Mapping of the Human Amniotic Membrane: In Situ Detection of Microvesicles Secreted by Amniotic Epithelial Cells.

The potential clinical applications of human amniotic membrane (hAM) and human amniotic epithelial cells (hAECs) in the field of regenerative medicine have been known in literature since long. However, it has yet to be elucidated whether hAM contains different anatomical regions with different plasticity and differentiation potential. Recently, for the first time, we highlighted many differences in terms of morphology, marker expression, and differentiation capabilities among four distinct anatomical regions of hAM, demonstrating peculiar functional features in hAEC populations. The aim of this study was to investigate in situ the ultrastructure of the four different regions of hAM by means of transmission electron microscopy (TEM) to deeply understand their peculiar characteristics and to investigate the presence and localization of secretory products because to our knowledge, there are no similar studies in the literature. The results of this study confirm our previous observations of hAM heterogeneity and highlight for the first time that hAM can produce extracellular vesicles (EVs) in a heterogeneous manner. These findings should be considered to increase efficiency of hAM applications within a therapeutic context.

Effects of H2O2 Treatment Combined With PI3K Inhibitor and MEK Inhibitor in AGS Cells: Oxidative Stress Outcomes in a Model of Gastric Cancer.

Gastric cancer is worldwide the fifth and third cancer for incidence and mortality, respectively. Stomach wall is daily exposed to oxidative stress and BER system has a key role in the defense from oxidation-induced DNA damage, whilst ErbB receptors have important roles in the pathogenesis of cancer. We used AGS cells as an aggressive gastric carcinoma cell model, treated with H2O2 alone or combined with ErbB signaling pathway inhibitors, to evaluate the effects of oxidative stress in gastric cancer, focusing on the modulation of ErbB signaling pathways and their eventual cross-talk with BER system. We showed that treatment with H2O2 combined with PI3K/AKT and MEK inhibitors influenced cell morphology and resulted in a reduction of cancer cell viability. Migration ability was reduced after H2O2 treatment alone or combined with MEK inhibitor and after PI3K/AKT inhibitor alone. Western blotting analysis showed that oxidative stress stimulated EGFR pathway favoring the MAPKs activation at the expense of PI3K/AKT pathway. Gene expression analysis by RT-qPCR showed ErbB2 and OGG1 increase under oxidative stress conditions. Therefore, we suggest that in AGS cells a pro-oxidant treatment can reduce gastric cancer cell growth and migration via a different modulation of PI3K and MAPKs pathways. Moreover, the observed ErbB2 and OGG1 induction is a cellular response to protect the cells from H2O2-induced cell death. In conclusion, to tailor specific combinations of therapies and to decide which strategy to use, administration of a chemotherapy that increases intracellular ROS to toxic levels, might not only be dependent on the tumor type, but also on the molecular targeting therapy used.

The Role of the CREB Protein Family Members and the Related Transcription Factors in Radioresistance Mechanisms.

In the framework of space flight, the risk of radiation carcinogenesis is considered a "red" risk due to the high likelihood of occurrence as well as the high potential impact on the quality of life in terms of disease-free survival after space missions. The cyclic AMP response element-binding protein (CREB) is overexpressed both in haematological malignancies and solid tumours and its expression and function are modulated following irradiation. The CREB protein is a transcription factor and member of the CREB/activating transcription factor (ATF) family. As such, it has an essential role in a wide range of cell processes, including cell survival, proliferation, and differentiation. Among the CREB-related nuclear transcription factors, NF-κB and p53 have a relevant role in cell response to ionising radiation. Their expression and function can decide the fate of the cell by choosing between death or survival. The aim of this review was to define the role of the CREB/ATF family members and the related transcription factors in the response to ionising radiation of human haematological malignancies and solid tumours.

Human amniotic fluid stem cells are able to form embryoid body-like aggregates which performs specific functions: morphological evidences.

