Fragile X mental retardation protein in intrahepatic cholangiocarcinoma: regulating the cancer cell behavior plasticity at the leading edge.

Intrahepatic cholangiocarcinoma (iCCA) is a rare malignancy of the intrahepatic biliary tract with a very poor prognosis. Although some clinicopathological parameters can be prognostic factors for iCCA, the molecular prognostic markers and potential mechanisms of iCCA have not been well investigated. Here, we report that the Fragile X mental retardation protein (FMRP), a RNA binding protein functionally absent in patients with the Fragile X syndrome (FXS) and also involved in several types of cancers, is overexpressed in human iCCA and its expression is significantly increased in iCCA metastatic tissues. The silencing of FMRP in metastatic iCCA cell lines affects cell migration and invasion, suggesting a role of FMRP in iCCA progression. Moreover, we show evidence that FMRP is localized at the invasive front of human iCCA neoplastic nests and in pseudopodia and invadopodia protrusions of migrating and invading iCCA cancer cells. Here FMRP binds several mRNAs encoding key proteins involved in the formation and/or function of these protrusions. In particular, we find that FMRP binds to and regulates the expression of Cortactin, a critical regulator of invadopodia formation. Altogether, our findings suggest that FMRP could promote cell invasiveness modulating membrane plasticity and invadopodia formation at the leading edges of invading iCCA cells.

NLRP3 Inflammasome: A New Pharmacological Target for Reducing Testicular Damage Associated with Varicocele.

Many bioactive natural compounds are being increasingly used for therapeutics and nutraceutical applications to counteract male infertility, particularly varicocele. The roles of selenium and Polydeoxyribonucleotide (PDRN) were investigated in an experimental model of varicocele, with particular regard to the role of NLRP3 inflammasome. Male rats underwent sham operation and were daily administered with vehicle, seleno-L-methionine (Se), PDRN, and with the association Se-PDRN. Another group of rats were operated for varicocele. After twenty-eight days, sham and varicocele rats were sacrificed and both testes were weighted and analyzed. All the other rats were challenged for one month with the same compounds. In varicocele animals, lower testosterone levels, testes weight, NLRP3 inflammasome, IL-1ß and caspase-1 increased gene expression were demonstrated. TUNEL assay showed an increased number of apoptotic cells. Structural and ultrastructural damage to testes was also shown. PDRN alone significantly improved all considered parameters more than Se. The Se-PDRN association significantly improved all morphological parameters, significantly increased testosterone levels, and reduced NLRP3 inflammasome, caspase-1 and IL-1ß expression and TUNEL-positive cell numbers. Our results suggest that NLRP3 inflammasome can be considered an interesting target in varicocele and that Se-PDRN may be a new medical approach in support to surgery.

Structural, ultrastructural, and morphometric study of the zebrafish ocular surface: a model for human corneal diseases?

PURPOSE: A morphological and morphometric study of the adult zebrafish ocular surface was performed to provide a comprehensive description of its parts and to evaluate its similarity to the human. MATERIALS AND METHODS: The eyes of adult zebrafish were processed for light, transmission and scanning electron microscopy, and for immunohistochemical stain of corneal nerves; a morphometric analysis was also performed on several morphological parameters. RESULTS: The corneal epithelium was formed by five layers of cells. No Bowman's layer could be demonstrated. The stroma consisted of lamellae of different thickness with few keratocytes. The Descemet's membrane was absent as the flat and polygonal endothelial cells directly adhered to the deepest corneal lamella. The immunohistochemical stain of neurofilaments failed to demonstrate corneal nerve fibers. The conjunctival epithelium was stratified, overlying the stroma formed by a subepithelial and a deep layer, this latter connected to the scleral cartilage. In the peripheral cornea and in the conjunctiva, many goblet and rodlet cells were observed. The morphometric analysis showed that the peripheral cornea epithelium was thicker when compared to the other parts of the ocular surface, with smaller superficial cells. Desmosomes and hemidesmosomes in the conjunctiva were significantly fewer in number than the other parts of the ocular surface. The stroma was thinner in the conjunctiva than in the cornea, while corneal lamellae were thicker in the intermediate stroma. CONCLUSIONS: The zebrafish ocular surface showed significant differences compared to the human, such as the absence of Bowman's layer, Descemet's membrane and corneal nerve fibers, the reduced stromal thickness, and the presence of rodlet cells. On the basis of these original findings, it is suggested that the use of the zebrafish as a model for studying normal or pathological human corneas should be undertaken with particular caution.

