform plays a fundamental role in maintaining the undifferentiated phenotype of murine erythroleukaemia cells.

Acetamides↗
PubMed1995

Extracellular release of the 'differentiation enhancing factor', a HMG1 protein type, is an early step in murine erythroleukemia cell differentiation.

Differentiation enhancing factor (DEF) is a 29 kDa protein expressed in murine erythroleukemia (MEL) cells and active in promoting a significant increase in the rate of hexamethylenebisacetamide induced differentiation of these cells. The factor was recently shown to possess an amino acid sequence identical to that reported for one of the HMG1 proteins, designated as 'amphoterin' on the basis of its highly dipolar sequence. In the present study, we have expressed DEF cDNA in an E. coli strain and found that the recombinant protein has functional properties identical to those observed with native DEF. Furthermore, we demonstrate that, following MEL cell stimulation with the chemical inducer, DEF is secreted in large amounts in the extracellular medium. In fact, the N-terminal sequence and the partial amino acid sequence of tryptic peptides from the secreted protein correspond to those of DEF isolated from the soluble fraction of resting MEL cells. These results are indicative for an extracellular localization as the site of action of DEF and suggest a novel function for proteins belonging to the HMG1 family. Finally, the early decay of DEF mRNA, in chemical induced MEL cells, support the hypothesis that the involvement of the enhancing factor occurs and is completed in the early phases of cell differentiation.

Animals↗
PubMed1995

Identity in molecular structure between "differentiation enhancing factor" of murine erythroleukemia cells and the 30 kD heparin-binding protein of developing rat brain.

A 29 kD protein previously isolated from murine erythroleukemia (MEL) cells and shown to enhance the rate of differentiation of these cells has now been demonstrated to possess an amino acid sequence identical to that reported for the 30 kD heparin-binding protein from developing rat brain, named amphoterin after its highly dipolar structure. The identity between the two proteins has been established on the basis of a strong heparin binding affinity and a complete homology in the amino acid sequences of N-terminal region as well as of several tryptic peptides. Furthermore, the cDNA encoding this protein has been isolated from MEL cell mRNA, by means of reverse transcriptase-polymerase chain reaction, and its sequence was found to correspond to that of amphoterin. The MEL cell differentiation enhancing factor, previously abbreviated as DEF, is again confirmed to reduce the latent period preceding the appearance of hexamethylenebisacetamide induced cell commitment and to stimulate the catalytic activity of alpha-protein kinase C. Thus, here we demonstrate that a protein expressed in MEL cells, whose sequence is identical to that previously reported for amphoterin, plays an essential role in promoting cell differentiation, thereby indicating a new relevant function of amphoterin.

Amino Acid Sequence↗
PubMed1995

Changes in calcium influx affect the differentiation of murine erythroleukaemia cells.

As indicated by direct evidence, obtained by altering the cell-membrane permeability for Ca2+ in murine erythroleukaemia (MEL) cells, calpain is the triggering factor which connects fluctuations of the intracellular Ca2+ concentrations to the decay of protein kinase C (PKC), as well as to the kinetics of cell differentiation induced by hexamethylenebisacetamide. Cell exposure to verapamil caused a profound decrease in the rate of PKC down-regulation and a slower initial rate of accumulation of mature erythroid cells, whereas addition of the Ca2+ ionophore A23187 produced opposite effects. The high susceptibility of PKC-delta to calpain degradation, at concentrations of Ca2+ much lower than those required for degradation of the other PKC isoforms, may be explained by the finding that this kinase isoform is predominantly associated with the cell membrane. The different cellular localizations, as well as the different susceptibilities to calpain digestion, further support the hypothesis that in MEL cells the various PKC isoforms play distinct biological functions that are critical for the maintenance of the undifferentiated state of the cell and for its commitment to terminal erythroid differentiation.

Acetamides↗
PubMed1995

Decreased endothelium-dependent relaxation in subarachnoid hemorrhage-induced vasospasm: role of ET-1.

The purpose of this study was to test whether endothelium-dependent relaxation is decreased during acute vasospasm following subarachnoid hemorrhage (SAH) and the mechanism underlying the decrease. Basilar artery in situ was 35% constricted 3 days following injection of autologous arterial blood into the rabbit cisterna magna compared with vessels from control rabbits. In situ suffusion with the endothelium-dependent relaxant, acetylcholine (ACh; 10 microM), relaxed resting and serotonin (5-HT)-contracted control vessels but not vasospastic and 5-HT-contracted vasospastic vessels. In contrast, the relaxant potency and efficacy of ACh was similar in control and vasospastic vessels contracted with 5-HT in vitro. In situ suffusion with the ETA-receptor antagonist, BQ-123 (1 microM), reversed the vasospasm by 51% and restored the magnitude of ACh relaxation of vasospastic and 5-HT-contracted vasospastic vessels to that of controls. ACh in situ and in vitro relaxed endothelin-1 (ET-1)-contracted control vessels to a smaller magnitude than 5-HT-contracted control vessels. These results suggest, in contrast to previous studies, that endothelium-dependent relaxation is decreased during acute vasospasm following SAH. The decreased endothelium-dependent relaxation is secondary to the underlying ET-1-mediated spasm. The inhibition of endothelium-dependent relaxation observed in situ following SAH cannot be demonstrated in vitro, presumably due to loss of the ET-1-mediated vasospasm.