Human second trimester Amniotic Fluid Stem Cells (hAFSCs) harbour the potential to differentiate into cells of each of the three germ layers and to form Embryoid Body (EB)-like aggregates, without inducing teratoma formation and with no ethical concerns. However, in spite of the number of reports on hAFSCs-EBs and their characterization, a thorough evaluation in light and electron microscopy of morphological and morphometric features of hAFSCs-EBs development in vitro has not been reported yet. Apart from a superficial layer of epithelial-like flat cells, displaying rare microvilli on the free surface, hAFSCs-EBs enclose inner material, abundant in vesicles and secretory granules, showing early characteristics of connective extracellular matrix dispersed among different types of inner cells. The observation of a number of microvesicles mainly represented by microparticles and, to a lower extent, by exosomes indicates the presence of a complex cellular communication system within this structure. According to morphological analysis, after 7 days of in vitro culture hAFSCs-EB appears as a well-organized corpuscle, sufficiently young to be a carrier of stemness and at the same time, when appropriately stimulated, able to differentiate. In fact, 7-day hAFSCs-EB represents itself an initial cellular transformation towards a specialized structure both in recording and in providing different stimuli from the surrounding environment, organizing structures and cells towards a differentiation fate.

Rat bone healing induced by natural nanocrystalline carbonated hydroxyapatite in combination with human amniotic fluid stem cells (AFSCs).

The present study was aimed at identifying a new scaffold/stem cell combination useful to treat large bone defects. Human amniotic fluid stem cells (AFSCs) were expanded in vitro, labeled with a fluorescent cell-permeable dye (PKH26) and transplanted in vivo in a femoral injured rat model. The femoral defect was left untreated (control rats) or filled with hydroxyapatite (HA; natural nanocrystalline carbonated hydroxyapatite-Orthoss®) scaffold alone or loaded with PKH26-labeled AFSCs. All animals were killed 3 weeks after implantation. Both gross anatomy and histological observations revealed a major bone regenerative response in rat specimens treated with HA scaffold, alone or supplemented with AFSCs. Samples injected with HA plus AFSCs displayed the presence of abundant fibrotic tissue, the formation of periosteal woven bone, and an increased presence of blood vessels in the bone marrow, with still fluorescent AFSCs in close proximity. These observations provide evidence that natural HA plus AFSCs represents a promising alternative therapeutic strategy to autologous bone grafting procedures.

Differential response to hepatic differentiation stimuli of amniotic epithelial cells isolated from four regions of the amniotic membrane.

Human Amniotic Epithelial Cells (hAEC) isolated from term placenta are a promising source for regenerative medicine. However, it has long been debated whether the hAEC population consists of heterogeneous or homogeneous cells. In a previous study, we investigated the characteristics of hAEC isolated from four different regions of the amniotic membrane finding significant heterogeneity. The aim of this study was to evaluate the hepatic differentiation capability of hAEC isolated from these four regions. Human term placentae were collected after caesarean section and hAEC were isolated from four regions of the amniotic membrane (R1-R4, according to their relative distance from the umbilical cord) and treated in hepatic differentiation conditions for 14 days. hAEC-derived hepatocyte-like cells showed marked differences in the expression of hepatic markers: R4 showed higher levels of Albumin and Hepatocyte Nuclear Factor (HNF) 4α whereas R1 expressed higher Cytochrome P450 enzymes, both at the gene and protein level. These preliminary results suggest that hAEC isolated from R1 and R4 of the amniotic membrane are more prone to hepatic differentiation. Therefore, the use of hAEC from a specific region of the amniotic membrane should be taken into consideration as it could have an impact on the outcome of therapeutic applications.

Mapping of the Human Placenta: Experimental Evidence of Amniotic Epithelial Cell Heterogeneity.

