Spontaneous Apoptotic Cell Research Articles

Phosphatidylserine released from apoptotic cells in tumor induces M2-like macrophage polarization through the PSR-STAT3-JMJD3 axis.

BackgroundUnderstanding how the tumor microenvironment is shaped by various factors is important for the development of new therapeutic strategies. Tumor cells often undergo spontaneous apoptotic cell death in tumor microenvironment, these apoptotic cells are histologically co‐localized with immunosuppressive macrophages. However, the mechanism by which tumor cell apoptosis modulates macrophage polarization is not fully understood. In this study, we aimed to explore the tumor promoting effects of apoptotic tumor cells and the signal pathways involved.MethodsApoptotic cells and macrophages in tumors were detected by immunohistochemical staining. Morphological analysis was performed with Giemsa staining. Lipids generated from apoptotic cells were detected by liquid chromatography‐mass spectrometry. Phosphatidylserine‐containing liposomes were prepared to mimic apoptotic cells. The expression of protein was determined by real‐time PCR, immunohistochemistry enzyme‐linked immunosorbent assay and Western blotting. Mouse malignant ascites and subcutaneous tumor models were designed for in vivo analysis. Transgenic mice with specific genes knocked out and inhibitors specific to certain proteins were used for the mechanistic studies.ResultsThe location and the number of apoptotic cells were correlated with that of macrophages in several types of carcinomas. Phosphatidylserine, a lipid molecule generated in apoptotic cells, induced polarization and accumulation of M2‐like macrophages in vivo and in vitro. Moreover, sustained administration of phosphoserine promoted tumor growth in the malignant ascites and subcutaneous tumor models. Further analyses suggested that phosphoserine induced a M2‐like phenotype in macrophages, which was related to the activation of phosphoserine receptors including T‐cell immunoglobin mucin 4 (TIM4) and the FAK‐SRC‐STAT3 signaling pathway as well as elevated the expression of the histone demethylase Jumonji domain‐containing protein 3 (JMJD3). Administration of specific inhibitors of these pathways could reduce tumor progression.ConclusionsThis study suggest that apoptotic cell‐generated phosphoserine might be a notable signal for immunosuppressive macrophages in tumors, and the related pathways might be potential therapeutic targets for cancer therapy.

Deficiency of Antioxidative Paraoxonase 2 (Pon2) Leads to Increased Number of Phenotypic LT-HSCs and Disturbed Erythropoiesis.

Background Long-term hematopoietic stem cells (LT-HSCs) reside in bone marrow niches with tightly controlled reactive oxygen species (ROS) levels. ROS increase results into LT-HSC differentiation and stem cell exhaustion. Paraoxonase 2 (PON2) has been shown to be important for ROS control. Objectives We investigate the effects of inactivation of the PON2 gene on hematopoietic cell differentiation and activity. Methods and Results In young mice with inactivated Pon2 gene (Pon2−/−, <3 months), we observed an increase of LT-HSCs and a reduced frequency of progenitor cells. In competitive transplantations, young Pon2−/− BM outcompeted WT BM at early time points. ROS levels were significantly increased in Pon2−/− whole BM, but not in Pon2−/− LT-HSCs. In more differentiated stages of hematopoiesis, Pon2 deficiency led to a misbalanced erythropoiesis both in physiologic and stress conditions. In older mice (>9 months), Pon2 depletion caused an increase in LT-HSCs as well as increased levels of granulocyte/macrophage progenitors (GMPs) and myeloid skewing, indicating a premature aging phenotype. No significant changes in ROS levels in old Pon2−/− LT- and short-term (ST-) HSCs were observed, but a significant reduction of spontaneous apoptotic cell death was measured. RNA-seq analysis in Pon2−/− LT-HSCs identified overrepresentation of genes involved in the C-X-C chemokine receptor type 4 (Cxcr4) signaling, suggesting compensatory mechanisms to overcome ROS-mediated accelerated aging in hematopoietic progenitor cells. Conclusions In summary, our current data indicate that PON2 is involved in the regulation of HSC functions.

