Biomedical subjects

S Capitani

Publications and source records attributed to S Capitani.

At least 91 records · Page 5Linked to original sources

PubMed1997

Nuclear lipid-dependent signal transduction in human osteosarcoma cells.

The enzymes and substrates involved in phosphoinositide signal transduction which have been detected in the nucleus of several cell types have been demonstrated to be responsive to agonists. The complexity of this aspect of inositide function has been previously analyzed in some cell models characterized by a mitogenic or differentiating response to specific factors. An interesting experimental model is represented by human derived osteosarcoma Saos-2 cells, characterized by the expression of high affinity receptors for interleukin 1 alpha (IL-1 alpha), which is one of the most potent stimulators of bone resorption. In particular, we investigated the earliest intracellular events following the binding of IL-1 alpha to its receptor, involving the inositide signal transduction pathway. Saos-2 cells present a partitioning of the phosphoinositidase (PLC) isoforms; in fact, the nucleus contains both PLC beta 1 and gamma 1, while the cytoplasm contains almost exclusively the gamma 1 isoform. IL-1 alpha evokes a rapid and transient increase of the PLC beta 1 activity in the nucleus, which causes the hydrolysis of phosphatidylinositol mono- and bis-phosphate. In response to IL-1 alpha, not only the canonical inositol lipid pathway appears to be involved; also the 3'-phosphorylated lipids generated by phosphatidylinositol 3-kinase (PI 3-K), which may act as second messengers, appear to be affected. In fact, Saos-2 cells present a nuclear PI 3-K activity which can be enhanced by the IL-1 alpha treatment. Among the possible targets of the second messengers released by the nuclear PLC beta 1 activation, we found that some protein kinase C isoforms, namely the epsilon and zeta, which are present within the nucleus, are activated after IL-1 alpha exposure. These activated PKC isoforms, in turn, could modulate the activity of the transcription factor NFkB, which, 5 min after IL-1 alpha treatment, has already translocated to the nucleus and bound to DNA to promote gene activation. The actual role of the inositide pathway in the Saos-2 cell function has also been investigated by utilizing cell clones transfected with the mouse sequence of the PLC beta 1.

Animals↗

PubMed1997

Changes of nuclear PI-PLC gamma1 during rat liver regeneration.

We have previously demonstrated that rat liver nuclei contain PI-PLC beta1 and gamma1 in the inner nuclear matrix and lamina associated with specific phosphodiesterase activity (Bertagnolo et al., 1995, Cell Signall. 7, 669-678). Since compensatory hepatic growth is an informative and well characterized model for natural cell proliferation, the presence of specific PI-PLC isoforms and their activity as well as PIP2 recovery were studied at various regenerating times, ranging from 3 to 22 h after partial hepatectomy. Three PI-PLC isoforms (beta1, gamma1, delta1) were examined in control and regenerating liver cells by using specific antibodies. By means of in situ immunocytochemistry and confocal microscopy, PI-PLC beta1 was found mainly in the nucleoplasm and this pattern was not modified after hepatectomy. On the contrary, the nuclear gamma1 isoform showed a marked decrease at 3 and 16 h after hepatectomy, but a clear increase at 22 h covering with bright intensity the whole nucleus. The PI-PLC delta1 isoform, which is exclusively cytoplasmic, was not altered during rat liver regeneration. By western blotting analysis on whole cell homogenates, none of the PI-PLC isozymes under study showed proliferation-linked modification. However, analyses of isolated nuclei identified changes in the nucleus associated PI-PLC gamma1 that paralleled the in situ observation whereas the beta1 isoform was unmodified at all the times examined. Nuclear phosphodiesterase activity on PIP2 was lower at 3 and 16 h, in comparison with sham operated rats, increased at 6 h and reached the highest value after 22 h. Consistently, the recovery of PIP2, obtained in conditions that optimise PIP-kinase activity, showed a marked decrease at 3 h and an increase up to 16 h of liver regeneration, followed by a further decrease at 22 h. These data are consistent with a close relationship between cell proliferation and the nuclear inositide cycle, depending, in rat liver, predominantly on the modulation of the gamma1 isoform of PI-PLC.

Animals↗

PubMed1997

The engagement of CD4 surface antigen in the HEL haemopoietic cell line up-regulates the transforming growth factor-beta1 (TGF-beta1) promoter activity.

