Lia Guidotti

Cyclooxygenase-2 expression is induced during human megakaryopoiesis and characterizes newly formed platelets

Cyclooxygenase (COX)-1 or -2 and prostaglandin (PG) synthases catalyze the formation of various PGs and thromboxane (TX) A2. We have investigated the expression and activity of COX-1 and -2 during human megakaryocytopoiesis. We analyzed megakaryocytes from bone marrow biopsies and derived from thrombopoietin-treated CD34+ hemopoietic progenitor cells in culture. Platelets were obtained from healthy donors and patients with high platelet regeneration because of immune thrombocytopenia or peripheral blood stem cell transplantation. By immunocytochemistry, COX-1 was observed in CD34+ cells and in megakaryocytes at each stage of maturation, whereas COX-2 was induced after 6 days of culture, and remained detectable in mature megakaryocytes. CD34+ cells synthesized more PGE2 than TXB2 (214 ± 50 vs. 30 ± 10 pg/106 cells), whereas the reverse was true in mature megakaryocytes (TXB2 8,440 ± 2,500 vs. PGE2 906 ± 161 pg/106 cells). By immunostaining, COX-2 was observed in <10% of circulating platelets from healthy controls, whereas up to 60% of COX-2-positive platelets were found in patients. A selective COX-2 inhibitor reduced platelet production of both PGE2 and TXB2 to a significantly greater extent in patients than in healthy subjects. Finally, we found that COX-2 and the inducible PGE-synthase were coexpressed in mature megakaryocytes and in platelets. We conclude that both COX-isoforms contribute to prostanoid formation during human megakaryocytopoiesis and that COX-2-derived PGE2 and TXA2 may play an unrecognized role in inflammatory and hemostatic responses in clinical syndromes associated with high platelet turnover.

Supravital exposure to propidium iodide identifies apoptosis on adherent cells.

BACKGROUND Several studies indicate that plasma membrane changes during apoptosis are a general phenomenon. Among the flow cytometric methods to measure apoptosis, the Annexin V assay that detects the membrane exposure of phosphatidylserine (PS) is one of the most commonly used. However, the various treatments used for the detachment of adherent cells generally interfere with the binding of Annexin V to membrane PS, making apoptosis measurement a technical problem. Materials and Methods Apoptosis of different cell lines was investigated by fluorescence microscopy and multiple flow assays designed to assess loss of membrane integrity, translocation of PS, DNA fragmentation, and light scatter changes. Results and Conclusions We show that supravital propidium iodide (PI) assay stains adherent apoptotic cells, allowing flow cytometric quantification. Moreover, supravital exposure to PI without prior permeabilization identifies apoptotic cells as well as Annexin V and permits the simultaneous surface staining by FITC- and PE-conjugated monoclonal antibodies. As in the case of necrotic or permeabilized cells, fluorescence microscopy has revealed that PI staining of apoptotic cells is localized in the nucleus. This suggests that the binding of PI to the DNA/RNA structures is stable enough to withstand the trypsinization and/or washing procedures necessary to detach adherent cells.

Ultrastructural characterization of maturation, platelet release, and senescence of human cultured megakaryocytes

The aim of this study was to evaluate the ultrastructural features of human megakaryocytes cultured in vitro. For this purpose, pluripotent CD34+ (cluster of differentiation 34) hematopoietic progenitor cells, obtained from the peripheral blood of healthy adult donors, were differentiated along the megakaryocytic lineage in liquid cultures by the addition of the megakaryocyte‐specific growth factor thrombopoietin (TPO, 100 ng/ml). After only 6–8 days, virtually all of the CD34‐derived cells expressed the early megakaryocytic CD61 antigen, while, after 15–16 days, most cells also expressed the late megakaryocytic CD42a antigen. Ultrastructural analysis of cells obtained after 7 days of culture showed aspects typical of developing megakaryocytes (MK), such as formation of platelet territories and cytoplasmic fragmentation. At later (15–16 day) culture times, two distinct cell populations were observed: fully developed megakaryocytes releasing platelets into the culture medium and senescent megakaryocytes, characterized by morphological features of apoptosis. Analysis of DNA fragmentation in these cells revealed that apoptosis in megakaryocytes occurred in the absence of the internucleosomic cleavage, which is characteristic of most, but not all, types of apoptosis in cells of hematopoietic origin. On the other hand, flow cytometry of the DNA content of senescent megakaryocytes showed a subdiploid peak that was likely due to a loss of micronuclei during processing. Anat Rec 258:90–99, 2000.