The human placenta is an important source of stem cells that can be easily collected without ethical concerns since it is usually discarded after childbirth. In this study, we analyzed the amniotic membrane (AM) from the human placenta with the aim of mapping different regions with respect to their morpho-functional features and regenerative potential. AMs were obtained from 24 healthy women, undergoing a caesarean section, and mapped into 4 different regions according to their position in relation to the umbilical cord: the central, intermediate, peripheral, and reflected areas. We carried out a multiparametric analysis focusing our attention on amniotic epithelial cells (AECs). Our results revealed that AECs, isolated from the different areas, are a heterogeneous cell population with different pluripotency and proliferation marker expression (octamer-binding transcription factor 4 [OCT-4], tyrosine-protein kinase KIT [c-KIT], sex determining region Y-box 2 [SOX-2], α-fetoprotein, cyclic AMP response element binding [CREB] protein, and phosphorylated active form of CREB [p-CREB]), proliferative ability, and osteogenic potential. Our investigation discloses interesting findings that could be useful for increasing the efficiency of AM isolation and application for therapeutic purposes.

Regulation of Cancer Cell Responsiveness to Ionizing Radiation Treatment by Cyclic AMP Response Element Binding Nuclear Transcription Factor.

Cyclic AMP response element binding (CREB) protein is a member of the CREB/activating transcription factor (ATF) family of transcription factors that play an important role in the cell response to different environmental stimuli leading to proliferation, differentiation, apoptosis, and survival. A number of studies highlight the involvement of CREB in the resistance to ionizing radiation (IR) therapy, demonstrating a relationship between IR-induced CREB family members' activation and cell survival. Consistent with these observations, we have recently demonstrated that CREB and ATF-1 are expressed in leukemia cell lines and that low-dose radiation treatment can trigger CREB activation, leading to survival of erythro-leukemia cells (K562). On the other hand, a number of evidences highlight a proapoptotic role of CREB following IR treatment of cancer cells. Since the development of multiple mechanisms of resistance is one key problem of most malignancies, including those of hematological origin, it is highly desirable to identify biological markers of responsiveness/unresponsiveness useful to follow-up the individual response and to adjust anticancer treatments. Taking into account all these considerations, this mini-review will be focused on the involvement of CREB/ATF family members in response to IR therapy, to deepen our knowledge of this topic, and to pave the way to translation into a therapeutic context.

Curcuma longa Is Able to Induce Apoptotic Cell Death of Pterygium-Derived Human Keratinocytes.

Pterygium is a relatively common eye disease that can display an aggressive clinical behaviour. To evaluate the in vitro effects of Curcuma longa on human pterygium-derived keratinocytes, specimens of pterygium from 20 patients undergoing pterygium surgical excision were collected. Pterygium explants were put into culture and derived keratinocytes were treated with an alcoholic extract of 1.3% Curcuma longa in 0.001% Benzalkonium Chloride for 3, 6, and 24 h. Cultured cells were examined for CAM5.2 (anti-cytokeratin antibody) and CD140 (anti-fibroblast transmembrane glycoprotein antibody) expression between 3th and 16th passage to assess cell homogeneity. TUNEL technique and Annexin-V/PI staining in flow cytometry were used to detect keratinocyte apoptosis. We showed that Curcuma longa exerts a proapoptotic effect on pterygium-derived keratinocytes already after 3 h treatment. Moreover, after 24 h treatment, Curcuma longa induces a significant increase in TUNEL as well as Annexin-V/PI positive cells in comparison to untreated samples. Our study confirms previous observations highlighting the expression, in pterygium keratinocytes, of nuclear VEGF and gives evidence for the first time to the expression of nuclear and cytoplasmic VEGF-R1. All in all, these findings suggest that Curcuma longa could have some therapeutic potential in the treatment and prevention of human pterygium.

Predictive value of microparticle-associated tissue factor activity for permeability glycoprotein-mediated multidrug resistance in cancer.