In order to investigate the effect of CD4 engagement on the transforming growth factor beta1 (TGF-beta1) promoter activity in haemopoietic progenitors, HEL cells were transiently transfected with a plasmid vector containing -453/+11 nucleotides of the TGF-beta1 promoter fused with the bacterial chloramphenicol acetyltransferase (CAT) gene and then treated with various agonists. Both cross-linked CD4mAb and envelope gp120 were able to significantly up-regulate CAT activity with respect to the levels of activation observed in HEL cells treated with cross-linked CD8 mAb or p24. By using deletion mutants of the TGFbeta1 promoter, we found that the minimal DNA sequence still responsive to cross-linked CD4 mAb or gp120 was located between nucleotides -453/-323 of the TGF-beta1 promoter, which contains two activating protein 1 (AP1) binding sites. In electromobility shift assays (EMSA) we could demonstrate that CD4 engagement of HEL cells induced a significant increase of AP1 binding activity at the nuclear level. Furthermore, the steady-state mRNA of endogenous TGF-beta1 showed a small but reproducible increase when HEL cells were treated with cross-linked CD4mAb or gp120. Altogether, these findings suggest that the engagement of CD4 in HEL cells modulates TGF-beta1 expression, acting predominantly at the transcriptional level.

Blotting, Northern↗

PubMed1997

The effect of follicle stimulating hormone therapy on human sperm structure (Notulae seminologicae 11).

The effects of follicle stimulating hormone (FSH) treatment on the quality of human spermatozoa were assessed by examining the ultrastructure and the function of infertile human spermatozoa using a previously-defined formula. Using the spermatozoa as an andrological monitor shows that the therapeutic effect of FSH depends on the type of sperm defect. The response to FSH is, in many cases, positive and can be evaluated by examining the state of the ejaculated spermatozoa. From an initial group of 81 patients, 15 were placebo-treated controls, and 19 were non-responders (mainly with microbially infected semen). Out of 47 responders, after therapy nine achieved improved sperm quality which approached the natural fertility threshold. These responders all had spermatozoa affected by immaturity or apoptosis (n = 27). The 20 microbially-infected responders also had immature spermatozoa and never achieved the quality level of natural fertility. Thus, a natural fertility level was only achieved by nine responders out of 27 (three with immature spermatozoa, and six with apoptotic spermatozoa). Using our method of sperm analysis, these patients' spermatozoa were clearly categorized before treatment as either immature or apoptotic. In consequence, the success of the therapy was predictable. The response of individual organelles to therapy was examined. Certain qualities of the acrosome, the chromatin, the mitochondria, and the axoneme appear to be sensitive to FSH. Most of the previous conflicting results reported in the literature may be due to a lack of relevant discrimination between the different defects present in the spermatozoa of the patients, without assessing the likelihood of their response.

Acrosome↗

PubMed1997

Nuclear scaffold proteins are differently sensitive to stabilizing treatment by heat or Cu++.

The distribution of three nuclear scaffold proteins (of which one is a component of a particular class of nuclear bodies) has been studied in intact K562 human erythroleukemia cells, isolated nuclei, and nuclear scaffolds. Nuclear scaffolds were obtained by extraction with the ionic detergent lithium diidosalicylate (LIS), using nuclei prepared in the absence of divalent cations (metal-depleted nuclei) and stabilized either by a brief heat exposure (20 min at 37C or 42C) or by Cu++ ions at 0C. Proteins were visualized by in situ immunocytochemistry and confocal microscopy. Only a 160-kD nuclear scaffold protein was unaffected by all the stabilization procedures performed on isolated nuclei. However, LIS extraction and scaffold preparation procedures markedly modified the distribution of the polypeptide seen in intact cells, unless stabilization had been performed by Cu++. In isolated nuclei, only Cu++ treatment preserved the original distribution of the two other antigens (M(r), 125 and 126 kD), whereas in heat-stabilized nuclei we detected dramatic changes. In nuclear scaffolds reacted with antibodies to 125 and 126-kD proteins, the fluorescent pattern was always disarranged regardless of the stabilization procedure. These results, obtained with nuclei prepared in the absence of Mg+2 ions, indicate that heat treatment per se can induce changes in the distribution of nuclear proteins, at variance with previous suggestions. Nevertheless, each of the proteins we have studied behaves in a different way, possibly because of its specific association with the nuclear scaffold.

