DNA Fragmentation In Apoptotic Cells Research Articles

Bone marrow stromal cells of bcl-2 transgenic mice express widespread Bcl-2 protein and reduce apoptosis in a serum-free medium

P225 Stem cell transplantation is a vibrant area of research with the goal of treatment of uncurable diseases. Bone marrow stromal cells (MSCs, including marrow stem and progenitor cells) have been employed to improve neurological functional recovery after stoke and injury. Grafted cell death is a common feature of intracerebral transplantation. Bcl-2 has been shown to protect a variety of cell types from apoptoic cell death. We tested the hypothesis that MSCs harvested from bcl-2 transgenic mice promote survival and decrease apoptosis of cells in vitro. MSCs were harvested from adult male C57BL/6 transgenic mice (n=4) overexpressing bcl-2, and from normal control C57BL/6 mice (n=4). Cells were cultured in regular medium, Iscove s Modified Dulbecco s medium (IMDM) supplemented with 10% fetal bovine serum (FBS); and then isolated by their adherence to the plastic dishes from non-adherent hematopoietic cells at 72 h of incubation. To examine the effect of bcl-2 on cell death induced by serum deprivation. MSCs were washed and incubated with the serum-free medium. Immunostaining and Western blot were used to identify Bcl-2 protein expression, and TUNEL staining was used to identify in situ DNA fragmentation of apoptotic cells. Immunocytochemical staining indicated that 100% of MSCs harvested from bcl-2 transgenic mice express Bcl-2 protein. Western blot showed that Bcl-2 protein was 8 times more intense in the MSCs harvested from transgenic mice than that from normal control mice at 12 days cultured in regular medium. After 48 h cultured in the serum-free medium, 5.8% of MSCs from bcl-2 transgenic mice were presented apoptoic morphology, compared with 39% of MSCs from normal controls. Our finding demonstrates that the overexpression of bcl-2 gene in the MSCs reduces apoptotic cell death.

Coadministration of bone marrow cells and an inhibitor of apoptosis (Z-VAD) promotes bone marrow cell survival and neurological functional recovery after focal cerebral ischemia in adult rat

P222 Most bone marrow cells (BMCs) transplanted into the ischemic brain of adult rodents die shortly after they are grafted, similar to the 90%-95% of the fetal cells that die after transplantation into Parkinson s patients. We tested whether coadministration of BMCs and Z-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD), an inhibitor of apoptosis, into the ischemic boundary zone of the striatum and the cortex in rat brain promotes BMC survival. Adult Wistar rats were subjected to transient (2 h) middle cerebral artery occlusion (MCAo). At 1 d after ischemia, saline (n=9, Group 1); BMCs (1x10 6 in 10 :l, n=8, Group 2); or BMCs with Z-VAD (50 nM/ml, n=4, Group 3) were injected into brain. BMCs were harvested from donor adult rats injected with bromodeoxyuridine (BrdU, as a tracer). Rats were subjected to rotarod-motor and adhesive-removal somatosensory functional tests before MCAo and at 1 and 7 d after MCAo. Rats in Group 3 exhibited significant improvement (10.3±2.6 seconds, p<0.05) on the adhesive-removal test at 7 d, compared with those in Group 1 (29.3±9.4 seconds) and Group 2 (21.3±5.5 seconds), respectively. Immunohistochemistry staining was employed to identify BrdU-BMCs, and TUNEL staining was used to identify in situ DNA fragmentation of apoptotic cells. Even though the infarct volume in Group 3 (29.9±9.2%) did not change significantly, compared with Group 1 (34.3±4.0%) and Group 2 (26.6±3.8%); the number of BrdU-BMCs (88,200±7,400, ∼8.8% of 10 6 transplanted cells) increased significantly (p<0.05) in rats in Group 3, compared with that in Group 2 (31,700±9,100, ∼3.2% of 10 6 cells) at 7 d. In the grafting areas, apoptotic cells were less clustered (<90 vs.>240 per region) and apoptotic cells were significantly decreased (12.3±1.7/mm 2 vs. 25.9±2.1/mm 2 , ∼47%, p<0.05). Our data suggest that intracerebral coadministration of bone marrow cells and inhibitors of apoptosis enhance cellular survival of bone marrow cells and improves neurological functional recovery after cerebral ischemia.

Gene expression of endothelin-1 in the porcine ovary: follicular development.