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 (α, βI, βII, γ, δ, ε, θ, η, ζ) 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‐α, ‐βI, ‐ε, ‐θ and ‐ζ isoforms after 30 min of PMA treatment, followed by a drastic decline of all but PKC‐ζ isoforms. Moreover, PKC‐δ 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.

Functional expression of TRAIL and TRAIL-R2 during human megakaryocytic development

The expression and function of surface TRAIL and TRAIL receptors were investigated in primary megakaryocytic cells, generated in serum‐free liquid phase from peripheral human CD34+ cells. The surface expression of both TRAIL and “death receptor” TRAIL‐R2 became detectable starting from the early phase of megakaryocytic differentiation (day 6 of culture) and persisted at later (days10–14) culture times. On the other hand, “death receptor” TRAIL‐R1, “decoy receptors” TRAIL‐R3, and TRAIL‐R4 were barely detectable or undetectable at any time point examined. Addition of recombinant TRAIL at day 6 of culture increased the rate of spontaneous apoptosis of CD34+/CD41dim megakaryoblasts and it significantly decreased the total output of mature megakaryocytic cells evaluated after additional 4–8 days of culture. Conversely, addition in culture of TRAIL‐R2‐Fc chimera, which blocked the interaction between endogenous TRAIL and TRAIL‐R2 on the surface of cultured megakaryocytic cells, increased the total megakaryocytic cell count. In addition, recombinant TRAIL promoted a small but reproducible increase of maturation in the surviving megakaryocytic cell population, evaluated by both phenotypic analysis and morphology. A similar pro‐maturation effect was observed when TRAIL was added to bone marrow‐derived CD61+ megakaryocytic cells. Thus, our data suggest a role of TRAIL as a regulator of megakaryocytopoiesis.

In vitro apoptotic cell death during erythroid differentiation

Erythropoiesis occurs in bone marrow and it has been shown that during in vivo erythroid differentiation some immature erythroblasts undergo apoptosis. In this regard, it is known that immature erythroblasts are FasL- and TRAIL-sensitive and can be killed by cells expressing these ligand molecules. In the present study, we have investigated the cell death phenomenon that occurs during a common unilineage model of erythroid development.Purified CD34+ human haemopoietic progenitors were cultured in vitro in the presence of SCF, IL-3 and erythropoietin. Their differentiation stages and apoptosis were followed by multiple technical approaches.Flow cytometric evaluation of surface and intracellular molecules revealed that glycophorin A appeared at day 3–4 of incubation and about 75% of viable cells co-expressed high density glycophorin A (Glybright) and adult haemoglobin at day 14 of culture, indicating that this system reasonably recapitulates in vivo normal erythropoiesis. Interestingly, when mature (Glybright) erythroid cells reached their higher percentages (day 14) almost half of cultured cells were apoptotic. Morphological studies indicated that the majority of dead cells contained cytoplasmic granular material typical of basophilic stage, and DNA analysis by flow cytometry and TUNEL reaction revealed nuclear fragmentation.These observations indicate that in vitro unilineage erythroid differentiation, as in vivo, is associated with apoptotic cell death of cells with characteristics of basophilic erythroblasts. We suggest that the interactions between different death receptors on immature basophilic erythroblasts with their ligands on more mature erythroblasts may contribute to induce apoptosis in vitro.

Microarray-based identification and RT-PCR test screening for epithelial-specific mRNAs in peripheral blood of patients with colon cancer.

BackgroundThe efficacy of screening for colorectal cancer using a simple blood-based assay for the detection of tumor cells disseminated in the circulation at an early stage of the disease is gaining positive feedback from several lines of research. This method seems able to reduce colorectal cancer mortality and may replace colonoscopy as the most effective means of detecting colonic lesions.MethodsIn this work, we present a new microarray-based high-throughput screening method to identifying candidate marker mRNAs for the early detection of epithelial cells diluted in peripheral blood cells. This method includes 1. direct comparison of different samples of colonic mucosa and of blood cells to identify consistent epithelial-specific mRNAs from among 20,000 cDNA assayed by microarray slides; 2. identification of candidate marker mRNAs by data analysis, which allowed selection of only 10 putative differentially expressed genes; 3. Selection of some of the most suitable mRNAs (TMEM69, RANBP3 and PRSS22) that were assayed in blood samples from normal subjects and patients with colon cancer as possible markers for the presence of epithelial cells in the blood, using reverse transcription – polymerase chain reaction (RT-PCR).ResultsOur present results seem to provide an indication, for the first time obtained by genome-scale screening, that a suitable and consistent colon epithelium mRNA marker may be difficult to identify.ConclusionThe design of new approaches to identify such markers is warranted.