The fragile X mental retardation protein regulates tumor invasiveness-related pathways in melanoma cells.

The fragile X mental retardation protein (FMRP) is lacking or mutated in patients with the fragile X syndrome (FXS), the most frequent form of inherited intellectual disability. FMRP affects metastasis formation in a mouse model for breast cancer. Here we show that FMRP is overexpressed in human melanoma with high Breslow thickness and high Clark level. Furthermore, meta-analysis of the TCGA melanoma data revealed that high levels of FMRP expression correlate significantly with metastatic tumor tissues, risk of relapsing and disease-free survival. Reduction of FMRP in metastatic melanoma cell lines impinges on cell migration, invasion and adhesion. Next-generation sequencing in human melanoma cells revealed that FMRP regulates a large number of mRNAs involved in relevant processes of melanoma progression. Our findings suggest an association between FMRP levels and the invasive phenotype in melanoma and might open new avenues towards the discovery of novel therapeutic targets.

Modifications in Human Oral Fibroblast Ultrastructure, Collagen Production, and Lysosomal Compartment in Response to Electronic Cigarette Fluids.

BACKGROUND: Many smokers have recently turned to electronic cigarettes (e-cigarettes) because they have been marketed as a cheaper, safer smokeless alternative to traditional cigarettes and a possible smoking cessation tool. Although the safety of these electronic devices is still not fully known, there is evidence of their cytotoxicity on cells belonging to the oral cavity. In a previous study by the authors, the increase of reactive oxygen production and Bax expression, followed by the occurrence of apoptosis, was demonstrated in human gingival fibroblasts (HGFs). The aim of this paper is to further investigate the effects of the e-cigarette liquids (with and without nicotine) on the same experimental model. METHODS: HGFs were treated with e-cigarette fluids containing nicotine (final concentration 1 mg/mL) and the equivalent volume of a fluid without nicotine, for periods ≤48 hours. Lactate dehydrogenase assay (LDH), electronic microscopy analysis, collagen I production, flow cytometry lysosome compartment evaluation, and western blotting light chain 3 (microtubule-associated protein 1A/1B-LC3) expression were performed. RESULTS: Fluids containing nicotine exerted cytotoxicity as demonstrated by increased levels of LDH, in parallel to the presence of numerous vacuoles in the cytoplasm, a decrease in collagen I production, and augmented LC3 II expression. Autophagic vesicles and more procollagen I molecules were present in the cytoplasm of fibroblasts exposed to nicotine-free fluids. In the same samples, time-dependent activation of the lysosomal compartment with no changes in LC3 expression was detected. CONCLUSION: E-cigarette fluids (with and without nicotine) trigger molecular and morphologic responses in oral fibroblasts, raising concerns about their role in the pathogenesis of oral diseases.

P-Selectin Sustains Extramedullary Hematopoiesis in the Gata1 low Model of Myelofibrosis.