We have investigated which follicular compartment and stage of follicular development are associated with endothelin-1 (ET-1) gene expression in the porcine ovary. The localization of mature ET-1 peptide and of its mRNA was determined by immunohistochemistry and by in situ hybridization. Stage of follicular development associated with ET-1 expression was investigated in terms of follicular class and occurrence of atresia. The latter was investigated by determining the occurrence of DNA fragmentation in apoptotic cells on adjacent sections to those used for ET-1 gene expression. Fifteen ovaries from 10 prepubertal pigs stimulated with gonadotropin were collected; a total of 1050 follicles were examined. Specific ET-1 immunoreactivity was restricted to the ovarian vasculature and to the granulosa cell compartment of antral follicles. The pattern of ET-1 mRNA expression was similar to that found for ET-1 immunoreactivity. Primordial, primary, and most secondary follicles did not express ET-1. The theca cell layer did not express ET-1 regardless of follicle developmental stage. ET-1 expression occurred with a significantly greater probability (P < 0.001 by the likelihood ratio test) in the granulosa cell compartment of antral follicles than in any other follicle class. Furthermore, in antral follicles, ET-1 expression occurred with a greater likelihood in large antral follicles than in small antral follicles (P < 0.001 by the likelihood ratio test). In small antral follicles, only 16.8% expressed ET-1; in contrast, 66.7% of large antral follicles exhibited ET-1 expression. It is interesting that in follicles in which ovulation had already occurred, intense ET-1 expression was found only in the prominent developing vasculature, the other cells present in the luteinized follicle did not display any ET-1 expression. The pattern of ET-1 gene expression observed in this study would be in agreement with our previous suggestion of a plausible physiological role for ET-1 in preventing premature progesterone production by granulosa cells of an antral follicle. The occurrence of atresia and expression of ET-1 in the same follicle was rare. Small and large antral follicles constituted 5.1% and 5.6%, respectively, of the examined follicles in this category. The majority of atretic follicles did not express ET-1 and, conversely, follicles that expressed ET-1 were not atretic. To the best of our knowledge, this is the first report in which large, nonatretic follicles are clearly identified as the population of follicles expressing ET-1. The results of this study delineate the follicular developmental stage and the compartment of when and where ET-1 may be physiologically meaningful.

An auxiliary mode of apoptotic DNA fragmentation provided by phagocytes

CAD (caspase-activated DNase) can cause DNA fragmentation in apoptotic cells. Transgenic mice that ubiquitously express a caspase-resistant form of the CAD inhibitor (ICAD) were generated. Thymocytes prepared from the mice were resistant to DNA fragmentation induced by a variety of stimuli. However, similar numbers of TUNEL-positive cells were present in adult tissues of transgenic and wild-type mice. Exposure to gamma-irradiation caused a striking increase in the number of TUNEL-positive cells in the thymus of wild-type, but not transgenic, mice. TUNEL-positive nuclei in transgenic mice were confined to thymic macrophages. When apoptotic thymocytes from the transgenic mice were cocultured with macrophages, the thymocytes underwent phagocytosis and their chromosomal DNA underwent fragmentation. This DNA fragmentation was sensitive to inhibitors that block the acidification of lysosomes. Hence, we conclude that the DNA fragmentation that occurs during apoptosis not only can result cell-autonomously from CAD activity but can also be attributed to a lysosomal acid DNase(s), most likely DNase II, after the apoptotic cells are engulfed.

Bone marrow stromal cells of bcl-2 transgenic mice express widespread Bcl-2 protein and reduce apoptosis in a serum-free medium

P225 Stem cell transplantation is a vibrant area of research with the goal of treatment of uncurable diseases. Bone marrow stromal cells (MSCs, including marrow stem and progenitor cells) have been employed to improve neurological functional recovery after stoke and injury. Grafted cell death is a common feature of intracerebral transplantation. Bcl-2 has been shown to protect a variety of cell types from apoptoic cell death. We tested the hypothesis that MSCs harvested from bcl-2 transgenic mice promote survival and decrease apoptosis of cells in vitro. MSCs were harvested from adult male C57BL/6 transgenic mice (n=4) overexpressing bcl-2, and from normal control C57BL/6 mice (n=4). Cells were cultured in regular medium, Iscove s Modified Dulbecco s medium (IMDM) supplemented with 10% fetal bovine serum (FBS); and then isolated by their adherence to the plastic dishes from non-adherent hematopoietic cells at 72 h of incubation. To examine the effect of bcl-2 on cell death induced by serum deprivation. MSCs were washed and incubated with the serum-free medium. Immunostaining and Western blot were used to identify Bcl-2 protein expression, and TUNEL staining was used to identify in situ DNA fragmentation of apoptotic cells. Immunocytochemical staining indicated that 100% of MSCs harvested from bcl-2 transgenic mice express Bcl-2 protein. Western blot showed that Bcl-2 protein was 8 times more intense in the MSCs harvested from transgenic mice than that from normal control mice at 12 days cultured in regular medium. After 48 h cultured in the serum-free medium, 5.8% of MSCs from bcl-2 transgenic mice were presented apoptoic morphology, compared with 39% of MSCs from normal controls. Our finding demonstrates that the overexpression of bcl-2 gene in the MSCs reduces apoptotic cell death.