Deficiency of PDK4 Sensitizes Mouse Liver to Diethylnitrosamine and Arsenic Toxicity Through Inducing Apoptosis

BackgroundPyruvate dehydrogenase kinase 4 (PDK4) is a metabolic switch that regulates glucose oxidation and the TCA cycle in the mitochondria. The liver detoxifies xenobiotics and is constantly challenged by various injuries. How the loss of this metabolic regulator, PDK4, contributes to liver injuries remains elusive. Previous studies from our lab have revealed that knockout of Pdk4 in mice resulted in increased cellular proliferation via E2F1 transcription factor‐mediated increase in cyclins.ObjectiveThe objective of this study was to elucidate a novel function of PDK4 in toxin‐induced liver injury.MethodsTwo week‐old wild‐type (WT) (C57BL/6J, male) and Pdk4−/− mice were i.p. injected with a single dose of diethylnitrosamine (DEN) (25 mg/kg body weight) or two month‐old mice were fed with arsenic at 50 ppm (one month) via distilled drinking water. DEN treated mouse samples were collected at day 6 after DEN injection due to unexpected death of Pdk4−/− mice. Liver tissues were collected after both treatments for western blot (WB), IHC, and qPCR. Pdk4 shRNA was used to generate Pdk4 knockdown cells for mitochondrial respiration analysis to measure cellular oxygen consumption rate and extracellular acidification rate (Seahorse Bioscience). Electron microscopy was applied in order to visualize mitochondria.ResultsTUNEL analysis revealed a significant level of spontaneous apoptotic cells in Pdk4−/− mouse livers, which increased with age in the absence of treatment indicating an age‐dependent progression. Microscopy and staining revealed extensive hepatic damage in DEN treated Pdk4−/− mouse livers while WT mouse livers showed no sign of damage which suggests loss of Pdk4 increases hepatic sensitivity to chemical toxicant DEN administration. TUNEL analysis of Arsenic treated mouse liver sections revealed increased apoptosis in Pdk4−/− mice compared to WT mice. Caspase‐3 activity was analyzed via western blot and activity assays revealing a caspase‐3 dependent induction of apoptosis in Pdk4−/− mouse livers. Mitochondrial respiration analysis revealed shPDK4 knockdown cells had significantly higher oxygen consumption rate and a higher respiratory capacity than control cells. Mitochondrial respiration analysis also revealed that shPDK4 knockdown cells had higher extracellular acidification rates suggesting increased glycolytic capacity.ConclusionsIn the present study, we showed that age‐dependent spontaneous hepatic apoptosis happened in systematic Pdk4 knockout (Pdk4−/−) mice and that PDK4‐deficiency increased the sensitivity of adult mice to arsenic insult, as well as juvenile mice to toxicity of DEN, which correlated with a lethal consequence and massive hepatic apoptosis and abnormal mitochondrial respiration.Support or Funding InformationNIH R01ES025909, R01DK104656, R01DK080440, R21AA022482, R21AA024935, VA Merit Award 1I01BX002634, P30 DK34989 (Yale Liver Center).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Establishment of a rapid drug screening system based on embryonic stem cells

Embryonic stem (ES) cells have the capacity for self-renewal and differentiation into three germ layers following formation of embryonic bodies (EB). To investigate toxicity of pharmaceutical compounds, five toxic chemicals, indomethacin, dexamethasone, hydroxyurea, 5-fluorouracil, and cytosine arabinoside were applied in mouse ES cells during formation of EBs. Using microscopic evaluation, the size of EBs was reduced in a dose-dependent manner by treatment with pharmaceutical chemicals. While apoptosis-related proteins, cleaved caspase-3 and PARP, were decreased in compound-exposed EBs, necrosis-related protein (Hmgb1) was present in culture media of EBs, indicating that detection of Hmgb1 can result in activation of necrosis by pharmaceutical compounds. While pharmaceutical compounds impaired the differentiation of mES cells linked with spontaneous apoptotic cell death, it was determined that cytotoxic cell damage is necrosis-dependent in mES cells. In addition, an apoptotic transcript (Noxa mRNA) in toxicant-exposed EBs was decreased in parallel with apoptosis-related proteins. Following impairment of apoptosis, differentiation-related markers including un-differentiation (Sox2), endoderm (Hnf4), mesoderm (Bmp4), and ectoderm (Pax6) also fluctuated by treatment with pharmaceutical compounds. Taken together, the data imply that exposure to pharmaceutical compounds results in increased cell death hindering the spontaneous apoptosis of cells to undergo differentiation. Using both characteristics of ES cells like self-renewal or cellular pluripotency and potentials of ES cells for evaluation in toxicity of various compounds, the current study was conducted for establishment of a novel drug screening system beyond hidden virtues of the well-known chemicals.

Nanoparticles – a Novel Approach to Chronic Lymphocytic Leukemia Treatment?

