Internucleosomal DNA Cleavage Research Articles

DNA inclusions within autolytic cytoplasmic vacuoles of hemopoietic stem cell line FDCP-Mix.

FDCP-Mix, a pluripotent routine hemopoietic stem cell line undergoes internucleosomal cleavage of DNA when induced to apoptosis either by drugs or by withdrawal of growth factor (IL-3), and also displays a pattern of nuclear morphology that is typical for apoptosis. However, increased autolytic activity in the cytoplasm precedes the nuclear changes. For etoposide-treated FDCP-Mix cells, mitochondria were identified as a target for autolytic digestion in large autolytic vacuoles, but during this period an increase in the number of mitochondria was observed. The autolytic vacuoles displayed variations in their content. Large, electron-dense inclusions resembling "condensed chromatin" could regularly be found in FDCP-Mix cells treated with low concentrations of etoposide (<4 microM). Confocal fluorescence microscopy and DNAse-gold labeling were employed to demonstrate the presence of DNA in the formation of the electron-dense inclusions within autolytic vacuoles. The identification of mitochondrial macroautophagy, the evidence for an etoposide-induced proliferation of mitochondria, and the fact that electron-dense inclusions are formed at a stage when the morphology of the nucleus is still not effected, suggests that the DNA within the autolytic vacuoles may be of mitochondrial origin.

Apoptosis of human seminoma cells upon disruption of their microenvironment

One of the main obstacles encountered when trying to culture human seminoma (SE) cells in vitro is massive degeneration of the tumour cells. We investigated whether dissociation of tumour tissue, to obtain single-cell suspensions for in vitro culture, results in the onset of apoptosis. Using morphological analysis and in situ end labelling, less than 4% of apoptotic tumour cells were detected in intact tissue from 11 out of 14 SEs. In these 11 tumours, apoptosis-specific DNA ladders, indicative of internucleosomal double-strand DNA cleavage, were not detected on electrophoresis gels. In contrast, three SEs with over 12% of apoptotic tumour cells in the intact tissue and all analysed (pure) SE cell suspensions, obtained after mechanical dissociation of intact tumour tissue, showed DNA ladders. Flow cytometric analysis of end labelled SE suspensions showed DNA breaks in up to 85% of the tumour cells. As indicated by cell morphology and DNA degradation, SE cells appear to rapidly enter the apoptotic pathway upon mechanical disruption of their microenvironment. No expression of p53 and of the apoptosis-inhibitor bcl-2 was detectable in intact SE tissue or cell suspensions. Our data suggest that abrogation of apoptosis might be crucial to succeed in culturing human SE cells in vitro.

ILA, a member of the human nerve growth factor/tumor necrosis factor receptor family, regulates T-lymphocyte proliferation and survival

ILA, a gene induced by lymphocyte activation, is a member of the human nerve growth factor tumor necrosis factor receptor family and the human homologue of murine 4-1BB. The present study analyzed the role of ILA in the regulation of human peripheral blood T-lymphocyte function. Antibodies raised against different fusion proteins recognized ILA on activated lymphocytes. These antibodies significantly increased anti-CD3--induced T-lymphocyte proliferation. When anti-CD3--stimulated cells were incubated on ILA-expressing CHO cells, proliferation was inhibited. CHO cells transfected with a control construct and not expressing ILA did not reduce T-cell proliferation. A purified fusion protein containing the extracellular domain of ILA and the constant domain of human IgG (ILA-IgG) also inhibited lymphocyte proliferation. Results obtained by 3H-thymidine incorporation were confirmed by cell cycle analysis that showed a decrease in the number of lymphocytes in S phase. Lymphocyte morphology in cultures with ILA-expressing CHO cells was suggestive of apoptosis. Flow cytometry on propidium iodide-stained cells showed a time-dependent increase in the number of hypodiploid apoptotic cells when lymphocytes were cultured on ILA-expressing CHO cells. Internucleosomal DNA cleavage was seen in these cultures, but not in the presence of ILA-negative CHO cells. Studies on the mechanism by which ILA regulates T-cell function showed that ILA-IgG inhibited anti-CD3-induced T-cell proliferation when presented in immobilized but not in soluble form. These results suggest that ILA may act by cross-linking its ligand and thereby inhibit T-cell proliferation.