Splenomegaly is a major manifestation of primary myelofibrosis (PMF) contributing to clinical symptoms and hematologic abnormalities. The spleen from PMF patients contains increased numbers of hematopoietic stem cells (HSC) and megakaryocytes (MK). These MK express high levels of P-selectin (P-sel) that, by triggering neutrophil emperipolesis, may cause TGF-ß release and disease progression. This hypothesis was tested by deleting the P-sel gene in the myelofibrosis mouse model carrying the hypomorphic Gata1(low) mutation that induces megakaryocyte abnormalities that recapitulate those observed in PMF. P-sel(null) Gata1(low) mice survived splenectomy and lived 3 months longer than P-sel(WT) Gata1(low) littermates and expressed limited fibrosis and osteosclerosis in the marrow or splenomegaly. Furthermore, deletion of P-sel disrupted megakaryocyte/neutrophil interactions in spleen, reduced TGF-ß content, and corrected the HSC distribution that in Gata1(low) mice, as in PMF patients, is abnormally expanded in spleen. Conversely, pharmacological inhibition of TGF-ß reduced P-sel expression in MK and corrected HSC distribution. Spleens, but not marrow, of Gata1(low) mice contained numerous cKIT(pos) activated fibrocytes, probably of dendritic cell origin, whose membrane protrusions interacted with MK establishing niches hosting immature cKIT(pos) hematopoietic cells. These activated fibrocytes were not detected in spleens from P-sel(null) Gata1(low) or TGF-ß-inhibited Gata1(low) littermates and were observed in spleen, but not in marrow, from PMF patients. Therefore, in Gata1(low) mice, and possibly in PMF, abnormal P-sel expression in MK may mediate the pathological cell interactions that increase TGF-ß content in MK and favor establishment of a microenvironment that supports myelofibrosis-related HSC in spleen.

Involvement of cyclic-nucleotide response element-binding family members in the radiation response of Ramos B lymphoma cells.

The aim of the present study was to investigate the role of Cyclic-nucleotide Response Element-Binding (CREB) family members and related nuclear transcription factors in the radiation response of human B lymphoma cell lines (Daudi and Ramos). Unlike the more radiosensitive Daudi cells, Ramos cells demonstrated only a moderate increase in early apoptosis after 3-5 Gy irradiation doses, which was detected with Annexin V/PI staining. Moreover, a significant and dose-dependent G2/M phase accumulation was observed in the same cell line at 24 h after both ionizing radiation (IR) doses. Western blot analysis showed an early increase in CREB protein expression that was still present at 3 h and more evident after 3 Gy IR in Ramos cells, along with the dose-dependent upregulation of p53 and NF-κB. These findings were consistent with real-time RT-PCR analysis that showed an early- and dose-dependent upregulation of NFKB1, IKBKB and XIAP gene expression. Unexpectedly, pre-treatment with SN50 did not increase cell death, but cell viability. Taken together, these findings let us hypothesise that the early induction and activation of NF-κB1 in Ramos cells could mediate necrotic cell death and be linked to other molecules belonging to CREB family and involved in the cell cycle regulation.

A novel interaction between megakaryocytes and activated fibrocytes increases TGF-ß bioavailability in the Gata1(low) mouse model of myelofibrosis.

Despite numerous circumstantial evidences, the pathogenic role of TGF-ß in primary myelofibrosis (PMF), the most severe of the Philadelphia-negative myeloproliferative neoplasms, is still unclear because of the modest (2-fold) increases in its plasma levels observed in PMF patients and in the Gata1(low) mouse model. Whether myelofibrosis is associated with increased bioavailability of TGF-ß bound to fibrotic fibres is unknown. Transmission electron-microscopy (TEM) observations identified that spleen from PMF patients and Gata1(low) mice contained megakaryocytes with abnormally high levels of TGF-ß and collagen fibres embedded in their cytoplasm. Additional immuno-TEM observations of spleen from Gata1(low) mice revealed the presence of numerous activated fibrocytes establishing with their protrusions a novel cellular interaction, defined as peripolesis, with megakaryocytes. These protrusions infiltrated the megakaryocyte cytoplasm releasing collagen that was eventually detected in its mature polymerized form. Megakaryocytes, engulfed with mature collagen fibres, acquired the morphology of para-apoptotic cells and, in the most advanced cases, were recognized as polylobated heterochromatic nuclei surrounded by collagen fibres strictly associated with TGF-ß. These areas contained concentrations of TGF-ß-gold particles ~1000-fold greater than normal and numerous myofibroblasts, an indication that TGF-ß was bioactive. Loss-of-function studies indicated that peripolesis between megakaryocytes and fibrocytes required both TGF-ß, possibly for inducing fibrocyte activation, and P-selectin, possibly for mediating interaction between the two cell types. Loss-of-function of TGF-ß and P-selectin also prevented fibrosis. These observations identify that myelofibrosis is associated with pathological increases of TGF-ß bioavailability and suggest a novel megakaryocyte-mediated mechanism that may increase TGF-ß bioavailability in chronic inflammation.