AbstractAbstract 4601Treatment of chronic lymphocytic leukemia (CLL), including conventional therapy based on alkylating agents, purine nucleoside analogues (cladribine, fludarabine (FA)) or monoclonal antibodies (rituximab (Rit), alemtuzumab), significantly improved overall and complete responses. Although there are many treatment possibilities, this disease still remains incurable. Therefore, searching for new therapeutical strategies in CLL is vital.Nanotechnology, a new and promising field of scientific research, may be of use in medicine and the pharmaceutical industry. Dendrimers, nanoparticles (polymers) of dendritic architecture, used as carriers of drugs, nucleic acid and photosensitizers for targeted delivery, contrast agents in magnetic resonance imaging (MRI) have already been reported. It seems likely that dendrimers themselves might be damaging for neoplastic cells.The aim of our study was to preliminarly assess the clinical value of treating CLL patients with poly(propylene imine) (PPI) dendrimers. The assessment was based on the in vitro induction of cytotoxicity and apoptosis by fourth generation PPI dendrimers, with the surface amino groups substituted with maltotriose [Mal-III] residues. PPI-G4-OS-Mal-III dendrimers have, in contrast to the parental cationic PPI-G4, a virtually neutral surface charge. Therefore, they are preferentially involved in H-bond driven interactions.The study was conducted in 15 untreated CLL patients (7 men, 8 women) who were diagnosed in accordance with IWCLL criteria and followed at the Hematology Department, Medical University, Lodz, Poland. The Ethics Committee of the Medical University of Lodz, Poland approved the study (RNN/75/10/KE). Informed consent was obtained from all patients involved in the study. The mean age of CLL patients was 63.80 yrs.Peripheral blood mononuclear cells (MNCs) were separated from EDTA fresh blood. MNCs were incubated with dendrimers at concentrations of 4, 6 and 8 mg/ml.The cultures without dendrimers served as controls.Dendrimers in which approximately 35% of peripheral amino groups were coated with Mal-III (Leibniz Institute of Polymer Research, Dresden, Germany) have been defined as PPI-G4-OS-Mal-III. The abbreviation OS describes the open shell structure of carbohydrate-modified dendrimers (Fig. 1). The molar mass of this PPI dendrimer was 31000 g/mol.Apoptosis was measured by the Annexin-V/Propidium Iodide test. A decrease in mitochondrial membrane potential (one of the earliest events in the apoptotic pathway) was evaluated by the CMXRos technique, using flow cytometry. The percentage of cells with lower mitochondrial membrane potential (MMP) (DYmlow/Gly-A−) was determined.The percentage of apoptotic MNCs induced by PPI-G4-OS-Mal-III after 24 h and 48 h incubations was significantly higher than the percentage of spontaneous apoptotic leukemic cells. PPI glycodendrimers induced MNC apoptosis to a greater degree after 48 h than 24 h. The largest differences were observed for Ann-V+JP+ cells at a concentration of 8 mg/ml (p = 0.007). The IC50 value after 48-h-incubation was calculated as 8.24 mg/ml.Moreover, the CMXRos technique revealed apoptosis induction by PPI-G4-OS-Mal-III at each examined concentration in comparison with control cultures (Table 1).Table 124hControlPPI-G4-OS-Mal-III 4 mg/mlPPI-G4-OS-Mal-III 6 mg/mlPPI-G4-OS-Mal-III 8 mg/mlFA 1.6 μMRit 10 μg/mlX23.751.060.164.462.928.048hX28.367.871.979.389.029.4X – mean percentage of cells with lower mitochondrial potential (DYmlow/Gly-A− [%])Finally, PPI-G4-OS-Mal-III was not observed to have any harmful effects on erythrocytes (RBCs) or platelets (PLTs). The studied dendrimer induced PLT aggregation at a concentration of 50 mg/ml. Hemolysis induced by the PPI-G4-OS-Mal-III dendrimer is not important from the biological point of view.To summarise, the PPI-G4-OS-Mal-III dendrimer demonstrated higher cytotoxicity towards CLL cells than healthy donor cells. Its potency to trigger apoptosis is similar to many PNAs and monoclonal antibodies widely used in CLL therapy. Thus, dendrimers are a potential tool for CLL treatment.The study was partially supported by Grant No. DEC-2011/01/B/NZ5/01371 from the National Science Centre, Poland. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

The use of sequential staining for detection of heterogeneous intracellular response of individual Jurkat cells to lysophosphatidylcholine