Mechanisms of death induced by cisplatin in proximal tubular epithelial cells: apoptosis vs. necrosis.

We have examined the mechanisms of cell death induced by cisplatin in primary cultures of mouse proximal tubular cells. High concentrations of cisplatin (800 microM) led to necrotic cell death over a few hours. Much lower concentrations of cisplatin (8 microM) led to apoptosis, which caused loss of the cell monolayer over several days. Necrosis was characterized by a cytosolic swelling and early loss of plasma membrane integrity. In contrast, early features of cells undergoing apoptosis included cell shrinkage and loss of attachment to the monolayers. Nuclear chromatin became condensed and fragmented in apoptosing cells. These features were absent in necrotic cells. DNA electrophoresis of cells exposed to 800 microM cisplatin yielded a "smear" pattern, due to random DNA degradation. In contrast, the DNA of apoptosing cells demonstrated a "ladder" pattern resulting from internucleosomal DNA cleavage. Antioxidants delayed cisplatin-induced apoptosis but not necrosis. Thus the mechanism of cell death induced by cisplatin is concentration dependent. Reactive oxygen species play a role in mediating apoptosis but not necrosis induced by cisplatin.

Rapid initiation of apoptosis by photodynamic therapy.

Photodynamic therapy (PDT) of neoplastic cell lines is sometimes associated with the rapid initiation of apoptosis, a mode of cell death that results in a distinct pattern of cellular and DNA fragmentation. The apoptotic response appears to be a function of both the sensitizer and the cell line. In this study, we examined photodynamic effects of several photosensitizers on murine leukemia P388 cells. Two drugs, a porphycene dimer (PcD) and tin etiopurpurin (SnET2), which localized at lysosomal sites, were tested at PDT doses that resulted in 50% loss of viability (LD50), measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. An oligonucleosomal pattern of DNA degradation was observed within 1 h after irradiation. Neither sensitizer antagonized PDT-mediated internucleosomal DNA cleavage by the other. Very high PDT doses with either agent abolished this rapid internucleosomal cleavage. Exposure of cells to high concentrations of either sensitizer in the dark also resulted in rapid DNA fragmentation to nucleosomes and nucleosome multimers; this effect was not altered by the antioxidant 6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid (trolox), although the latter could protect cells from cytotoxicity and apoptotic effects caused by LD50 PDT doses. Photodamage from two cationic sensitizers, which localized at membrane sites, caused rapid DNA cleavage to 50 kb particles; however, no further fragmentation was detected after 1 h under LD10, LD50 or LD95 PDT conditions. Moreover, the presence of either cationic sensitizer inhibited the rapid internucleosomal cleavage induced by SnET2 or PcD photodamage. The site of photodynamic action may therefore be a major determinant of the initiation and rate of progression of apoptosis.

Retroviral Vector-Mediated Transfer of the Tumor Necrosis Factor α Gene Into Human Cancer Cells Restores an Apoptotic Cell Death Program and Induces a Bystander-Killing Effect

Tumor necrosis factor alpha (TNFalpha) may induce tumor cell death by apoptosis, the physiologic program of cell death usually lost during neoplastic progression. However, many tumor cells are resistant to its effect unless high doses are administered. By retroviral vector-mediated gene transfer, we have transduced the TNFalpha gene into the DNA of human tumor cells to investigate whether the indefinite neoplastic cell proliferation could be blocked and the lost physiologic program of cell death restored. Evidence is provided that high-TNFalpha-producing clones generated from a human lymphoma T-cell line (ST4) can undergo apoptosis following transduction of the TNFalpha gene. Internucleosomal DNA cleavage was documented by May-Grünwald-Giemsa and by propidium iodide staining, as well as by gel electrophoresis. The induced apoptotic phenomenon is TNFalpha-mediated, since it can be reverted following incubation with anti-TNFalpha monoclonal antibodies (MoAbs), and it occurs with cytokine levels released in the supernatant by the engineered cells much lower(>100 times) than those required to promote the same effect on parental ST4 cells following administration of exogenous recombinant TNFalpha. The process is associated with a downregulation of the apoptosis-preventing gene, bcl-2, while the expression of bax and p53, genes usually involved in promoting apoptosis, persists. Mixed-culture experiments performed coincubating TNFalpha-transduced and untransduced ST4 cells allowed documentation of a bystander-killing effect on the parental cells. This phenomenon still occurred at transduced to parental cell ratios as low as 1:20 and was blocked in the presence of an anti-TNFalpha MoAb. These findings indicated that TNFalpha may play a regulatory role in the proliferation of human tumor cells, and suggest potential new antitumor therapeutic strategies based on the direct delivery of the TNFalpha gene into cancer cells.