Abnormal P-selectin localization during megakaryocyte development determines thrombosis in the gata1low model of myelofibrosis.

Patients with primary myelofibrosis have increased risk for bleeding and thrombosis. It is debated whether propensity to thrombosis is due to increased numbers of platelet microparticles and/or to pathological platelet-neutrophil interactions. Platelet neutrophil interactions are mediated by P-selectin and even though the megakaryocytes of myelofibrosis patients express normal levels of P-selectin, it remains abnormally localized to the demarcation membrane system rather than being assembled into the α-granules in platelets. Mice carrying the hypomorphic Gata1(low) mutation express the same megakaryocyte abnormalities presented by primary myelofibrosis patients, including abnormal P-selectin localization to the DMS and develop with age myelofibrosis, a disease that closely resembles human primary myelofibrosis. Whether these mice would also develop thrombosis has not been investigated as yet. The aim of this study was to determine whether Gata1(low) mice would develop thrombosis with age and, in this case, the role played by P-selectin in the development of the trait. To this aim, Gata1(low) mice were crossed with P-sel(null) mice according to standard genetic protocols and Gata1(low)P-sel(wt), Gata1(low)P-sel(null) and Gata1(WT)P-sel(null) or Gata1(wt)P-sel(wt) (as controls) littermates obtained. It was shown that platelet counts, but not hematocrit, are reduced in Gata1(low) mice. Moreover, platelet microparticles are reduced in Gata1(low) mice and P-selectin positive platelet microparticles were not found. To determine the phenotypic implications of the different mutations, bleeding time was estimated by a tail cut procedure. Mutant mice were sacrificed and presence of thrombosis was determined by immunohistological staining of organs. Gata1(low) mice with or without the P-selectin null trait had a prolonged bleeding time compared to wild type mice. However, in Gata1(low) mice significantly higher frequency of thrombotic events was seen in adult and old Gata1(low) mice compared to Gata1(low)P-sel(null) mice. Thus, presence of the P-selectin null trait rescued Gata1(low) mice from the thrombotic phenotype, but did not change the level of platelet microparticles. Taken together these data indicate that abnormal localization of P-selectin, induced by the Gata1(low) mutation, and thus, increased pathological interactions with leucocytes, is responsible for the increased presence of thrombosis seen in these mice.

Characterization of the TGF-ß1 signaling abnormalities in the Gata1low mouse model of myelofibrosis.

Primary myelofibrosis (PMF) is characterized by fibrosis, ineffective hematopoiesis in marrow, and hematopoiesis in extramedullary sites and is associated with abnormal megakaryocyte (MK) development and increased transforming growth factor (TGF)-ß1 release. To clarify the role of TGF-ß1 in the pathogenesis of this disease, the TGF-ß1 signaling pathway of marrow and spleen of the Gata1(low) mouse model of myelofibrosis (MF) was profiled and the consequences of inhibition of TGF-ß1 signaling on disease manifestations determined. The expression of 20 genes in marrow and 36 genes in spleen of Gata1(low) mice was altered. David-pathway analyses identified alterations of TGF-ß1, Hedgehog, and p53 signaling in marrow and spleen and of mammalian target of rapamycin (mTOR) in spleen only and predicted that these alterations would induce consequences consistent with the Gata1(low) phenotype (increased apoptosis and G1 arrest both in marrow and spleen and increased osteoblast differentiation and reduced ubiquitin-mediated proteolysis in marrow only). Inhibition of TGF-ß1 signaling normalized the expression of p53-related genes, restoring hematopoiesis and MK development and reducing fibrosis, neovascularization, and osteogenesis in marrow. It also normalized p53/mTOR/Hedgehog-related genes in spleen, reducing extramedullary hematopoiesis. These data identify altered expression signatures of TGF-ß1 signaling that may be responsible for MF in Gata1(low) mice and may represent additional targets for therapeutic intervention in PMF.