Antigens, Nuclear↗

PubMed1997

Different concentrations of Mg++ ions affect nuclear matrix protein distribution during thermal stabilization of isolated nuclei.

The nuclear matrix, a proteinaceous network believed to be a scaffolding structure determining higher-order organization of chromatin, is usually prepared from intact nuclei by a series of extraction steps. In most cell types investigated the nuclear matrix does not spontaneously resist these treatments but must be stabilized before the application of extracting agents. Incubation of isolated nuclei at 37C or 42C in buffers containing Mg++ has been widely employed as stabilizing agent. We have previously demonstrated that heat treatment induces changes in the distribution of three nuclear scaffold proteins in nuclei prepared in the absence of Mg++ ions. We studied whether different concentrations of Mg++ (2.0-5 mM) affect the spatial distribution of nuclear matrix proteins in nuclei isolated from K562 erythroleukemia cells and stabilized by heat at either 37C or 42C. Five proteins were studied, two of which were RNA metabolism-related proteins (a 105-kD component of splicing complexes and an RNP component), one a 126-kD constituent of a class of nuclear bodies, and two were components of the inner matrix network. The localization of proteins was determined by immunofluorescent staining and confocal scanning laser microscope. Mg++ induced significant changes of antigen distribution even at the lowest concentration employed, and these modifications were enhanced in parallel with increase in the concentration of the divalent cation. The different sensitivity to heat stabilization and Mg++ of these nuclear proteins might reflect a different degree of association with the nuclear scaffold and can be closely related to their functional or structural role.

Antigens, Nuclear↗

PubMed1997

Submicroscopic mathematical evaluation of spermatozoa in assisted reproduction. 3. Partial zona dissection (PZD) (Notulae seminologicae 12).

This paper belongs to a series of application of the Baccetti's et al. (1995) formula to the submicroscopical mathematical examination of the human spermatozoa used for assisted reproduction. The present experiment concerns partial zona dissection, a technique requiring a careful evaluation of sperm quality in order to predict the success of the program. Our results demonstrate that the sperm submicroscopic characters introduced in the formula are clearly correlated with the result of PZD. In fact the two numbers concerning the sperm ultrastructural quality (percentage and total number of spermatozoa free from defects in the ejaculate) obtained in successful and unsuccessful PZD groups, showed a large difference (P < 0.01). The most important characteristics seem to be the quality of the acrosomal complex, the condition of the chromatin and the mitochondrial assembly. All these characteristics are expressed with largely different means in successful and unsuccessful ejaculates (from P < 0.05 to P < 0.01). A comparison with the results previously obtained in ICSI (Strehler et al., 1995) and IVF (Piomboni et al., 1996) shows that sperm quality is significantly more implicated in the success of IVF than of PZD or ICSI.

Female↗

PubMed1997

Submicroscopic mathematical evaluation of spermatozoa in assisted reproduction. 4. The bovine fertilization (Notulae seminologicae 10).

In this paper we apply a modification of the formula of Baccetti et al. (1995) in the evaluation of submicroscopical characteristics of bull spermatozoa used in assisted reproduction. In the present experiment sperm quality is proposed as a useful parameter in predicting the success of fertilization. Our results demonstrate that the percentage of spermatozoa devoid of submicroscopic defects, according to the particular Bayesan formula proposed by us, is clearly correlated with the result of artificial insemination. In fact, the parameter concerning sperm quality obtained in variously successful donors shows a large correlation with fertility power. The synthetic parameters observed are therefore a good tool in the prediction of sperm power in artificial fertilization. The evaluation is mainly concerned with the quality of the acrosomal characters, the status of the chromatin, the shape of mitochondria, the position of the postacrosomal sheath, the perinuclear space and the axonemal pattern. All these characters are expressed with different means in ejaculates. All these data confirm that submicroscopic-mathematical evaluation offers a convincing and reliable diagnosis based upon sperm structure and functions such as acrosomal reaction and cell motility. It has been also demonstrated that sperm quality is a major factor in the success of artificial insemination and it is clearly revealed in the integrity of most of sperm organelles.