Role of c-myc in Tamoxifen-Induced Apoptosis in Estrogen-Independent Breast Cancer Cells

The antiestrogen tamoxifen (TAM) is effective in the treatment of estrogen receptor (ER)-positive as well as some ER-negative breast cancers. However, the precise mechanism of action of TAM, especially in estrogen-independent cells, remains unclear. Previous work by our laboratory has demonstrated that TAM induces the morphologic and biochemical changes that are characteristic of apoptosis in both ER-positive and ER-negative cells. We compared the effect of TAM at a clinically achievable concentration on cell growth and apoptosis with the effect of TAM on c-myc (also known as C-MYC) messenger RNA (mRNA) and protein expression in ER-negative MDA-231 cells. MDA-231 cells were treated for up to 72 hours with 1.0 microM TAM alone or in the presence of 50 microM c-myc antisense or nonsense oligonucleotides. c-myc mRNA expression was determined by northern blot analysis, protein expression by western blot analysis, cell growth inhibition counts, and DNA cleavage by agarose gel electrophoretic analysis. Differences between the mean values from different treatment groups were compared with the use of the two-sided Wilcoxon Ranksum test. TAM treatment for 72 hours increased c-myc mRNA five-fold (from a relative radiolabeled hybridization signal intensity of 17 +/- 4 up to 93 +/- 10; P<.05) and c-MYC protein threefold (from a relative immunofluorescence signal intensity of 28 +/- 7 up to 83+/-21; P< .05). The induction of c-myc by TAM was accompanied by internucleosomal DNA cleavage characteristic of apoptotic cell death. Addition of c-myc antisense oligonucleotide (5'CACGTTGAGGGGCAT-3') to MDA-231 cells resulted in a nearly twofold decrease of basal c-myc mRNA (P< .05) and a sevenfold decrease of basal c-Myc protein (P< .05) expression. Addition of c-myc antisense oligomer also antagonized the TAM-induced increase in c-myc mRNA (P< .05) and protein expression (P< .05) and inhibited TAM-induced cytostasis (P< .01) and apoptosis. In parallel experiments, addition of the nonsense oligomer had no effect on any of the measured parameters. These results indicate that the effects of TAM on ER-negative MDA-231 cells may be mediated through c-myc overexpression. c-myc may play a critical role in the growth and progression of MDA-231 breast cancer cells.

Apoptosis of macrophages during the resulution of muscle inflammation.

We tested the hypothesis that apoptosis contributes to the depletion of macrophages expressing the ED1 or ED2 antigen during the resolution of rat muscle inflammation. Muscle inflammation was induced by subjecting rat soleus muscle to 10 days of unloading followed by periods of muscle reloading. Terminal deoxynucleotidyl transferase- mediated deoxyuridine triphosphate (dUTP) labeling of apoptotic nuclei showed that apoptotic inflammatory cells increase in concentration within necrotic fibers and in the connective tissue at 2 days following muscle injury caused by increased loading. Four days following injury, the apoptotic cells within damaged fibers returned to control levels, and at 7 days following injury apoptotic cells in the connective tissue returned to control concentrations. No preferential, internucleosomal cleavage of DNA from inflamed muscle was observed, although there was greater fragmentation of DNA in inflamed muscle than in controls. Double labeling studies show that cells expressing either ED1 or ED2 antigen can undergo apoptosis in vivo. The time course of apoptosis and concentration of apoptotic cells within damaged muscle fibers indicates that apoptosis contributes to returning ED1+ cells to control concentration during the resolution of inflammation. However, apoptosis of ED2+ cells during the first week following injury is not sufficient to return ED2+ cell concentrations to control values.