Induction of apoptosis in mouse thymocytes by microcolin A and its synthetic analog

Microcolin A (Mic-1), a marine-derived compound, has been shown to be a novel antiproliferative and immunosuppressive agent. We investigated the ability of Mic-1 and its chemosynthetic analog, microcolin A3 (Mic-3), to induce apoptosis in murine thymocytes. Following incubation of the cells with Mic-1 (10–100 nM) or Mic-3 (10–100 nM), internucleosomal DNA fragmentation in apoptotic cells was detected by agarose gel electrophoresis and the diphenylamine (DPA) assay; the presence of hypodiploid nuclei assessed by propidium iodide (PI) staining; and the percentages of apoptotic and necrotic cells quantified by morphological observation and fluorescein labeled annexin-V binding. Our results show that both Mic-1 and Mic-3 are potent inducers of apoptosis in thymocytes depending on drug concentration and time of exposure, with Mic-3 being more potent than Mic-1 in the induction of apoptosis. Furthermore, flow cytometric analysis using monoclonal antibodies specific to thymocyte subpopulations showed that the proportion of the early immature CD 4 +CD 8 + T-cell subpopulation in thymocytes was selectively decreased by both agents with a corresponding increase of other subpopulations, indicating that CD 4 +CD 8 + T cells are the most likely targets of Mic-1 and Mic-3. These in vitro results suggest that the antiproliferative and immunosuppressive properties of both compounds are possibly associated with apoptosis-inducing events and imply that they may have additional potential value as antineoplastic agents.

Apoptosis and necrosis: Mechanisms of cell death induced by cyclosporine A in a renal proximal tubular cell line

The mechanisms of cyclosporine (CsA)-induced nephrotoxicity are not fully understood. While hemodynamic changes may be involved in vivo, there is also some evidence for tubular involvement. We previously showed direct toxicity of CsA in the LLC-PK1 renal tubular cell line. In the current study we examined mechanisms (apoptosis or necrosis) of cell death induced by CsA in the LLC-PK1 renal proximal tubular cell line. The possible role of the Fas (APO-1/CD95) antigen-Fas ligand system in the mediation of CsA-induced cell death was also investigated. Cells were treated with CsA (0.42 nM to 83 microM) for 24 hours and alterations in DNA and protein synthesis and membrane integrity were examined. Flow cytometry was used to investigate: (i) alterations in the DNA content and cell cycle; (ii) the forward (FSC) and side (SSC) light scattering properties (indicators of cell size and granularity, respectively); (iii) the externalization of phosphatidylserine (PS) as a marker of early apoptosis using FITC-annexin V binding; and (iv) expression of the apoptotic Fas protein. DNA fragmentation in apoptotic cells was also determined by the TUNEL assay. CsA (all doses) caused a block in the G0/G1 phase of the cell cycle as indicated by a decrease in DNA synthesis and supported by an increase in the % of cells in the G0/G1 phase with concurrent decreases of those in the S and G2/M phases. The effect on protein synthesis appeared to be much less. Lower doses of CsA (4.2 nM) caused the appearance of a "sub-G0/G1" peak, indicative of reduced DNA content, on the DNA histogram that was paralleled by a reduction in cell size and an increased cell granularity and an increase in FITC-annexin V binding. DNA fragmentation was evident in these cells as assessed using the TUNEL assay. Higher doses of CsA increased cell size and decreased cell granularity and reduced membrane integrity. Expression of Fas, the cell surface molecule that stimulates apoptosis, was increased following low dose CsA exposure. These results indicate that CsA is directly toxic to LLC-PK1 cells with reduced DNA synthesis and cell cycle blockade. The mode of cell death, namely apoptosis or necrosis, is dose dependent. Fas may be an important mediator of CsA induced apoptosis in renal proximal tubular cells.