Animals↗

PubMed1996

Extracellular human immunodeficiency virus type-1 Tat protein activates phosphatidylinositol 3-kinase in PC12 neuronal cells.

We have here investigated the effect of the regulatory Tat protein of the human immunodeficiency virus type 1 (HIV-1) on the PI 3-kinase catalytic activity in PC12 rat pheochromocytoma cells. After as early as 1 min from the beginning of the treatment with recombinant HIV-1 Tat protein, a significant increase in the tyrosine phosphorylation levels of the p85 regulatory subunit of PI 3-kinase was noticed in 48 h serum-starved PC12 cells. Moreover, the addition of Tat to PC12 cells induced a great increase in PI 3-kinase immunoprecipitated with an anti-phosphotyrosine antibody with a peak of activity (19-fold increase with respect to the basal levels) after a 15-min treatment. This increase in PI 3-kinase activity was significantly higher in PC12 cell cultures supplemented with Tat protein than in cultures stimulated by 100 ng/ml nerve growth factor (NGF; 8-fold increase with respect to the basal levels). Further experiments showed that Tat protein was able to specifically activate PI 3-kinase at picomolar concentrations. In fact: (i) maximal activation of PI 3-kinase was observed at concentrations as low as 1 ng/ml and was specifically blocked by anti-Tat neutralizing antibody; (ii) a Tat-dependent activation was also observed in experiments in which PI 3-kinase activity was evaluated in either anti-Tyr(P) or anti-p85 immunoprecipitates; (iii) 100 nM wortmannin completely blocked the Tat-mediated increase in PI 3-kinase activity both in vitro and in vivo. Our data strongly support the concept that extracellular Tat acts as a cell stimulator, inducing intracellular signal transduction in uninfected cells.

Animals↗

PubMed1996

Pleiotropic effects of immobilized versus soluble recombinant HIV-1 Tat protein on CD3-mediated activation, induction of apoptosis, and HIV-1 long terminal repeat transactivation in purified CD4+ T lymphocytes.

CD3 mAb and HIV-1 Tat protein co-immobilized on plastic were able to induce a strong proliferation of resting human CD4 T cells, cultured in a serum-free chemically defined medium. Blocking studies performed with heparin or peptides containing the RGD sequence demonstrated that the heparin-binding basic domain of Tat plays a predominant role in CD4+ T cell activation. Moreover, the enhanced proliferative response of CD4+ T cells to immobilized Tat appeared to be mediated by alpha 5, beta 1, and alpha v subunits of surface integrin receptors. In contrast, soluble Tat showed a dose-dependent inhibitory activity on the proliferative response of resting CD4+ T cells stimulated by CD3 mAb co-immobilized with Tat or fibronectin, but not with CD28 mAb. In transient transfection assays performed with an HIV-1 long terminal repeat (LTR)-chloramphenicol acetyltransferase (CAT) plasmid CD3 mAb co-immobilized with Tat or fibronectin or CD28 mAb significantly stimulated CAT activity over the background. On the other hand, while immobilized Tat alone had no effects on LTR transactivation, soluble Tat was able to transactivate LTR-CAT in a dose-dependent manner. When CD4+ T cells activated by CD3 mAb co-immobilized with Tat were recovered, cultured for 7 days with 25 U/ml recombinant IL-2, and given an additional activation signal by recross-linking CD3 mAb, a marked increase of apoptosis was observed with respect to cells not subjected to CD3 mAb recross-linking. While co-immobilized Tat plus CD3 mAb did not show any significant effect on activation-induced cell death, high concentrations of soluble Tat synergized with immobilized CD3 mAb in the induction of apoptosis.

Adult↗

PubMed1996

Changes of nuclear protein kinase C activity and isotype composition in PC12 cell proliferation and differentiation.