Effect of a Nitric Oxide Synthase Inhibitor, S-Ethylisothiourea, on Cultured Cells and Cardiovascular Functions of Normal and Lipopolysaccharide-Treated Rabbits

Nitric oxide (NO) is synthesized from L-arginine by three isoforms of NO synthase (NOS). It is essential to suppress the function of the inducible isoform (macNOS) for amelioration of some inflammatory diseases in which the cytotoxic effect of NO is involved. S-Ethylsiothiourea (S-EIU) was reported to be a potent and specific inhibitor of macNOS. We also confirmed that it rather specifically inhibited the activity of the purified macNOS and the formation of nitrite by RAW264.7 cells compared to NG-monomethyl-L-arginine (L-NMA) and NG-nitro-L-arginine (L-NNA), the other isoforms being less effective. S-EIU suppressed the release of nitrite and lactate dehydrogenase from rat vascular smooth muscle cells treated with interleukin-1 beta and forskolin more potently than L-NMA or L-NNA. S-EIU also slightly suppressed internucleosomal DNA cleavage in pancreatic beta-cells induced by NO produced by macNOS. Intravenous administration of either S-EIU at 0.1 mg/kg/min or L-NMA at 1 mg/kg/min increased the blood pressure but decreased the heart rate in normal rabbits, while aminoguanidine at 1 mg/kg/min affected neither cardiovascular function. These inhibitors at these doses caused recovery of the blood pressure in lipopolysaccharide-treated rabbits that exhibited lowered blood pressure similar to that in the case of septic shock. Although S-EIU seemed not to be an adequate inhibitor for therapeutic use in vivo due to its side effects on cardiovascular functions, it is one of the most potent inhibitors of macNOS among reported inhibitors in vitro.

Effect of Protease Inhibitors on Early Events of Apoptosis

Proteolysis is an early event of apoptosis which appears to be associated with activation of the endonuclease which is responsible for internucleosomal DNA cleavage. The present study was designed to reveal the possible role of proteolysis in other early events, such as chromatin condensation, nuclear breakdown, and destabilization ofin situDNA double-stranded structure. Apoptosis of human leukemic HL-60 cells and rat thymocytes was induced by different agents, including DNA topoisomerase inhibitors, an RNA antimetabolite, and the glucocorticosteroid, prednisolone. DNA degradation was evaluated by pulsed field and conventional gel electrophoresis and by the presence ofin situDNA strand breaks. DNA stability was estimated by the measure of its sensitivityin situto denaturation. Chromatin condensation, nuclear breakdown, and other morphological changes were monitored by interference contrast and UV microscopy following cell staining with the DNA-specific fluorochrome 4′,6-diamidino-2-phenylindole. Several irreversible or reversible serine protease inhibitors prevented internucleosomal DNA degradation, nuclear breakdown, and destabilization of DNA double-stranded structure. The effective inhibitors, however, did not prevent the onset of chromatin condensation, nor the loss of the fine structural framework, nor the initial step of DNA cleavage generating DNA fragments of ≥50 kb in size. The data indicate that in both cell systems the activity of proteases sensitive to the inhibitors tested is needed for internucleosomal DNA cleavage to occur. The data also suggest that these proteases may be involved in dissolution of the nuclear envelope. Because nuclear matrix proteins and histones stabilize DNAin situ,and the decrease in DNA stability which occurs during apoptosis is precluded by the inhibitors, it is likely that serine proteases may degrade DNA stabilizing proteins. The activity of these proteases, however, appears needed neither for DNA cleavage to ≥50-kb fragments nor for the onset of chromatin condensation which is associated with dissolution of the structural framework of the nucleus.

The Sigma Receptor Ligand, Reduced Haloperidol, Induces Apoptosis and Increases Intracellular-Free Calcium Levels [Ca2+]iin Colon and Mammary Adenocarinoma Cells

The sigma receptor ligand reduced haloperidol (50 and 100μM), potently inhibited cell proliferation, and induced apoptosis in WIDr colon and MCF-7 adenocarcinoma cell lines. Apoptosis was confirmed after drug treatment cells by the presence of nuclear fragmentation after staining of the cells with Hoechst 33258 and cellular DNA fragmentation ELISA and by condensation of the heterochromatin using transmission electron microscopy. However, internucleosomal DNA cleavage was not detected using gel electrophoresis. Reduced haloperidol (100 μM) increased the intercellular free calcium levels [Ca2+]iin both cell lines, which was independent of extracellular calcium, suggesting that the rise in [Ca2"]iwas from intracellular stores and that an increase in [Ca2+]imay act as a "trigger" for apoptosis in these cell lines.