Living cells are known to exhibit great morphological, functional, spatial and temporal heterogeneity. Hence, the study of cells in a bulk, whether this bulk is homogenous or heterogeneous, does not provide sufficiently detailed or interpretable results. An advantageous approach would rather be a comprehensive study of cell biological activity in single isolated living cells. In this study, we present an imaging approach for studying pre-apoptotic and very early apoptotic events, during cell death induced by lysophosphatidylcholine (LPC) at the single cell level. The aim of this study is to investigate intracellular events, such as the mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) formation, before and immediately after LPC introduction to the lymphocytes at the level of individual cells. A new protocol of sequential staining was developed to study the relation between early apoptosis signs (PS externalization), MMP changes and intracellular ROS production rates at an individual Jurkat cell resolution. Simultaneous kinetic assessments of MMP, intracellular ROS levels and phosphatidylserine (PS) externalization were performed at a single cell resolution, using Optical LiveCell™ Array technology and image analysis. The parameters were measured and analyzed both before and during exposure to inducers in a Jurkat cell population, including three groups of single cells: spontaneous apoptotic cells, induced apoptotic cells and fully functional living cells. Exogenous LPC caused a heterogeneous intracellular response among Jurkat cells immediately after its introduction. Subgroups of cells with opposite changes of MMP and different kinetics of ROS increase, were revealed within the whole cell population. The subset of apoptosis-induced Jurkat cells, which became apoptotic within 3h after the LPC introduction, exhibited higher initial MMP compared to fully functional or spontaneous apoptotic cells. LPC-induced apoptosis was accompanied by a concomitant increase in intracellular ROS levels.In the present study, a method is described to assess the intracellular events in cells which were initially different in their physiological status. The individual T lymphocytes (Jurkat cells) in vitro have various susceptibilities to LPC effects at the very early stage of contact with the inducer. The apoptotic effect of LPC in individual Jurkat cells is associated with a relatively higher initial MMP before the introduction of the inducer and with a faster ROS formation within the affected cells. Such divergence may be significant in regulating the balance of lymphocyte subsets in pathological sites, either maintaining or preventing the inflammation components of atherosclerosis.We conclude that the presented approach provides the researcher, not only with the cell retaining methodology, but with opportunities to observe and find the distinctive cell subsets within the whole cell population as well, thus helping to define more exactly the role and importance of such sub-populations in physiological or pathological conditions.

Cellular characteristics of head and neck cancer stem cells in type IV collagen-coated adherent cultures

Although head and neck squamous carcinoma cancer stem cells (HNSC-CSCs) can be enriched in serum-free suspension cultures, it is difficult to stably expand HNSC-CSC lines in suspension due to spontaneous apoptosis and differentiation. Here, we investigated whether HNSC-CSCs can be expanded without loss of stem cell properties by adherent culture methods. Cell culture plates were coated with type IV collagen, laminin, or fibronectin. We examined cancer stem cell traits of adherent HNSC-CSCs grown on these plates using immunocytochemistry for stem cell marker expression and analyses of chemo-resistance and xenograft tumorigenicity. We also assessed the growth rate, apoptosis rate, and gene transduction efficiency of adherent and suspended HNSC-CSCs. HNSC-CSCs grew much faster on type IV collagen-coated plates than in suspension. Adherent HNSC-CSCs expressed putative stem cell markers (OCT4 and CD44) and were chemo-resistant to various cytotoxic drugs (cisplatin, fluorouracil, paclitaxel, and docetaxel). Adherent HNSC-CSCs at the limiting dilution (1000 cells) produced tumors in nude mice. Adherent HNSC-CSCs also showed less spontaneous apoptotic cell death and were more competent to lentiviral transduction than suspended HNSC-CSCs. In conclusion, compared to suspension cultures, adherence on type IV collagen-coated culture plates provides better experimental conditions for HNSC-CSC expansion, which should facilitate various refined cellular studies.

Optic atrophy 3 as a protein of the mitochondrial outer membrane induces mitochondrial fragmentation

The optic atrophy 3 (OPA3) gene, which has no known homolog or biological function, is mutated in patients with hereditary optic neuropathies. Here, we identified OPA3 as an integral protein of the mitochondrial outer membrane (MOM), with a C-terminus exposed to the cytosol and an N-terminal mitochondrial targeting domain. By quantitative analysis, we demonstrated that overexpression of OPA3 significantly induced mitochondrial fragmentation, whereas OPA3 knockdown resulted in highly elongated mitochondria. Cells with mitochondria fragmented by OPA3 did not undergo spontaneous apoptotic cell death, but were significantly sensitized to staurosporine- and TRAIL-induced apoptosis. In contrast, overexpression of a familial OPA3 mutant (G93S) induced mitochondrial fragmentation and spontaneous apoptosis, suggesting that OPA3 mutations may cause optic atrophy via a gain-of-function mechanism. Together, these results indicate that OPA3, as an integral MOM protein, has a crucial role in mitochondrial fission, and provides a direct link between mitochondrial morphology and optic atrophy.