To establish whether protein kinase C was involved in the nuclear events underlying cell differentiation and proliferation, rat pheochromocytoma PC12 cells, serum-starved for 24 h, were treated with either differentiating doses of nerve growth factor or high serum concentrations, which represented a powerful mitogenic stimulus. Western blot analysis with isoform-specific antibodies, performed on whole cell homogenates, cytoplasms, and purified nuclei, showed that PKC isotypes alpha, beta I, beta II, delta, epsilon, eta, and zeta were expressed in PC12 cells and that all of them, except for beta I, were found at the nuclear level, variably modulated depending on the cell treatment. Compared to serum-stimulated cells, in which an early (1 day) and marked rise of protein kinase C activity was followed by a plateau, nerve growth factor-treated cells showed a progressive increase of protein kinase C activity coincident with the onset and maintenance of the differentiated phenotype. Western blot analysis of nuclei isolated from fully differentiated cells demonstrated an increase of protein kinase C alpha, paralleled by enhanced phosphotransferase activity along with the nerve growth factor treatment, and complete loss of the delta isotype. In contrast, in nuclei of proliferating PC12 cells, after an early but modest increase at 1 day of mitogenic stimulation, protein kinase C activity reached a plateau. Isotype-specific analysis indicated a concomitant increase of protein kinase C beta II, delta, and zeta and the appearance of protein kinase C epsilon and eta at the nuclear level. Considering the relative intensity of the cytoplasmic and nuclear immunoreactive bands under the three conditions examined, clear-cut translocation to the nucleus occurred for PKC epsilon and eta in serum-stimulated cells. Additional nuclear accumulation of PKC by translocation from the cytoplasm was prominently induced for the zeta isoform after mitogenic stimulation and for PKC alpha during prolonged NGF treatment. Our data suggest that nuclear translocation and selective activation of distinct protein kinase C isoforms play a relevant role in the control of proliferation and differentiation of the same cell type and that nuclear protein kinase C is crucial to the induction and persistence of the differentiated neuronal phenotype of PC12 cells.

Amino Acid Sequence↗

PubMed1996

Low nanogram range quantitation of diglycerides and ceramide by high-performance liquid chromatography.

A method for ceramide (CER) and diradylglycerol (DG) determination after normal-phase HPLC separation was developed. The free oxydril group of ceramide and diradylglycerol is coupled to the carboxylic group of the fluorescent label (+)-6-methoxy-alpha-methyl-2 naphthaleneacetic acid (NAP), using as catalytic agents 4-dimethylaminopyridine and N,N'-dicyclohexylcarbodiimide. The use of NAP-free acid instead of the halide-activated form ensures higher stability of the reagent, lower reaction temperatures, and improved yield and reproducibility. The yield of the reaction is greater than 90% after a period of 3 h at the temperature of -20 degrees C. Over 85% of the starting material is recovered at the end of HPLC separation. The lower detection limit is below 5 ng for CER and 150 ng for DG. Under the conditions employed in the assay, no significant hydrolysis of triglycerides, sphingolipids, or phospholipids occurs and the esterification reaction is not affected by components of crude lipid extracts. Since separation and/or purification steps are not required, cellular levels of CER and DG can be easily and rapidly measured.

Cell Line↗

PubMed1996

Inhibition of purified CD34+ hematopoietic progenitor cells by human immunodeficiency virus 1 or gp120 mediated by endogenous transforming growth factor beta 1.

Human CD34+ hematopoietic progenitor cells, stringently purified from the peripheral blood of 20 normal donors, showed an impaired survival and clonogenic capacity after exposure to either heat-inactivated human immunodeficiency virus (HIV) 1 (strain IIIB) or cross-linked envelope gp120. Cell cycle analysis, performed at different times in serum-free liquid culture, showed an accumulation in G0/G1 in HIV-1- or gp120-treated cells and a progressive increase of cells with subdiploid DNA content, characteristic of apoptosis. In blocking experiments with anti-transforming growth factor (TGF) beta 1 neutralizing serum or TGF-beta 1 oligonucleotides, we demonstrated that the HIV-1- or gp120-mediated suppression of CD34+ cell growth was almost entirely due to an upregulation of endogenous TGF-beta 1 produced by purified hematopoietic progenitors. Moreover, by using a sensitive assay on the CCL64 cell line, increased levels of bioactive TGF-beta 1 were recovered in the culture supernatant of HIV-1/gp120-treated CD34+ cells. Anti-TGF-beta 1 neutralizing serum or TGF-beta 1 oligonucleotides were also effective in inducing a significant increase of the plating efficiency of CD34+ cells, purified from the peripheral blood of three HIV-1-seropositive individuals, suggesting that a similar mechanism may be also operative in vivo. The relevance of these findings to a better understanding of the pathogenesis of HIV-1-related cytopenias is discussed.