Tumor cell resistance to apoptosis due to a defect in the activation of sphingomyelinase and the 24 kDa apoptotic protease (AP24).

Signal transduction pathways involved in apoptotic cell death are poorly understood, although recent studies have implicated sphingomyelin hydrolysis and generation of the second messenger, ceramide. Previous work in this laboratory demonstrated that a serine protease termed AP24 was activated by TNF or UV light and induced DNA fragmentation in isolated nuclei. This study extended these findings to examine the role of these enzymes in apoptosis of the U937 cell line and the mechanism of resistance of its variant, U9-TR. Although this subclone was selected by growth in TNF, it was unexpectedly found to resist apoptosis induced by UV light, but was still sensitive to anti-Fas-induced DNA fragmentation. Here we show that in contrast to normal U937 cells, UV light and TNF both failed to activate neutral or acidic sphingomyelinase or AP24 in the U9-TR variant. However, anti-Fas activated both neutral and acidic sphingomyelinase in the variant comparable to that seen in parental U937. The U9-TR variant could be sensitized to TNF or UV light activation of both sphingomyelinase and DNA fragmentation by the protein phosphatase inhibitors okadaic acid and calyculin A. Furthermore, exogenous bacterial-derived sphingomyelinase caused U9-TR activation of AP24 and DNA fragmentation comparable to that in the parental U937. Exposure of permeabilized U937 cells to ceramide caused internucleosomal DNA cleavage that was blocked by an inhibitor of AP24. Taken altogether, these findings demonstrate that TNF or UV light activate sphingomyelinase that leads to the generation of ceramide resulting in activation of AP24 and DNA fragmentation in sensitive cells. A selective defect in signals leading to sphingomyelinase activation can confer resistance to apoptosis even though the variant is still sensitive to downstream apoptotic signals such as nuclear DNA fragmentation by activated exogenous AP24.

Apoptotic cell death in the rat adrenal gland: an in vivo and in vitro investigation.

Adrenocortical cell apoptosis was studied by using an established in vivo model, the hypophysectomized rat, and an in vitro model, viz., rat adrenal glands in short-term organ culture. In vivo, apoptosis (biochemical autoradiographic analysis of internucleosomal DNA cleavage) was weak and not apparent until 12-24 h after hypophysectomy. In situ histochemical localization of 3'-end DNA strand breaks revealed that apoptosis in vivo occurred nearly exclusively in subpopulations of zona reticularis cells. Adrenocorticotropic hormone (ACTH) maintenance completely blocked these indices of apoptosis. By contrast, apoptosis (DNA fragmentation) in cultured rat adrenal glands without ACTH was extensive and relatively rapid, being apparent after 1 h and increasing with the duration of incubation. ACTH attenuated (by 44%) but did not completely block apoptosis in vitro. Thus, ACTH appears to be the sole pituitary hormone that forestalls apoptosis of terminally differentiated adrenocortical (zona reticularis) cells. However, the discrepancy between in vitro and in vivo models in terms of the magnitude and rate of DNA fragmentation suggests that, in vivo, other factors finely regulate the magnitude of adrenocortical apoptotic cell death.