Multidrug resistance (MDR) protein 1, which is also known as permeability glycoprotein (Pgp), and tissue factor (TF) are recurrently overexpressed on the surface of cancer cells, likely in response to stimuli such as chemotherapy. Microparticles (MPs) released from cancer cells into the bloodstream express tumour markers on their surface that may be useful as predictive biomarkers for evaluating disease progression. The present study measured the level of TF/factor VII (FVII)-dependent coagulation of MPs isolated from the plasma of cancer patients with various tumours, who were undergoing chemotherapy. Furthermore, Pgp expression on the surface of MPs was evaluated by immunohistochemistry. A total of 50 cancer patients, as well as 10 healthy volunteers, were enrolled in the present study. MP-associated TF/FVII-dependent coagulation pathways were evaluated as the effect of an anti-FVII antibody on the time to thrombin generation, as compared with controls treated with saline. The significantly lengthened times of coagulation [obtained in 20/50 samples (36.5 ± 16%) after treatment with anti-FVIIa when compared with controls] suggest the presence of TF activity is associated with circulating MPs. Furthermore, the 20 MP/TF-positive samples were associated with Pgp overexpression on their surface. Conversely, in the remaining samples (n=30), treatment with the anti-FVIIa antibody did not significantly lengthen the time to clotting (<10%), and Pgp overexpression was not detected. In addition, in the control samples from healthy individuals, Pgp expression at the plasma membrane and clotting in the presence of the anti-FVII antibody were not observed, indicating the absence of MPs. The present study demonstrated that MPs in the blood of cancer patients promoted fibrin generation via TF/FVII-dependent pathways, thus suggesting that the evaluation of MP-TF activity may have a predictive value for Pgp-mediated MDR in various cancer types. Although further studies are required, the measurement of plasma MP-associated TF activity as a predictive biomarker may provide novel therapeutic perspectives to improve the prognosis and effectiveness of anti-cancer drugs in patients who are at a high-risk of Pgp-mediated MDR.

DNA Repair and Cytokines: TGF-ß, IL-6, and Thrombopoietin as Different Biomarkers of Radioresistance.

Double strand breaks (DSBs) induced by radiotherapy are highly cytotoxic lesions, leading to chromosomal aberrations and cell death. Ataxia-telangiectasia-mutated (ATM)-dependent DNA-damage response, non-homologous end joining, and homologous recombination pathways coordinately contribute to repairing DSBs in higher eukaryotes. It is known that the expression of DSB repair genes is increased in tumors, which is one of the main reasons for radioresistance. The inhibition of DSB repair pathways may be useful to increase tumor cell radiosensitivity and may target stem cell-like cancer cells, known to be the most radioresistant tumor components. Commonly overexpressed in neoplastic cells, cytokines confer radioresistance by promoting proliferation, survival, invasion, and angiogenesis. Unfortunately, tumor irradiation increases the expression of various cytokines displaying these effects, including transforming growth factor-beta and interleukin-6. Recently, the capabilities of these cytokines to support DNA repair pathways and the ATM-dependent DNA response have been demonstrated. Thrombopoietin, essential for megakaryopoiesis and very important for hematopoietic stem cell (HSC) homeostasis, has also been found to promote DNA repair in a highly selective manner. These findings reveal a novel mechanism underlying cytokine-related radioresistance, which may be clinically relevant. Therapies targeting specific cytokines may be used to improve radiosensitivity. Specific inhibitors may be chosen in consideration of different tumor microenvironments. Thrombopoietin may be useful in fending off irradiation-induced loss of HSCs.

Ultrastructural regenerating features of nasal mucosa following microdebrider-assisted turbinoplasty are related to clinical recovery.

BACKGROUND: The nasal mucosa plays a key role in conditioning the inhaled air and in regulating the immune response. These functions led many authors to recommend mucosal sparing techniques for the surgical management of inferior turbinate hypertrophy. However, the histological modifications of chronic diseases retain the inflammatory activity and prevent the nasal physiology restoration. It has been proved that the basal cells of the nasal mucosa are able to proliferate and to repair after cold-knife incision. The aim of this study was to demonstrate that the healing process after removal of the inferior turbinate mucosa with cold techniques results in a complete structural restoration. METHODS: A prospective study was performed in 18 patients who underwent Microdebrider inferior turbinoplasty (cold technique). Subjective and objective improvement of nasal patency was evaluated with visual analogue scale, rhinomanometry, videoendoscopy and mucociliary transport test. Pre- and post-operative biopsy specimens were taken from 7 patients to evaluate the healing process. Two samples were taken from two healthy patients as control. The specimens were processed for transmission electron microscopy analysis. RESULTS: Videoendoscopy showed reduction of lower turbinate after surgery. Nasal patency augmented and no adverse consequences were observed. After 4 months the nasal mucosa showed normal appearance, with restoration of the pseudostratified ciliated pattern, intercellular connections and normal cellular morphology. Fibrosis and submucosal edema disappeared. At longer time after operation (4 years) clinical improvement was confirmed. CONCLUSIONS: The total removal of the nasal mucosa with cold techniques results in a complete restoration of the normal structure and permanent resolution of the chronic inflammation typical of hypertrophic rhinopathy.