Acetylcholine↗
PubMed1994

Differential expression of protein kinase C isoform genes in three murine erythroleukemia cell variants: implication for chemical induced differentiation.

The presence of alpha, delta, epsilon, theta, and zeta protein kinase C isoforms in DS19 murine erythroleukemia cells has been established in this study. In addition, the mRNA levels of these isozymes have been measured by quantitative reverse transcriptase-polymerase chain reaction. Isoform delta has been found to be the most abundant isotype, whereas isoform zeta resulted to be present in only few copies. Furthermore, the expression levels of all five protein kinase C isozymes have been studied in three cell clones, derived from parental DS19 cells and characterized by different susceptibilities to differentiation. This comparative analysis indicated that the calcium-independent isozymes (delta, epsilon, zeta, and theta) display significantly higher expression levels in cells less prone to differentiation. On the other hand, the mRNA levels of the only calcium-dependent isoform present (alpha) fluctuate poorly from one cell clone to the other, but are the highest in the cell clone characterized by the fastest rate of differentiation. This study represents the first complete characterization of the basal levels of specific protein kinase C isotypes in different murine erythroleukemia cell clones and provides further evidence for the role of individual isozymes in the early events that trigger chemical induced murine erithroleukemia cell differentiation.

Animals↗
PubMed1994

Modulation of the intracellular Ca(2+)-dependent proteolytic system is critically correlated with the kinetics of differentiation of murine erythroleukemia cells.

Calpain has been identified as the intracellular proteinase that catalyzes the selective down-regulation of protein kinase C (PKC) isoforms, occurring in the early stages of commitment to terminal erythroid differentiation of murine erythroleukemia (MEL) cells induced by hexamethylenebisacetamide. This conclusion has been reached through direct experiments performed with two MEL cell clones, one characterized by a high and the other by a low rate of differentiation. In both cell types, introduction of an anti-calpain antibody resulted in a significant delay in the onset of down-regulation of PKC isoforms, and in an increase in the latent period that precedes differentiation. Both cell lines also displayed reduced rates of PKC decay and accumulation of mature erythroid cells. Furthermore, in the fast-responding clone, calpastatin, the natural calpain-inhibitor protein, was found to be almost completely absent, resulting in activation and expression of proteolytic activity of calpain even at micromolar concentrations of Ca2+, a condition not sufficient to trigger calpain activation in the slowly responding clone which contains high levels of calpastatin. The fast-responding MEL cell clone, enriched with calpastatin, displayed a lower rate of cell differentiation, with a kinetics almost identical to that observed following introduction of the anti-calpain antibody. It is proposed that Ca(2+)-dependent proteolysis plays a crucial role for the progress of MEL cell differentiation through the specific degradation of PKC isozymes.

Acetamides↗
PubMed1994

Protein kinase C isoforms in murine erythroleukemia cells and their involvement in the differentiation process.

In addition to alpha, delta and epsilon-protein kinase C, murine erythroleukemia cells contain zeta-PKC and also a c-PKC isoform, named alpha 1, which shows cross-reactivity with an anti-alpha-PKC antipeptide antibody. In a C44 MEL cell clone, characterized by a high rate of differentiation, both c-PKC forms are expressed at a level higher than that of the N23 MEL cell clone which differentiates at a low rate and contains higher levels of epsilon-PKC and particularly of the delta-PKC isozyme. In the course of MEL cell differentiation, delta-PKC in N23 cells and alpha 1-PKC in C44 cells are rapidly down-regulated and the overall process is almost completed before cell commitment. Of the other three PKC isozymes present in both clones, only alpha-PKC is down-regulated to a significant extent. It is proposed that modulation of the signal delivered by each PKC isozyme is one of the biochemical mechanisms involved in MEL cell differentiation.

Acetamides↗
PubMed1993

Differentiation of HL60 promyelocytic cells is promoted by a 'differentiation enhancing factor' produced by erythroleukemia cells.

A differentiation enhancing factor isolated from murine erythroleukemia cells is also a potent enhancer of the differentiation of HL60 human promyelocytic leukemia cells, induced by retinoic acid and by phorbol ester. This stimulating effect is the result of a large increase in the sensitivity of HL60 cells for retinoic acid and for phorbol 12-myristate 13-acetate (20-fold and 40-fold, respectively). Accelerated differentiation induced by the protein factor, and monitored by the appearance of marker enzymes, is accompanied by a large increase in the fluctuation of the levels of protein kinase C (PKC) isozymes in HL60 cells. These results provide further support for the role of this new protein factor in cell differentiation and indicate that other cell types are susceptible to its biological effect.