Selective modulation of the cyclin B/CDK1 and cyclin D/CDK4 complexes during in vitro human megakaryocyte development

Mammalian megakaryocyte development is characterized by a progressive accumulation of cells exhibiting a polylobated nucleus with a polyploid DNA content. In this study human megakaryocytes were obtained from CD34+ haemopoietic progenitors by in vitro liquid culture in the presence of 100 ng/ml of recombinant thrombopoietin (TPO). Ultrastructural examination of polyploid megakaryocytes showed the presence of a large number of centrioles, the breakdown of the nuclear envelope, and the progressive chromatin condensation, all aspects characteristic of mitosis. At both indirect immunofluorescence and Western blot analyses, cyclin B and its related cyclin‐dependent kinase (CDK)1, which forms the mitosis promoting factor (MPF), showed an increased expression in maturating megakaryoblasts and megakaryocytes (day 8 of culture) with respect to freshly isolated CD34+ progenitors. This expression tended to decline in fully developed megakaryocytes (day 15 of culture). The amount of cyclin D and of the related CDK4, governing the G1 phase of the cell cycle, increased during megakaryocyte development, maintaining high levels of expression also in mature megakaryocytes. These results indicate that megakaryocyte polyploidization depends on a true, although incomplete, mitotic process, and that cyclin D/CDK4 probably plays a crucial role throughout megakaryocytopoiesis.

Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines.

BackgroundEGFR is frequently overexpressed in colon cancer. We characterized HT-29 and Caco-2, human colon cancer cell lines, untreated and treated with cetuximab or gefitinib alone and in combination with EGF.MethodsCell growth was determined using a variation on the MTT assay. Cell-cycle analysis was conducted by flow cytometry. Immunohistochemistry was performed to evaluate EGFR expression and scanning electron microscopy (SEM) evidenced the ultrastructural morphology. Gene expression profiling was performed using hybridization of the microarray Ocimum Pan Human 40 K array A.ResultsCaco-2 and HT-29 were respectively 66.25 and 59.24 % in G0/G1. They maintained this level of cell cycle distribution after treatment, suggesting a predominantly differentiated state. Treatment of Caco-2 with EGF or the two EGFR inhibitors produced a significant reduction in their viability. SEM clearly showed morphological cellular transformations in the direction of cellular death in both cell lines treated with EGFR inhibitors. HT-29 and Caco-2 displayed an important reduction of the microvilli (which also lose their erect position in Caco-2), possibly invalidating microvilli absorption function. HT-29 treated with cetuximab lost their boundary contacts and showed filipodi; when treated with gefitinib, they showed some vesicles: generally membrane reshaping is evident. Both cell lines showed a similar behavior in terms of on/off switched genes upon treatment with cetuximab. The gefitinib global gene expression pattern was different for the 2 cell lines; gefitinib treatment induced more changes, but directly correlated with EGF treatment.In cetuximab or gefitinib plus EGF treatments there was possible summation of the morphological effects: cells seemed more weakly affected by the transformation towards apoptosis. The genes appeared to be less stimulated than for single drug cases.ConclusionThis is the first study to have systematically investigated the effect of cetuximab or gefitinib, alone and in combination with EGF, on human colon cancer cell lines. The EGFR inhibitors have a weaker effect in the presence of EGF that binds EGFR. Cetuximab treatment showed an expression pattern that inversely correlates with EGF treatment. We found interesting cyto-morphological features closely relating to gene expression profile. Both drugs have an effect on differentiation towards cellular death.

HIV‐1 gp120 induces the activation of both c‐fos and c‐jun immediate‐early genes in HEL megakaryocytic cells

We have previously demonstrated that the addition in culture of recombinant HIV‐1 IIIB envelope gp120 affects the survival/growth of pluripotent haemopoietic progenitors, and, in particular, of those committed towards the megakaryocytic lineage. To characterize some of the molecular mechanisms involved in this phenomenon, we investigated the expression of members of the activating protein‐1 (AP‐1) complex in the HEL megakaryoblastic cell line. Following the treatment of HEL cells with recombinant IIIB envelope gp120, we noticed: (i) increased levels of endogenous c‐fos and c‐jun mRNA and proteins, (ii) activation of both c‐fos and c‐jun promoters, and (iii) a very rapid stimulation of a MAPK/ERK pathway.