Molecular and phenotypic characterization of human amniotic fluid-derived cells: a morphological and proteomic approach.

Mesenchymal Stem Cells derived from Amniotic Fluid (AFMSCs) are multipotent cells of great interest for regenerative medicine. Two predominant cell types, that is, Epithelial-like (E-like) and Fibroblast-like (F-like), have been previously detected in the amniotic fluid (AF). In this study, we examined the AF from 12 donors and observed the prevalence of the E-like phenotype in 5, whereas the F-like morphology was predominant in 7 samples. These phenotypes showed slight differences in membrane markers, with higher CD90 and lower Sox2 and SSEA-4 expression in F-like than in E-like cells; whereas CD326 was expressed only in the E-like phenotype. They did not show any significant differences in osteogenic, adipogenic or chondrogenic differentiation. Proteomic analysis revealed that samples with a predominant E-like phenotype (HC1) showed a different profile than those with a predominant F-like phenotype (HC2). Twenty-five and eighteen protein spots were differentially expressed in HC1 and HC2 classes, respectively. Of these, 17 from HC1 and 4 from HC2 were identified by mass spectrometry. Protein-interaction networks for both phenotypes showed strong interactions between specific AFMSC proteins and molecular chaperones, such as preproteasomes and mature proteasomes, both of which are important for cell cycle regulation and apoptosis. Collectively, our results provide evidence that, regardless of differences in protein profiling, the prevalence of E-like or F-like cells in AF does not affect the differentiation capacity of AFMSC preparations. This may be valuable information with a view to the therapeutic use of AFMSCs.

Human second trimester amniotic fluid cells are able to create embryoid body-like structures in vitro and to show typical expression profiles of embryonic and primordial germ cells.

Human amniotic fluid-derived stem cells (AFSCs) represent a novel class of broadly multipotent stem cells sharing characteristics of both embryonic and adult stem cells. However, both the origin of these cells and their actual properties in terms of pluripotent differentiation potential are still debated. In order to verify the presence of features of pluripotency in human second trimester AFSCs, we have investigated the ability of these cells to form in vitro three-dimensional aggregates, known as embryoid bodies (EBs), and to express specific genes of embryonic stem cells (ESCs) and primordial germ cells (PGCs). EBs were obtained after 5 days of AFSC culture in suspension and showed positivity for alkaline phosphatase (AP) staining and for specific markers of pluripotency (OCT4 and SOX2). Moreover, EB-derived cells showed the expression of specific transcripts of the three germ layers. RT-PCR analysis, carried out at different culture times (second, third, fourth, fifth, and eighth passages), revealed the presence of specific markers of ESCs (such as FGF4 and DAPPA4), as well as of markers typical of PGCs and, in particular, genes involved in early stages of germ cell development (Fragilis, Stella, Vasa, c-Kit, Rnf17). Finally, the expression of genes related to the control of DNA methylation (DNMT3A, DNMT3b1, DNMT1, DNMT3L, MBD1, MBD2, MBD3, MDB4, MeCP2), as well as the lack of inactivation of the X-chromosome in female samples, was also demonstrated. Taken together, these data provide further evidence for the presence of common features among human AFSCs, PGCs, and ESCs.

Stem cell ageing and apoptosis.