Altered Expression of NDRG2 in the Testes of Experimental Rat Model of Cryptorchidism

To examine the expression of N-Myc downstream regulated gene 2 (NDRG2) in the cryptorchid testes and its potential association with germ cell apoptosis in a rat model of cryptorchidism. Groups of pubertal Sprague-Dawley rats were subjected to surgical procedure for the induction of experimental unilateral cryptorchidism. The weight of the abdominal and scrotal testes was measured and their apoptotic cells and NDRG2 expression were examined longitudinally by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, immunohistochemistry, and immunofluorescence analyses. The expression of NDRG2 and p53 in the purified germ cells and Leydig cells was determined by Western blot assay. Compared with the control scrotal testes, the mean weights of the abdominal testes were significantly reduced at 7 and 14 days after surgery, accompanied by significantly increased numbers of apoptotic germ cells. Although the NDRG2 expression was detected in Leydig cells and spontaneous apoptotic germ cells in the scrotal testes, it was significantly upregulated predominately in apoptotic spermatocytes and round spermatids in the abdominal testes. However, the NDRG2 expression was downregulated in Leydig cells in the abdominal testes. Western blot analysis revealed that the expression of NDRG2 and p53 was upregulated significantly in purified germ cells 7 days after surgery, whereas NDRG2 was downregulated in the purified Leydig cells from the abdominal testes 7 and 14 days after surgery. The upregulated expression of NDRG2 was associated with germ cell apoptosis in the cryptorchid testes, suggesting that the NDRG2 may be a positive regulator for germ cell apoptosis in rat cryptorchidism.

The influence of LIF (Leukemia Inhibitory Factor) on the functional status of R1 mouse embryonic stem cells

The influence of cytokine LIF (Leukemia Inhibitory Factor) on the viability, and proliferation of mouse R1 line embryonic stem cells (ESC) and their distribution by cell cycle stages has been investigated. LIF (5-20 ng/ml) increased growth of colonies and maintained high proliferative and pluripotent properties of R1. LIF was also involved into the inhibition of spontaneous cell differentiation and apoptotic cell death; it also decreased the rations of S/G2+M cell cycle and doubling-time of cell population.

Human Flt3 Is Expressed at the Hematopoietic Stem Cell and the Granulocyte/Macrophage Progenitor Stages to Maintain Cell Survival

FLT3/FLK2, a member of the receptor tyrosine kinase family, plays a critical role in maintenance of hematopoietic homeostasis, and the constitutively active form of the FLT3 mutation is one of the most common genetic abnormalities in acute myelogenous leukemia. In murine hematopoiesis, Flt3 is not expressed in self-renewing hematopoietic stem cells, but its expression is restricted to the multipotent and the lymphoid progenitor stages at which cells are incapable of self-renewal. We extensively analyzed the expression of Flt3 in human (h) hematopoiesis. Strikingly, in both the bone marrow and the cord blood, the human hematopoietic stem cell population capable of long-term reconstitution in xenogeneic hosts uniformly expressed Flt3. Furthermore, human Flt3 is expressed not only in early lymphoid progenitors, but also in progenitors continuously along the granulocyte/macrophage pathway, including the common myeloid progenitor and the granulocyte/macrophage progenitor. We further found that human Flt3 signaling prevents stem and progenitors from spontaneous apoptotic cell death at least through up-regulating Mcl-1, an indispensable survival factor for hematopoiesis. Thus, the distribution of Flt3 expression is considerably different in human and mouse hematopoiesis, and human FLT3 signaling might play an important role in cell survival, especially at stem and progenitor cells that are critical cellular targets for acute myelogenous leukemia transformation.

Serum Cytochrome C as a Tumor Marker in Leukemia and Lymphoma.

When cells become apoptotic, cytochrome c (Cyt c) is translocated from the mitochondria to the cytosol. It has been reported that Cyt c released to the cytosol is further released from the cells into the extracellular medium. Since tumor cells show an accelerated cell cycle with rapid cell growth accompanied by various grades of cell death including apoptosis, sera from patients with malignancies may contain increased levels of Cyt c. We thus investigated serum Cyt c as a tumor marker in hematological malignancies. Cyt c concentration was measured by our original electrochemiluminescence immunoassay. The measurement range of this assay was 5 to 3,000 ng/ml in serum. We confirmed that cell lines triggered by inducers such as anti-Fas/CD95 or TNF-related apoptosis-inducing ligand (TRAIL) released Cyt c into the culture medium within several hours. The serum Cyt c concentration in healthy adults showed a distribution from 14.6 to 36.1 ng/ml (median value: 25.1). Unexpectedly, there was no significant difference between healthy adults and patients with AML, CML, ALL, CLL, multiple myeloma (MM), non-Hodgkin Lymphoma (NHL) or adult T cell leukemia/lymphoma (ATLL), and the median values in these diseases were distributed between 19.7 and 42.4. Of note, however, there were some patients whose Cyt c concentrations were extremely high up to 2,533.2 ng/ml. Values over 50ng/ml were observed in three of the 10 patients with AML (30%), three of the 6 patients with ALL (50%), five of the 21 patients with ATLL (23.8%) and one of the 6 patients with NHL (16.7%). In contrast, there were no patients with CML, CLL or MM who showed values higher than 50 ng/ml. On subtype-analysis in ATLL, the five patients who showed more than 50 ng/ml all had aggressive subtypes, and none of the patients with indolent subtypes showed values higher than 50 ng/ml There was a significant positive correlation between Cyt c concentration and absolute ATLL cell counts in the peripheral blood. More interestingly, parallel analysis using serum and bone marrow specimens in AML and ALL indicated that bone marrow contained more than 10 times higher Cyt c than serum. These results indicate that certain rapidly growing tumor cells exhibit increased spontaneous apoptotic cell death, and serum and/or bone marrow Cyt c concentration can be a reliable marker to estimate the extent. Then, we examined serial serum Cyt c levels during chemotherapy to see whether they reflect the effect of chemotherapy. There was a clear correlation between serum Cyt c and the effect of chemotherapy; Cyt c increased several times soon after the start of chemotherapy, then returned to normal levels after completion. Although serum LDH activity also showed similar variation, the response of serum Cyt c was much quicker and sharper than that of LDH activity. In conclusion, serum Cyt c serves as a marker not only for spontaneous tumor cell death but also for monitoring chemotherapeutic response. Monitoring Cyt c provides new insight for understanding the pathophysiology of hematological malignancies.