Antigens, CD34↗

PubMed1996

Submicroscopic mathematical evaluation of spermatozoa in assisted reproduction. 2. In vitro fertilization. (Notulae seminologicae. 7)

This paper belongs to a series of applications of the Baccetti et al. formula (1) to the submicroscopical mathematical examination of human spermatozoa used for assisted reproduction. The present experiment concerns IVF, a technique requiring careful evaluation of sperm quality to predict the success of the program. Our results demonstrate that the sperm submicroscopic characters introduced in the formula are clearly correlated with the result of IVF. In fact the two numbers concerning sperm quality (percentage of spermatozoa free from structural defects and total number in the ejaculate of spermatozoa free from defects) obtained in successful and unsuccessful IVF groups, showed a large difference. The t distribution in both cases reached a significance of 0.005. The synthetic parameters obtained are therefore a good tool in the prediction of sperm power in in vitro insemination techniques. The most important characteristics seem to be the quality of the acrosomal complex, the status of the chromatin, the shape of mitochondria, the axonemal pattern, and the membrane integrity. All these characteristics are expressed with largely different means in successful and unsuccessful ejaculates (t distribution significant at 0.005). All these data confirm that submicroscopic mathematical diagnosis offers a convincing evaluation of sperm structure and function, involving all organelles, including acrosome function and cell motility. It is also demonstrated that sperm quality is a major factor in the success of IVF and that it is clearly revealed by the integrity of the majority of the sperm organelles.

Acrosome↗

PubMed1996

Inositol lipid cycle and autonomous nuclear signalling.

The involvement of phospholipids and in particular polyphosphoinositides in cellular signalling has been documented in detail in the last 20 years. In addition to the plasma membrane localization also the nucleus is shown to be a site for both synthesis and hydrolysis of the phosphorylated forms of phosphatidylinositol. Previous observation have established that the nucleus possesses a specific PLC for inositol lipids, i.e., the PLC beta 1 isoform, which undergoes rapid and transient activation after IGF-I stimulation of quiescent Swiss 3T3 cells and is down-regulated after treatment of Friend erythroleukemia cells with DMSO. Here we have reviewed: (i) the potential of nuclear PLC beta 1 to be a target for anti-cancer drug, (ii) the capability of this PLC isoform, when activated by IGF-I, to be a key signalling molecule in the onset of DNA synthesis, via DAG generation and PKC alpha translocation to the nucleus, (iii) the chromosome mapping of PLC beta 1 gene. The differentiation program of Friend cells can be activated by other agents besides DMSO including tiazofurin, an anti-tumor drug, also capable of affecting the nuclear inositol lipid cycle. Tiazofurin induces a lowering of the activity of PLC beta 1 due to down regulation of this isoform as revealed by both Western blotting and Northern blotting analyses. Using Swiss 3T3 cells stably transformed with an antisense PLC beta 1 construct, the knock-out of the PLC beta 1 gene induces both a loss of PLC beta 1 expression, as determined by Western blots, and a loss of the mitogenic responsiveness to IGF-I. These events show a direct relationship between nuclear PLC beta 1 evoked signals and IGF-I induced cell proliferation. Finally, the assignment of the PLC beta 1 gene to the band q35-36 of rat chromosome 3 paves the way for further genetic studies given the fact that the region where PLC beta 1 gene maps is a hot spot for genetic alterations in a number of experimentally induced rat tumors. Taken as a whole, these results assign a key role to the regulation of nuclear PLC activity and expression both in growth-factor activated mitogenesis and in in vitro erythroid differentiation.

Antineoplastic Agents↗

PubMed1996

PMA-induced megakaryocytic differentiation of HEL cells is accompanied by striking modifications of protein kinase C catalytic activity and isoform composition at the nuclear level.