Ageing has been defined as the process of deterioration of many body functions over the lifespan of an individual. In spite of the number of different theories about ageing, there is a general consensus in identifying ageing effects in a reduced capacity to regenerate injured tissues or organs and an increased propensity to infections and cancer. In recent years the stem cell theory of ageing has gained much attention. Adult stem cells residing in mammalian tissues are essential for tissue homeostasis and repair throughout adult life. With advancing age, the highly regulated molecular signalling necessary to ensure proper cellular, tissue, and organ homeostasis loses coordination and leads, as a consequence, to a compromised potential of regeneration and repair of damaged cells and tissues. Although a complete comprehension of the molecular mechanisms involved in stem cell ageing and apoptosis is far to be reached, recent studies are beginning to unravel the processes involved in stem cell ageing, particularly in adult skeletal muscle stem cells, namely satellite cells. Thus, the focus of this review is to analyse the relationship between stem cell ageing and apoptosis with a peculiar attention to human satellite cells as compared to haematopoietic stem cells. Undoubtedly, the knowledge of age-related changes of stem cells will help in understanding the ageing process itself and will provide novel therapeutic challenges for improved tissue regeneration.

HIF-1alpha cytoplasmic accumulation is associated with cell death in old rat cerebral cortex exposed to intermittent hypoxia.

Intermittent hypoxia, followed by reoxygenation, determines the production of reactive oxygen species (ROS), which may lead to accelerated aging and to the appearance of age-related diseases. The rise in ROS levels might constitute a stress-stimulus activating specific redox-sensitive signalling pathways, so inducing either damaging or protective functions. Here, we report that in old rat cerebral cortex exposed to hypoxia, the accumulation in the cytoplasm of hypoxic inducible factor 1alpha (HIF-1alpha)--the master regulator of oxygen homeostasis--concomitant with p66(Shc) activation and reduced IkBalpha phosphorylation is associated with tissue apoptosis or necrosis. In young cerebral cortex, we hypothesize that the hypoxic damage may be reversible, based on our demonstration of elevated HIF-1alpha levels, combined with a low level of IkBalpha phosphorylation, a decrease in IAP-1 and a lack of major change in Bcl2 family proteins. These observations are associated with a low level of cell death induced by hypoxia, suggesting that HIF-1alpha activation in cortical neurons may produce rescue proteins in response to intermittent hypoxia.

Increased and pathologic emperipolesis of neutrophils within megakaryocytes associated with marrow fibrosis in GATA-1(low) mice.

Deletion of megakaryocytic-specific regulatory sequences of GATA-1 (Gata1(tm2Sho) or GATA-1(low) mutation) results in severe thrombocytopenia, because of defective thrombocytopoiesis, and myelofibrosis. As documented here, the GATA-1(low) mutation blocks megakaryocytic maturation between stage I and II, resulting in accumulation of defective megakaryocytes (MKs) in the tissues of GATA-1(low) mice. The block in maturation includes failure to properly organize alpha granules because von Willebrand factor is barely detectable in mutant MKs, and P-selectin, although normally expressed, is found frequently associated with the demarcation membrane system (DMS) instead of within granules. Conversely, both von Willebrand factor and P-selectin are barely detectable in GATA-1(low) platelets. Mutant MKs are surrounded by numerous myeloperoxidase-positive neutrophils, some of which appear in the process to establish contact with MKs by fusing their membrane with those of the DMS. As a result, 16% (in spleen) to 34% (in marrow) of GATA-1(low) MKs contain 1 to 3 neutrophils embedded in a vacuolated cytoplasm. The neutrophil-embedded GATA-1(low) MKs have morphologic features (high electron density and negativity to TUNEL staining) compatible with those of cells dying from para-apoptosis. We suggest that such an increased and pathologic neutrophil emperipolesis may represent one of the mechanisms leading to myelofibrosis by releasing fibrogenic MK cytokines and neutrophil proteases in the microenvironment.

Molecular and morphological modifications occurring in rat heart exposed to intermittent hypoxia: role for protein kinase C alpha.