Growth hormone and cell survival in the neural retina: caspase dependence and independence

Growth hormone has recently been shown to be expressed in the retinal ganglion cells of embryonic chicks, in which it induces cell survival during neurogenesis. The mechanism of this action has been examined in neural retina explants from 6-day-old and 8-day-old embryos that were incubated for 48 h in 10 M growth hormone, to reduce the number of spontaneous apoptotic cells. This anti-apoptotic action was accompanied by a reduction in caspase-3 expression and, at embryonic day 8, by reduced expression of apoptosis inducing factor-1, which is caspase independent. These actions were specific, as other genes involved in apoptotic signaling (bcl-2, bcl-x, bid and inhibitor of apoptosis protein-1) were unaffected. These results therefore demonstrate caspase-dependent and caspase-independent pathways in growth hormone-induced retinal cell survival.

Survivin expression and its relationship with apoptosis and prognosis in nasal and paranasal sinus carcinomas

Conclusions. The inverse correlation found in this study between survivin expression and the level of spontaneous apoptosis in nasal and paranasal sinus carcinoma (NPSC) suggests that survivin may play an important role in the tumorigenesis of NPSC. Survivin expression may provide a novel predictive indicator for disease-free survival after surgical resection and postoperative radiochemotherapy in NPSC. Objectives. NPSC is the third commonest neoplasm in otolaryngology. Little is known about the distribution of inhibitors of apoptosis in NPSC and how they correlate with the prognoses of patients. The objectives of this study were to assess the protein expression of a new inhibitor of apoptosis, survivin, and its correlation with spontaneous apoptosis, tumor clinicopathological parameters and prognosis in NPSC. Material and methods. A total of 48 patients (23 females, 25 males; age range 20–71 years) with NPSC treated by means of radical operations between 1991 and 2000 were studied retrospectively. Spontaneous apoptotic cells were visualized by means of terminal deoxynucleotide transferase-mediated dUTP nick-end labeling in combination with hematoxylin–eosin and propidium iodide staining. Immunohistochemistry using the streptavidin–peroxidase method was used to assess the expression of survivin. Results. Survivin expression was positive in 23/48 samples (47.92%) but no staining was present in normal tissues adjacent to the tumors. The expression of survivin correlated negatively with the apoptotic index (r= − 0.393; p=0.006). Patients with positive expression of survivin had a lower apoptotic index than those with negative expression (p<0.05). The expression of survivin in advanced-stage tumors (T3 + T4) was higher than that in early ones (T1 + T2) (p<0.05). Patients with negative expression of survivin had a much better prognosis than those with positive expression (p=0.0346). Multivariate analyses revealed that expression of survivin and histological type were independent prognostic factors for overall survival.

Hematological Malignancies

B-cell chronic lymphocytic leukemia (CLL) has been traditionally described as a disease characterized by an accumulation of quiescent small lymphocytes with decreased susceptibility to apoptotic cell death. However, small numbers of "atypical" lymphocytes and prolymphocytes (PL) are frequently observed in the bone marrow (BM) of patients with CLL. In this study, we examined BM biopsy and aspirate specimens obtained from seven patients with atypical CLL. Using a double labeling (Ki-67 + /CD20 + ) immunohistochemical method, we found that an appreciable number of the atypical CLL cells expressed the proliferation-associated protein Ki-67. Because CLL is characterized by a slow change in the peripheral blood (PB) lymphocyte count, we reasoned that a subpopulation of CLL cells probably undergoes spontaneous apoptosis. Using Western blot analysis, we observed expression of procaspase-9, procaspase-10, and poly(ADP-ribose) polymerase by the neoplastic cells in all seven cases of CLL, and procaspase-3 and procaspase-8 expression in six neoplasms. We also detected cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase in four and five CLL cases, respectively. To determine whether CLL cells undergo spontaneous apoptosis, we performed the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay using BM biopsy specimens. We found TUNEL-positive lymphocytes in areas infiltrated by CLL. In summary, our data show that subpopulations of B-lymphocytes are proliferating or undergoing spontaneous apoptotic cell death in patients with atypical CLL.