Apoptosis in the brains of infants suffering intrauterine cerebral injury.

This study addressed the hypothesis that in human infants severe in utero insults induce a significant proportion of brain cells to undergo apoptosis. Morphologic criteria were used to quantify apoptosis and necrosis in the cingulate gyrus of two groups of infants: six infants who died after severe birth asphyxia with hypoxic-ischemic encephalopathy, and six others who suffered unexpected and apparently sudden intrauterine death at or close to term. The fraction of apoptotic cells was much higher than basal levels determined in animal experiments, and within both groups increased in proportion to the severity of injury as determined by total cell death (p < 0.05). The mean fraction of apoptotic cells was similar in asphyxiated infants, 8.3% (95% confidence interval for the population, 3.7-12%), and in stillbirths, 6.7% (0.2-13.6%). In the asphyxiated group, 20.8% (11-30.6%) of cells were necrotic, but significantly less necrosis, 3% (0.4-5.6%), was seen in stillborn infants (p < 0.05). Cell death was apoptotic after birth asphyxia in 26% (1-51%) and 78% (41-100%) in stillborn infants. In situ end labeling studies confirmed the presence of DNA fragmentation in apoptotic cells. These results demonstrate that infants who die after intrauterine insults, both those with evidence of delayed cerebral injury after hypoxia-ischemia and those without, have a significant number of cells in the brain with the morphologic characteristics of apoptosis. They confirm that apoptosis contributes significantly to cerebral damage in the perinatal period.

Fas and Fas ligand expression in the salivary glands of patients with primary Sjögren's syndrome

To assess the role of Fas-mediated apoptosis in the salivary glands of patients with primary Sjögren's syndrome (SS). Expression of Fas, Fas ligand (FasL), and bcl-2 in salivary gland biopsy material was detected in situ by immunohistochemical staining and reverse transcriptase-polymerase chain reaction. DNA fragmentation in apoptotic cells was assessed by the enzymatic incorporation of labeled nucleotides (digoxigenin-dUTP). The acinar epithelial cells in SS were Fas+ and FasL+, and these cells died by apoptosis. The majority of infiltrating lymphocytes in SS were Fas+ and bcl-2+, while few lymphocytes expressed FasL. In situ detection of apoptosis showed minimal cell death of lymphocytes, particularly in dense periductal foci. Lymphocytic cell death was significantly lower (P < 0.0001) in these foci compared with that in the interstitium. Infiltrating lymphocytes in the focal lesions of the salivary glands of patients with SS are blocked in their ability to commit to apoptosis, even though they may express Fas. The presence of bcl-2 in these cells may explain their inability to undergo apoptosis. The acinar epithelial cells, in contrast, may undergo Fas-mediated apoptosis. These results suggest that the Fas death pathway may be an important mechanism leading to the glandular destruction found in SS.

Apoptosis in mesangial cells induced by ionizing radiation and cytotoxic drugs

Mesangial proliferation contributes to the pathogenesis of many forms of glomerulonephritis. To evaluate the role of apoptosis on the pharmacologic effects of cytotoxic drugs and ionizing radiation, we studied their effects on cultured rat mesangial cells (MC), whose apoptotic response to these drugs is unknown. Mesangial cells were cultured with or without stimuli to induce apoptosis and were harvested at 24 and 48 hours. MC morphology was examined by light microscopy, in situ end labeling technique using terminal deoxy-transferase (TUNEL) and by electrophoresis of extracted total cellular DNA. MCs exposed to cytotoxic drugs or irradiation demonstrated statistically significant increases in apoptotic cells identified by light microscopy. DNA fragmentation of apoptotic cells was also visualized as characteristic staining by the TUNEL method and statistically significant increases in apoptotic cell number in cells exposed to cytotoxic drugs and irradiation were noted compared to control cultures. In general, the number of TUNEL positive cells was greater than that of morphologically apoptotic cells. DNA extracted from these cells also showed the characteristic ladder pattern of internucleosomal chromatin cleavage of 180 bp fragments on agarose gel electrophoresis. To further analyze whether MC apoptosis induced by these drugs alters the cell cycle, 3H-thymidine incorporation rates were measured in both the cell culture monolayer and in those cells shed into the supernatant when cultured with or without cyclophosphamide (N = 5). 3H-thymidine incorporation corrected for total cellular DNA showed a similar pattern in both control and cyclophosphamide treated groups, suggesting that cyclophosphamide did not alter the mesangial cell cycle. Considering that the dosage of the cytotoxic drugs utilized in these experiments in nearly the therapeutic plasma level in humans, these results suggest that cytotoxic drugs used to treat glomerular disease can induce apoptotic mesangial cell death and may operate in part via this mechanism.