Characteristics of cancer cell death after exposure to cytotoxic drugs in vitro

The characteristics of cell death were investigated after exposure of CCRF-CEM.f2 cells to five drugs over a broad concentration range; these were the glucocorticoid dexamethasone (DXM), the mitotic inhibitor vincristine (VIN) and three antimetabolites, methotrexate (MTX), 5'-fluoro-2'-deoxyuridine (FUdR) and 5'-fluorouracil (5-FU). Drug-treated cells were monitored for cell death mechanisms at different times by examining the pattern of DNA degradation, cell morphology and flow cytometric profile, together with effects on cell growth over 72 h. At growth-inhibitory drug concentrations, the first changes were cell cycle perturbations detectable after 4-6 h of drug exposure. The appearance of features characteristic of apoptotic cell death was noted after all drug treatments in the CCRF-CEM.f2 cell line, but the pattern and kinetics varied considerably. VIN induced apoptotic changes by 12 h, while DXM treatment caused apoptosis only after 48 h. Both MTX and FUdR induced morphological changes characteristic of apoptosis at least 24 h before internucleosomal DNA cleavage, which was detectable only after 48 h. In contrast, 5-FU did not cause internucleosomal DNA cleavage by 48 h at any concentration, despite the presence of morphologically apoptotic cells 24 h earlier. These data suggest that disruption of the cell cycle caused by drug treatment may be the common trigger initiating the drug-specific apoptotic sequence of dying cells.

Short exposures to tunicamycin induce apoptosis in SV40-transformed but not in normal human fibroblasts.

When SV40-transformed fibroblasts (line 90VAVI) were exposed to tunicamycin, an inhibitor of N-linked glycosylation, an extensive cell death occured compared with untransformed fibroblasts. A considerable cell loss was obtained within 24 h after tunicamycin addition, and after 72 h there were hardly any virus-transformed cells alive. A 2-h pulse treatment with tunicamycin was found to be almost as effective as a continuous 48-h treatment in killing the cells. Even such a short exposure as 7 min resulted in a drastically decreased cell viability (54%). The morphology of the dying tunicamycin-treated 90VAVI cells suggested that they were undergoing apoptosis. This was also supported by the appearance of nuclear condensation, as assayed by propidium iodide uptake, which was detectable within 2 h after tunicamycin addition. Furthermore, analysis of DNA from tunicamycin-treated 90VAVI cells by field inversion gel electrophoresis revealed DNA degradation into 50 kbp fragments within 2 h, and conventional agarose gel electrophoresis showed 'DNA laddering', indicating internucleosomal DNA cleavage, detectable after 36 h. Together with the finding that tunicamycin within seconds caused an elevation of [Ca2+]i, a well documented early feature of apoptosis in many experimental systems, these results strongly suggest that tunicamycin-induced cell death in 90VAVI is due to apoptosis. The short tunicamycin exposure required to trigger cell death in 90VAVI indicates that the apoptotic process is irreversibly induced soon after its addition. It seems unlikely that the pool of one or several specific N-linked glycoproteins could be depleted during such a short period. Instead the overall accumulation of unglycosylated proteins in ER might contribute to the apoptotic response in 90VAVI. Tunicamycin also killed and induced DNA degradation in the breast cancer cell line MDA-231.

Apoptotic Death of Cerebellar Neurons in Primary Culture Prepared from Neonatal Rats Exposed to Methylmercury in Utero.

A single dose of methylmercury at 10 or 15 mg Hg/kg body weight (p.o.) does not have an apparent adverse effect on adult rats. However, when primary cerebellar cells were prepared from neonatal rats exposed to methylmercury in utero by administering methylmercury to pregnant rats on day 18 of gestation, cerebellar neurons died after a certain lag time with morphological changes characteristic to apoptosis : formation of apoptotic vesicles, disappearance of neurites and condensation of nuclear chromatin and internucleosomal cleavage of chromatin DNA. The apoptotic death of cerebellar neurons required diffusible factor (s) released from the methylmercury-loaded cells, which can also induce the death of cerebellar neurons from control animals. Cerebellar weight gain during the 10 d after birth was also retarded by the prenatal exposure to methylmercury. These results indicate that prenatal exposure to methylmercury at maternal doses which do not induce apparent abnormality in adult rats brings about an accumulation of methylmercury in cerebellar neurons sufficient to induce apoptotic death of those cells, which may be a cause of retardation of cerebellar development during early postnatal stage.