Disruption of Drosophila Rad50 causes pupal lethality, the accumulation of DNA double-strand breaks and the induction of apoptosis in third instar larvae

The Rad50/Mre11/Nbs1 protein complex has a crucial role in DNA metabolism, in particular in double-strand break (DSB) repair through homologous recombination (HR). To elucidate the role of the Rad50 protein complex in DSB repair in a multicellular eukaryote, we generated a Rad50 deficient Drosophila strain by P-element mediated mutagenesis. Disruption of Rad50 causes retarded development and pupal lethality. To investigate the mechanism of pupal death, brains and wing imaginal discs from third instar larvae were studied in more detail. Wing imaginal discs from Rad50 mutant larvae displayed a 3.5-fold increase in the induction of spontaneous apoptotic cells in comparison to their heterozygous siblings. This finding correlates with increased levels of phosphorylated histone H2Av, indicating an accumulation of DSBs in Rad50 mutant larvae. A 45-fold increase in the frequency of anaphase bridges was detected in the brains of Rad50 deficient larvae, consistent with a role for Rad50 in telomere maintenance and/or replication of DNA. The induction of DSBs and defects in chromosome segregation are in agreement with a role of Drosophila Rad50 in repairing the DSBs that arise during replication.

Increase in apoptotic polymorphonuclear neutrophils in peripheral blood after intramammary infusion of Escherichia coli lipopolysaccharide

A transient increase in apoptotic polymorphonuclear neutrophils (PMNs) as revealed by the terminal deoxynucleotidyl, transferase-mediated dUTP nick end labeling (TUNEL) technique in bovine jugular and milk vein blood was observed 4 h after intramammary infusion of Escherichia coli lipopolysaccharide (LPS) (jugular vein; before infusion 10.1%, 4 h 58.3%: milk vein; before infusion 13.2%, 4 h 76.6%) decrease in PMA-induced oxidative bursts of PMNs was also observed during the same period and continued until 8 h after the infusion. TUNEL-positive cells showed an intention of a Comet tail as detected by a single-cell gel electrophoresis assay (Comet assay) and the morphological apoptotic future, though DNA fragmentation was not clearly detected. A definite decrease in peripheral PMNs and a marked increase in PMNs in the LPS-infused teat cistern were observed during the same period. The migration of milk vein blood-derived PMN and the expression of adhesion receptors (L-selectin and CD18) on PMN were suppressed, accompanied by an increase in apoptotic cells. TUNEL-positive PMN observed in normal animals showed a reduced migration capacity. The increase in apoptotic PMNs observed in the LPS-infused cattle was thought to be due to the remaining intravenous spontaneous apoptotic cells existing under the normal condition (the aging cell), and this increase appeared to lower the expression of adhesion receptors and the migration capacity. Decreased PMA-induced oxidative burst activity in PMN was thought to be derived from these aging cells and immature band cells appearing in the circulation as a subsequent event of leukopenia and/or severe stress associated with mastitis. The results from the present study indicate the possibility that the function of PMN in the circulation at early stages of bovine mastitis is regulated by the kinetics of PMN aging.

Induction of CD95 ligand expression on T lymphocytes and B lymphocytes and its contribution to apoptosis of CD95-up-regulated CD4+ T lymphocytes in macaques by infection with a pathogenic simian/human immunodeficiency virus.

Using an established SIV/HIV-C2/1-infected cynomolgus monkey model displaying stable CD4+ T cell depletion, the kinetics of apoptosis and the levels of expression of CD95 membrane-associated CD95L on lymphocytes were investigated to test the involvement of the CD95/CD95L system in CD4+ T lymphocyte loss in vivo. Rapid depletion of CD4+ T cells occurred up to 2 weeks after infection, with chronic CD4+ T lymphopenia thereafter. During the initial CD4+ T cell loss, which was accompanied by viraemia, about 90% of the peripheral CD4+ T cell subset underwent spontaneous apoptotic cell death during 24 h of culture. Increased expression of CD95 was observed on both CD4+ and CD8+ T cell subsets, with CD95 expression on CD8+ cells declining rapidly, but high CD95 expression being maintained on CD4+ cells. Since CD95L was expressed on CD8+ T cells, B cells and to a lesser extent on CD4+ T cells, this suggests that CD95-mediated apoptosis might be controlled in an autocrine/paracrine fashion.