We investigated whether members of the protein kinase C (PKC) family of enzymes could play a role in the nuclear events involved in megakaryocytic differentiation. PKC activity was analysed using a serine substituted specific peptide, which enabled us to evaluate the whole catalytic activity in the pluripotent haemopoietic HEL cell line treated with 10(-7)M phorbol myristate acetate (PMA) or haemin. In parallel, the subcellular distribution of different PKC isoforms (alpha, beta I, beta II, gamma, delta, epsilon, theta, eta, zeta) was evaluated by Western blot. PKC catalytic activity in the nuclei of HEL cells showed a peak after acute (30 min) treatment with PMA, followed by a significant (P < 0.05) decline after prolonged exposure (72 h) to the same agonist, when most HEL cells had acquired a differentiated megakaryocytic phenotype. Western blot analysis of nuclear lysates consistently showed a significant increase of PKC-alpha, -beta I, -epsilon, theta and -zeta isoforms after 30 min of PMA treatment, followed by a drastic decline of all but PKC-zeta isoforms. Moreover, PKC-6 delta appeared in HEL nuclei only after 72 h of exposure to PMA. On the other hand, neither the catalytic activity nor the immunoreactivity of the different PKC isoforms showed remarkable variations in nuclei of HEL cells induced to differentiate along the erythroid lineage with 10(-7)M haemin. The possible implications of these findings for a better understanding of the molecular events underlying the process of megakaryocytic differentiation are discussed.

Blotting, Western↗

PubMed1996

Impaired survival of bone marrow GPIIb/IIa+ megakaryocytic cells as an additional pathogenetic mechanism of HIV-1-related thrombocytopenia.

Glycoproteic (GP) IIb/IIIa+ megakaryocytic cells were purified from the bone marrow (BM) of 15 HIV-1 seropositive thrombocytopenic patients, eight HIV-1 seronegative patients affected by immune thrombocytopenic purpura (ITP) and 14 HIV-1 seronegative normal donors. The presence of apoptosis was evaluated in freshly isolated GPIIb/IIIa+ cells by flow cytometry after propidium iodide staining and electron microscopy. GPIIb/IIIa+ cells from HIV-1 seropositive thrombocytopenic patients showed a significant (P < 0.0001) increase of apoptosis with respect to both HIV-1 seronegative ITP patients and normal donors. Moreover, the degree of apoptosis in bone marrow GPIIb/IIIa+ cells purified from HIV-1 seropositive thrombocytopenic patients was inversely (P < 0.01) related to the count of circulating platelets, whereas it did not show any significant correlation with the absolute number of circulating CD4 T cells, the CD4/CD8 ratio or the presence of proviral gag DNA sequences. Therefore neither an advanced stage of HIV-1 disease nor a direct infection with HIV-1 seems to play a primary role in the impaired survival of BMGPIIb/IIIa+ megakaryocytic cells. These findings strengthen the notation that, besides the immune targeting of peripheral platelets, an impairment of the bone marrow megakaryocyte compartment may also contribute to the pathogenesis of HIV-1 related thrombocytopenia.

Apoptosis↗

PubMed1996

Nuclear translocation of protein kinase C-alpha and -zeta isoforms in HL-60 cells induced to differentiate along the granulocytic lineage by all-trans retinoic acid.

We investigated whether members of the protein kinase C (PKC) family of enzymes were involved in the nuclear events underlying granulocytic differentiation induced by 10(-6) M all-trans retinoic acid (ATRA) in HL-60 cells. PKC activity was analysed by using a serine substituted specific peptide which enabled the evaluation of the whole catalytic activity of both Ca2+ -dependent and Ca2+ -independent PKC isoforms. In parallel, the subcellular distribution of various PKC isoforms was evaluated by Western blot, immunoprecipitation and in situ immunocytochemistry analyses. The level of PKC catalytic activity in the nuclei of HL-60 cells significantly (P < 0.01) and progressively increased from 1 h of ATRA treatment onwards. Consistently, PKC-alpha and -zeta showed a striking and selective accumulation inside the nucleus upon treatment with ATRA. On the other hand, PKC-beta I and -beta II, the only two other isoforms present at nuclear level, did not show any significant modification upon ATRA treatment. The remaining PKC isoforms were not detectable inside the nucleus and showed only modest and non-significant variations, also in whole cell homogenates, upon ATRA treatment, except PKC-delta which showed a progressive down-regulation. Our data suggest that a selective nuclear translocation of PKC-alpha and -zeta might be involved in the process of granulocytic differentiation induced by ATRA in HL-60 cells.

Blotting, Western↗