Removal of the spleen in mice alters the cytokine expression profile of the marrow micro-environment and increases bone formation.

Splenectomized mice express progressively increased numbers of platelets in the blood and reduced numbers of megakaryocytes in the marrow with age. The megakaryocytes in the marrow of these animals express reduced levels of Gata1, a transcription factor necessary for their maturation. In addition, the marrow from these animals expresses greater levels of cytokines (TGF-beta, PDGF-alpha, and VEGF) known to be produced at high levels by megakaryocytes expressing reduced levels of Gata1. This high level of cytokine expression is in turn associated with active osteoblast proliferation localized to areas of the femur, where megakaryocytes expressing reduced Gata1 levels are also found. These results confirm the role of megakaryocytes as regulator of bone formation in mice and suggest that a cross-talk between the spleen and marrow may regulate the total numbers of hemopoietic niches present in an animal.

Ionizing radiation induces apoptotic signal through protein kinase Cdelta (delta) and survival signal through Akt and cyclic-nucleotide response element-binding protein (CREB) in Jurkat T cells.

Although ionizing radiation induces a loss of proliferative capacity as well as cell death by apoptosis and necrosis, cells can oppose the damaging effects by activating survival signal pathways. Here we report the effect of 1.5- and 6-Gy doses of ionizing radiation on apoptotic protein kinase Cdelta (PKCdelta) and survival cyclic-nucleotide response element-binding protein (CREB) signal in Jurkat T cells. Cell cycle analysis, performed by flow cytometry, showed a significant G2M arrest 24 h after exposure to 6 Gy. This arrest was accompanied by dead cells, which increased in number up to 7 days, when cell viability was further reduced. The response was apparently promoted by caspase-3-mediated PKCdelta activation, and thus apoptosis. Moreover, the presence of viable cells up to 7 days in samples exposed to 6 Gy is explained by Akt activation, which may influence the nuclear transcription factor CREB, leading to resistance to ionizing radiation. Thus, the knowledge of apoptotic and survival pathways activated in tumor cells may help in establishing specific therapies by combining selective inhibitors or stimulators of key signaling proteins with conventional chemotherapy, hormone therapy, and radiotherapy.

The JAK2V617 mutation induces constitutive activation and agonist hypersensitivity in basophils from patients with polycythemia vera.

BACKGROUND: The JAK2V617F mutation has been associated with constitutive and enhanced activation of neutrophils, while no information is available concerning other leukocyte subtypes. DESIGN AND METHODS: We evaluated correlations between JAK2V617F mutation and the count of circulating basophils, the number of activated CD63(+) basophils, their response in vitro to agonists as well as the effects of a JAK2 inhibitor. RESULTS: We found that basophil count was increased in patients with JAK2V617F -positive myeloproliferative neoplasms, particularly in those with polycythemia vera, and was correlated with the V617F burden. The burden of V617F allele was similar in neutrophils and basophils from patients with polycythemia vera, while total JAK2 mRNA content was remarkably greater in the basophils; however, the content of JAK2 protein in basophils was not increased. The number of CD63(+) basophils was higher in patients with polycythemia vera than in healthy subjects or patients with essential thrombocythemia or primary myelofibrosis and was correlated with the V617F burden. Ultrastructurally, basophils from patients with polycythemia vera contained an increased number of granules, most of which were empty suggesting cell degranulation in vivo. Ex vivo experiments revealed that basophils from patients with polycythemia vera were hypersensitive to the priming effect of interleukin-3 and to f-MLP-induced activation; pre-treatment with a JAK2 inhibitor reduced polycythemia vera basophil activation. Finally, we found that the number of circulating CD63(+) basophils was significantly greater in patients suffering from aquagenic pruritus, who also showed a higher V617F allele burden. CONCLUSIONS: These data indicate that the number of constitutively activated and hypersensitive circulating basophils is increased in polycythemia vera, underscoring a role of JAK2V617F in these cells' abnormal function and, putatively, in the pathogenesis of pruritus.