Exposure of rats to intermittent hypoxia determines different responses at tissue and cell level. Heart mainly undergoes the effects of hypoxic injury and its response is determined both by the relationship between oxygen supply and demand and by its functional state. Since molecular mechanisms mediate cells sensing and response to low O(2) concentration, here we explore the role played by Protein Kinase C alpha (PKC alpha) in the signal transduction mechanisms leading to the occurrence of morphological responses in rat neonatal, young and old heart subjected to intermittent hypoxia. Along with a key role for hypoxia inducible factor and vascular endothelial growth factor in the occurrence of continuous state of dynamic adaptation of vasculature, PKC alpha presumably phosphorylates IkBalpha in rat normoxic and hypoxic neonatal hearts, supporting the hypothesis of a rescue strategy carried out against hypoxia, together with an hypertrophic response. In hypoxic young heart PKC alpha activation, paralleled by sustained Bax homodimerization and caspase-3 activation, along with reduced p-IKBalpha and Inhibiting Apoptosis Protein (IAP) expression, suggests that the young early and deeply undergoes the effects of lowered oxygen tension. In addition, since no modifications concerning PKC alpha driven signalling system are evidenced in both the experimental conditions, we suggest an oxygen impaired sensing during ageing.

Effect of chronic hypoxia on inducible nitric oxide synthase expression in rat myocardial tissue.

The purpose of our study was to evaluate the effect of chronic exposure to low cellular oxygen tension (90% N2 and 10% O2 for 14 days) in inducing apoptosis and activation of transcription and translation of inducible nitric oxide (NO) synthase (iNOS) in rat hearts tissue. Rats were divided into four groups: normoxic, hypoxic, rats maintained in normoxic condition for 7 days and subjected to hypoxic conditions for another 7 days, and rats maintained in hypoxic condition for 7 days and subjected to normoxic conditions for another 7 days. At the 7th and 14th days, five rats from each group were sacrificed. Immunohistochemical and Western blot analysis were performed on myocardial tissue to reveal the presence of iNOS. Expression of iNOS was determined by RT-PCR. Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling and by detection of internucleosomal DNA fragmentation by electrophoresis. Electrophoretic analysis of DNA showed oligonucleosomal fragmentation in the hypoxic groups, but no ladder was observed in the other groups. This data was confirmed through end labeling with streptavidin-biotin (biotin d-UTP). iNOS expression was evaluated through immunohistochemical techniques (Ab anti-iNOS) and Western blotting, and the results were quantified with a computerized imaging analysis. The expression of iNOS protein was greater in the hypoxic groups; in the normoxic groups, only a nonspecific background was detected. This data was supported with results obtained through RT-PCR, which showed the specific transcription of mRNA for iNOS in the same experimental conditions. In addition, the iNOS activity was also evaluated and was found to be more active in the hypoxic groups (0.1 +/- 0.01 vs 0.02 +/- 0.003). The present study shows that exposure to low oxygen tension is capable of inducing programmed cell death and activating iNOS.

NF-kappaB activation plays an antiapoptotic role in human leukemic K562 cells exposed to ionizing radiation.

Exposure of cells to ionizing radiation (IR) determines cellular lesions, such as DNA and membrane damage, which involve a coordinate network of signal transduction pathways responsible for resistance to or delay of apoptosis, depending on cell type and administered dose. Since, after IR exposure, the apoptotic profile appeared different in the two chosen cell lines K562 and Jurkat along with caspase-3 activation, we paid attention to the influence exerted by Protein kinase C delta on transcription factor NF-kappaB activation. Interestingly, K562 resist to IR carrying out a survival strategy which includes PKC delta/NF-kappaB pathway activation, probably mediated by novel IKKs and a role for PI-3-kinase in activating PKC delta at Thr 505 by PDK-1 phosphorylation is suggested. In addition, since caspase-3 is not activated in these cells upon ionizing radiation exposure, it could be supposed that NF-kappaB antagonizes apoptosis induction interfering with pathways which lead to caspase activation, may be by inducing expression of IAP, caspases 3, 7, 9, inhibitor. Thus NF-kappaB activation explains the resistance displayed by K562 to IR and drug potential interference directed to this protein could overcome apoptosis resistance in clinical settings.