Cell Differentiation↗
PubMed1993

Characterization of the biological role of murine erythroleukemia cells "differentiation enhancing factor" using antisense oligodeoxynucleotides.

On the basis of the amino acid sequence of isolated tryptic peptides, it has been established that the differentiation enhancing factor, produced and active on murine erythroleukemia (MEL) cells, possesses a unique sequence, with no similarity to that of known proteins. Accordingly, this factor can be defined as a novel biologically active peptide. An antisense oligodeoxynucleotide, deduced from the sequence of a non-decapeptide (produced by tryptic digestion of the factor), decreases the rate and the extent of MEL cell differentiation, induced by hexamethylenebisacetamide. In these cells the amount of the factor is reduced to one third of that constitutively present in untreated cells. Exogenous addition of the factor restores cell inducibility to normal values. Taken together, these results demonstrate the presence in MEL cells of a new factor, structurally and functionally unrelated to any of the known biologically active peptides, and suggest its crucial role in the promotion of an initial signal, in chemically induced erythroid differentiation.

Amino Acid Sequence↗
PubMed1993

Role of delta-PKC on the differentiation process of murine erythroleukemia cells.

In murine erythroleukemia (MEL) cells the length of the latent period before the onset of hexamethylenebisacetamide induced terminal erythroid differentiation is inversely correlated to the intracellular level of delta-PKC. This is supported by the following experimental evidence. V3.17[44] MEL cell line, characterized by a very high rate of differentiation, contains an amount of delta-PKC protein one third lower than that present in the N23 MEL cell line, characterized by a very low rate of differentiation. A similar difference in the amount of delta-PKC mRNA is present in the two cell lines. In N23 cells, following addition of HMBA, the amount of delta-PKC protein and delta-PKC mRNA is down-regulated to one third its original value, which now corresponds to that constitutively present in V3.17[44] cells. Furthermore, in these cells the levels of delta-PKC protein and of its specific mRNA are unaffected by treatment with HMBA. Following introduction of homologous purified delta-PKC both MEL cell variants display a longer latent period before the onset of differentiation from 50 to 75 hours in N23 cell line and from 20 to 40 hours in V3.17[44] cells, respectively. Taken together, these results suggest that a delta-PKC related signal plays a negative role in the early stages of MEL cell differentiation and that the level of the kinase is controlled through a down-regulation process upon exposure to the chemical inducer.

Acetamides↗
PubMed1991

Differentiation of murine erythroleukemia cells by hexamethylenebisacetamide involves secretion and binding to membranes of a differentiation enhancing factor.

A protein factor previously shown to enhance terminal differentiation of transformed erythroid cells is synthesized by murine erythroleukemia cells and secreted in the early stages of differentiation induced by hexamethylenebisacetamide (HMBA). Secretion also occurs, constitutively, in the absence of inducer, from a murine erythroleukemia cell variant characterized by an accelerated response to HMBA. The protein factor binds to intact cells following addition of HMBA and enhances translocation of protein kinase C to the nuclear fraction. These results strongly support an important role for this natural protein factor in cell differentiation.

Acetamides↗
PubMed1990

A vincristine-resistant murine erythroleukemia cell line secretes a differentiation enhancing factor.

A clone of vincristine resistant murine erythroleukemia cells V3.17[44], characterized by high sensitivity to terminal erythroid differentiation induced by hexamethylene bisacetamide, secretes into the extracellular medium a protein factor which partially reduces the latent period before commitment and accelerates the expression of the terminal differentiated phenotype in a slow responding murine erythroleukemia N23 cell variant. This differentiation enhancing factor increases the rate of protein kinase C down-regulation which occurs at slower rate during cell differentiation. The activity of the factor is detected either by coculture of the two cell line variants or by addition of conditioned medium from V3.17[44] cells to a culture of N23 cells in the presence of the inducer. In addition to being secreted by V3.17[44] cells, this factor can also be detected in the cytoplasm of both V3.17[44] and N23 cells, associated with a particulate fraction from which it can be released by sonication.

Animals↗
PubMed1990

Murine erythroleukemia cell differentiation: possible involvement of a calcium dependent neutral proteinase.

Murine erythroleukemia cells contain a single type of calpain classified, on the basis of its calcium requirement, as a type I proteinase. The enzyme is practically inactive at concentrations of calcium below 10 microM and expresses maximal activity in the presence of 0.12-0.15 mM Ca2+. The affinity for Ca2+ cannot be reduced by exposure of the enzyme to conditions known to promote autoproteolysis of calpain. Expression of catalytic activity at lower concentrations of Ca2+, is promoted by the interaction with phospholipid vesicles or plasma membranes. Conditions that promote activation of calpain, induce also the self-inactivation of the enzyme. During terminal differentiation of murine erythroleukemia cells induced by HMBA, the intracellular level of calpain activity undergoes significative reduction. Similar decrease in calpain activity progressively occurs during the loss of sensitivity to HMBA as a result of density growth arrest.

Acetamides↗