Gata1 expression driven by the alternative HS2 enhancer in the spleen rescues the hematopoietic failure induced by the hypomorphic Gata1low mutation.

Rigorously defined reconstitution assays developed in recent years have allowed recognition of the delicate relationship that exists between hematopoietic stem cells and their niches. This balance ensures that hematopoiesis occurs in the marrow under steady-state conditions. However, during development, recovery from hematopoietic stress and in myeloproliferative disorders, hematopoiesis occurs in extramedullary sites whose microenvironments are still poorly defined. The hypomorphic Gata1(low) mutation deletes the regulatory sequences of the gene necessary for its expression in hematopoietic cells generated in the marrow. By analyzing the mechanism that rescues hematopoiesis in mice carrying this mutation, we provide evidence that extramedullary microenvironments sustain maturation of stem cells that would be otherwise incapable of maturing in the marrow.

Increased differentiation of dermal mast cells in mice lacking the Mpl gene.

Thrombopoietin interactions with its receptor, Mpl, play an important role in the regulation of hematopoietic stem/progenitor cell proliferation and differentiation. In this study, we report that the mast cell restricted progenitor cells (MCP) and the mast cell precursors in the bone marrow of wild-type mice express Mpl on their surface. Furthermore, targeted deletion of the Mpl gene in mice decreases the number of MCP while increasing the number of mast cell precursors present in the marrow and spleen. It also increases the number of mast cells present in the dermis, in the peritoneal cavity, and in the gut of the mice. In addition, serosal mast cells from Mpl(null) mice have a distinctive differentiation profile similar to that expressed by wild-type dermal mast cells. These results suggest that not only does ligation of thrombopoietin with the Mpl receptor exert an effect at the mast cell restricted progenitor cell level, but also plays an unexpected yet important role in mast cell maturation.

Thrombopoietin inhibits murine mast cell differentiation.

We have recently shown that Mpl, the thrombopoietin receptor, is expressed on murine mast cells and on their precursors and that targeted deletion of the Mpl gene increases mast cell differentiation in mice. Here we report that treatment of mice with thrombopoietin or addition of this growth factor to bone marrow-derived mast cell cultures severely hampers the generation of mature cells from their precursors by inducing apoptosis. Analysis of the expression profiling of mast cells obtained in the presence of thrombopoietin suggests that thrombopoietin induces apoptosis of mast cells by reducing expression of the transcription factor Mitf and its target antiapoptotic gene Bcl2.

Role of thrombopoietin in mast cell differentiation.

Mast cells are important elements of the body response to foreign antigens, being those represented either by small molecules (allergic response) or harbored by foreign microorganisms (response to parasite infection). These cells derive from hematopoietic stem/progenitor cells present in the marrow. However, in contrast with most of the other hematopoietic lineages, mast cells do not differentiate in the marrow but in highly vascularized extramedullary sites, such as the skin or the gut. Mast cell differentiation in the marrow is activated as part of the body response to parasites. We will review here the mast cell differentiation pathway and what is known of its major intrinsic and extrinsic control mechanisms. It will also be described that thrombopoietin, the ligand for the Mpl receptor, in addition to its pivotal rule in the control of thrombocytopoiesis and of hematopoietic stem/progenitor cell proliferation, exerts a regulatory function in mast cell differentiation. Some of the possible implications of this newly described biological activity of thrombopoietin will be discussed.

Cyclic nucleotide response element binding (CREB) protein activation is involved in K562 erythroleukemia cells differentiation.