Human papillomavirus infection among Egyptian females with cervical carcinoma: relationship to spontaneous apoptosis and TNF-α

Objectives: This study was designed to detect HPV type-16 in Cervical carcinoma (CC) tissue specimens. The results were correlated with clinicopathological parameters of the carcinoma, with spontaneous apoptosis and with immunoreactivity to TNF-α antibodies Methods: Fresh frozen tissue specimens representing 30 cases of cervical carcinoma as well as 20 normal cervical tissues (NCT) were the subjects of this study. HPV-16 DNA was detected by Polymerase Chain Reaction (PCR). The occurrence of spontaneous apoptotic cell death was analyzed by the apoptosis assay. Apoptotic cells were also counted by light microscopy and the apoptotic index (AI) was calculated. Electron microscopy was used to confirm the morphology of apoptotic cells. TNF-α was quantified using EIA kit. Results: HPV-16 DNA was more frequent in CC than in NCT. No correlation was observed between HPV infection and grade, stage or pathologic type of CC. The occurrence of spontaneous apoptosis was significantly higher in CC than in NCT, where it was correlated to advanced tumor stage and tumor pathology being more in adenocarcinoma (AC) than in squamous cell carcinoma (SCC). Moreover, AI was negatively correlated to HPV-16 infection. TNF-α levels were significantly higher in CC vs. NCT, where they were positively correlated to advanced tumor stage. TNF-α levels were correlated to DNA fragmentation and AI (r = 0.47 and 0.57 respectively). A cut-off value for TNF-α was calculated to be 9.1 pg/mg protein (using ROC curve). At the determined cut-off point the sensitivity was 70% and the specificity was 80%. Conclusion: HPV infection, high levels of TNF-α and spontaneous apoptosis were strongly associated with malignant phenotype of cervical tissues. Rate of spontaneous apoptosis was higher in AC compared to SCC. On the other hand, HPV negativity was correlated with AI. Moreover, TNF-α and apoptotic cell death were correlated to each other as well as to tumor progression. No correlation was detected between TNF-α and HPV-16 infection.

Homozygous C1qa Deficiency Alters Pulmonary Clearance of Apoptotic Cells

Clearance of dying (apoptotic) cells from inflamed tissues is an integral step in the control of inflammation, and defective processing of autoantigens present in apoptotic cells may contribute to the initiation of autoimmune syndromes. The complement system is one mechanism that has been implicated in apoptotic cell removal. C1q, the first component of the classical complement cascade, binds to apoptotic cells, potentially facilitating their removal by phagocytes. In support of this hypothesis, mice homozygous for C1qa deficiency (C1qa−/−) spontaneously accumulate apoptotic cells in the kidney, and do not effectively clear apoptotic cells during thioglycolate-induced peritonitis. Interestingly, humans homozygous for C1qa−/− develop an early and severe form of systemic lupus erythematosus. We sought to determine whether C1qa−/− delayed resolution of pulmonary inflammation through defective removal of apoptotic cells. Baseline differences between C1qa−/− mice and control mice were assessed by CBCs, BAL WBC subsets, and apoptotic cells. Inflammation in C1qa−/− mice was evaluated in BAL by measurement of WBC subsets at 24, 48, and 72 h posttreatment with 20 μg of intratracheal endotoxin lipopolysaccharide [LPS]. Spontaneous clearance of apoptotic cells in BAL was assessed at these same time points by nuclear morphology, terminal deoxynucleotidyl nick-end labeling, and propidium iodide/annexin V staining. LPS-treated, C1qa−/− mice were also challenged intratracheally with apoptotic human polymorphonuclear leukocytes (PMNs) at 72 h, and clearance was ascertained. No differences were found in baseline blood cell counts, BAL WBC subsets, or BAL apoptotic cells in C1qa−/− mice vs control mice. LPS increased pulmonary PMN, but not macrophage, influx at 48 h and 72 h in C1qa−/− mice compared to control mice. BAL from LPS-challenged C1qa−/− mice and control mice contained only small percentages of spontaneous apoptotic cells (1 to 3%), and no differences were observed between groups. However, when C1qa−/− mice were challenged intratracheally with apoptotic human PMNs, clearance was decreased. These results demonstrate that C1qa−/− impairs clearance of apoptotic cells and enhances pulmonary inflammation. The impact of dysregulated apoptotic cell removal on interstitial lung disease is as yet unknown, but may provide a basis for ongoing inflammation.EST