Cellular depletion of p56lck during thymocyte apoptosis

The src-related protein tyrosine kinase p56lck is thought to be important in regulating maturation and functional responsiveness of T cells and thymocytes. In the present studies we report that expression of p56lck is suppressed during apoptosis. Using primary cultures of rat thymocytes, we found that agents that are effective in inducing apoptosis, including okadaic acid, dexamethasone, and antibodies to the CD3 receptor, also deplete cells of p56lck. This process is rapid, occurring within 24 h, and is not due to cytotoxicity. Inhibition of DNA fragmentation in apoptotic cells with the endonuclease inhibitor ZnCl2 failed to prevent depletion of p56lck, suggesting that it was not a consequence of the DNA degradation process. Using the thymic lymphoma cell line LSTRA, apoptosis was also associated with cellular depletion of p56lck. In contrast to thymocytes, this process required 48-72 h possibly because these cells overexpress p56lck. Although at this time we are uncertain as to the precise role of p56lck in the process of apoptosis, our results indicate that changes in the expression of this protein in thymocytes is an important marker of programmed cell death.

Activin A Induces Apoptotic Cell Death

Activins, members of a family of the transforming growth factor β (TGFβ), are involved in the regulation of multiple biological events. We found a novel effect of activin A on hybridoma and myeloma cell lines. Activin A exhibited a cytotoxic effect on interleukin-6 (IL-6)-dependent B9 cells and induced a significant increase in the proportion of fragmented DNA. 89 cells exposed to activin A released high amounts of lactate dehydrogenase (LDH) and exhibited the typical ladder pattern of DNA fragmentation of apoptotic cells. IL-6 did not prevent apoptosis of B9 cells induced by activin A. The cytotoxicity of activin A to B9 cells was suppressed by follistatin. On the other hand, TGFβ showed no cytotoxic effect on 89 cells. These findings indicate that apoptosis induced by activin A could be one of the mechanisms to prevent uncontrolled cell growth.

Nitric Oxide Synthase Induces Macrophage Death by Apoptosis

Stimulation processes effective for macrophage (MΦ) cytostasis induction also led to a L-arginine-dependent MΦ cell death by apoptosis in parallel to nitrite and citrulline production. Resident MΦ stimulated with LPS plus IFN-γ and MDP produced high amounts of nitrite and underwent apoptosis. Inflammatory MΦ treated with LPS or IFN-γ alone produced low levels of nitrite and were not apoptotic, whereas a synergistic effect was observed, the combined treatment leading to high amounts of nitrite and to apoptosis. Primed MΦ obtained from concanavalinA-treated mice, after stimulation with LPS alone, released high amounts of nitrite and exhibited the typical ladder pattern of DNA fragmentation of apoptotic cells. In the three cases the L-arginine-dependent nitrite production was inhibited by N G-monomethyl-L-arginine. a competitive inhibitor of NO synthase, which moreover, also inhibited cell apoptosis. These findings and kinetics studies suggest the involvement of NO synthase in apoptosis induction.

A quantitative assay for fragmented DNA in apoptotic cells

Cleavage of cellular chromatin at internucleosomal sites is a characteristic change of DNA integrity in cells undergoing apoptosis. We have developed an assay for quantitation of internucleosomal DNA fragmentation in apoptotic cells. This technique involves purification of cellular DNA, dephosphorylation of the DNA ends, labeling of DNA with 32P at the 5′-end, gel electrophoresis through agarose, and quantitation of the radioactivity in DNA bands. This assay, which is about 1000- to 2000-fold more sensitive than visualization of DNA bands by ethidium staining, allows the detection of DNA fragments at picogram levels. A method for quantitatively determining the number of fragmented DNA strands is also described. Application of this new assay to evaluate the time course of internucleosomal DNA fragmentation was demonstrated in apoptotic cells induced by an anticancer nucleoside analogue.