K562 are human erythroleukemia cells inducible to differentiate into megakaryocytic or erythroid lineage by different agents. Cyclic nucleotide Response Element Binding (CREB) protein, a nuclear transcription factor which mediates c-AMP signaling, is a potential candidate involved in the occurrence of erythroid differentiation and adaptive response. Here we investigated signaling events in K562 cells induced with 30 microM hemin to undergo erythroid differentiation. CREB activation was detected early 1 h after hemin treatment and up to 4 and 6 days of treatment, when K562 terminal differentiation occurs together with caspase-3 maximal activation and PARP degradation. It was interesting to note that after hemin treatment in the presence of SB203580, p38 MAP kinase specific inhibitor, a reduced rate of CREB phosphorylation as well as a lower percentage of CD71/Gly+ (Glycophorin A) cells were detectable, demonstrating the p38 MAP kinase dependency of these phenomena. All in all these results document a novel relationship between CREB activation and differentiation-related apoptotic cell death and assign a role to p38 MAP kinase pathway in determining these events in K562 erythroleukemia cells.

Cyclic nucleotide Response Element Binding protein (CREB) activation promotes survival signal in human K562 erythroleukemia cells exposed to ionising radiation/etoposide combined treatment.

Anticancer therapy addresses the destruction of tumour cells which try to counteract the effect of drugs and/or ionising radiation. Thus the knowledge of the threshold over which the cells do not resist such agents could help in the setting up of therapy protocols. Since a key role was assigned to Cyclic nucleotide Response Element Binding protein (CREB) multigenic family (which is composed of several nuclear transcription factors involved in c-AMP signalling in cell differentiation, proliferation, apoptosis, survival and adaptive response and in hematopoiesis and acute leukemias), attention was paid to the activation of Erk cascade and of the downstream kinases and transcription factors such as p90RSK and CREB. K562 erythroleukemia cell survival to 1.5 Gy ionising radiation with or without etoposide treatment seemed to involve Erk phosphorylation which, regulating p90 RSK, should activate CREB. In parallel, p38 MAP kinase activity down-modulation, along with low caspase-3 activity, and no modification of Bax and Bcl2 levels, supported such evidence. Thus, endogenous CREB activation, triggering a potent survival signal in K562 cells exposed to 1.5 Gy with or without etoposide, led us to suggest that using specific inhibitors against CREB, such as modified phosphorothionate oligodeoxynucleotides (ODN) corresponding to CREB-1 sequence, anticancer therapy efficacy could be improved.

Levels of apoptosis in human granulosa cells seem to be comparable after therapy with a gonadotropin-releasing hormone agonist or antagonist.

OBJECTIVE: To compare levels of apoptosis in granulosa cells from women treated with the gonadotropin-releasing hormone (GnRH) agonist triptorelin or the GnRH antagonist cetrorelix. DESIGN: Randomized, prospective study. SETTING: University hospital. PATIENT(S): Thirty-two women undergoing assisted reproduction techniques after ovulation induction with recombinant follicle-stimulating hormone (FSH) plus GnRH agonist or antagonist. INTERVENTION(S): Granulosa cells were isolated from follicular aspirates after oocyte removal. MAIN OUTCOME MEASURE(S): Apoptosis was assessed with Annexin V binding assay, terminal deoxynucleotidyl transferase (TdT)-mediated nick-end labeling (TUNEL) assay, flow cytometric analysis of DNA, and ultrastructural analysis of cell morphology in transmission electron microscopy. Serum and follicular hormonal levels were also determined. RESULT(S): Annexin V binding and TUNEL assays revealed comparable percentages of apoptosis in the two groups under investigation. Analysis of DNA histograms revealed a similar cell cycle distribution in the two groups. Ultrastructural analysis only occasionally displayed patterns of chromatin margination in apoptotic cells. The mean concentrations of all the follicular fluid steroid hormones evaluated (E2, T, and P) were significantly lower in the GnRH antagonist-treated group. CONCLUSION(S): Therapy with a GnRH agonist or antagonist is associated with comparable levels of apoptosis in granulosa cells.