Canalicular Fragmentation of Apoptotic Human Endothelial Cells

Apoptosis of endothelial cells (EC) is responsible for the removal of blood vessels during vascular remodelling. Cultured human umbilical vein EC (HUVEC) undergo apoptosis if deprived of either serum or adhesion. Apoptotic HUVEC rapidly loose adhesion and in this paper we describe the ultrastructure of detached apoptotic HWEC and human microvascular EC (HMEC). These cells displayed the formation of apoptotic bodies, nuclear condensation and nuclear fragmentation typical of apoptosis in other cell types. Ultrastructural changes occurred in parallel with the internucleosomal DNA cleavage characteristic of apoptosis. An important difference between apoptotic HWEC and other reported cells was the formation of vesicle-like canalicular structures confluent with the plasma membrane surface. These structures formed an interconnecting network throughout the apoptotic cell. Apoptotic HMEC also displayed this canalicular pattern. Continuity of the plasma membrane surface of apoptotic EC with these canaliculi was est...

Apoptosis of human glioma cells in response to calphostin C, a specific protein kinase C inhibitor.

Calphostin C acts at the regulatory domain as a highly selective inhibitor of protein kinase C (PKC), and staurosporine acts at the catalytic domain as a nonspecific PKC inhibitor. The authors investigated the capacity of calphostin C and staurosporine to promote apoptotic fragmentation of DNA in four human glioma cell lines. The exposure of glioma cell lines to 100 nM calphostin C for 2 to 8 hours induced a decrease in particulate PKC activities and exposure for 16 to 24 hours produced a concentration-dependent increase in internucleosomal DNA cleavage on agarose gel electrophoresis. In addition, the human glioma cells showed the classic morphological features of apoptosis: cell shrinkage, nuclear condensation, and the formation of apoptotic bodies. A 24-hour exposure to staurosporine failed to induce internucleosomal DNA fragmentation at concentrations generally used to achieve maximum inhibition of enzyme activity (50 nM) but promoted fragmentation at considerably higher concentration (more than 200 nM). Deoxyribonucleic acid fragments obtained from cells exposed to 100 nM calphostin C for 16 to 24 hours possessed predominantly 5'-phosphate termini, consistent with the action of a Ca++/Mg(++)-dependent endonuclease. Northern and Western blot analyses revealed that the exposure to 100 nM calphostin C for 4 hours failed to alter bcl-2 transcript and protein, but exposure for more than 8 hours decreased the amount of bcl-2 transcript and protein. Together, these observations suggest that calphostin C is capable of inducing apoptotic DNA fragmentation and cell death in a highly concentration dependent manner in human glioma cells and that the apoptosis is closely associated with the decrease in transcription and translation of bcl-2.

Changes of Mitochondrial Mass in the Hemopoietic Stem Cell Line FDCP-Mix after Treatment with Etoposide: A Correlative Study by Multiparameter Flow Cytometry and Confocal and Electron Microscopy

FDCP-Mix, a pluripotent murine hemopoietic stem cell line which undergoes typical internucleosomal cleavage of DNA when induced to apoptosis by either drugs or withdrawal of growth factor (interleukin-3) was studied after treatment with the topoisomerase II inhibitor etoposide (0.5-4 μ M). An increase in autolytic activity was the major early morphological change within the cytoplasm, with mitochondria as the main target for autolytic digestion. Despite this macroautophagy, thin sections showed a high number of mitochondria, suggesting mitochondrial proliferation as a result of drug treatment. This observation of an increase in the number of mitochondria was confirmed by flow cytometric studies of mitochondrial overall mass. Multiparameter flow cytometry of cells double stained with propidium iodide and nonyl-acridine orange gave an accurate assay for mitochondrial mass in relation to cell cycle stages. The increase in mitochondrial mass was found in all cell cycle stages. The results suggest a drug-induced proliferation of mitochondria separate from the processes involved in the doubling of mitochondrial mass during the cell cycle and a decline of mitochondria in the later stages of apoptosis.

Relationship between tamoxifen-induced transforming growth factor beta 1 expression, cytostasis and apoptosis in human breast cancer cells.

Previously we have shown that tamoxifen (TAM) induces morphological and biochemical changes typical of apoptosis in oestrogen receptor (ER)-positive MCF-7 or ER-negative MDA-231 human breast cancer cells. In this study the effects of TAM on expression of transforming growth factor beta 1 (TGF-beta 1) were correlated with the effects on cell cycle kinetics and apoptosis. TAM had similar biphasic effects on both cell lines. Short-term (< 6 h) TAM incubation resulted in a slight decrease in TGF-beta 1 protein despite an increase in TGF-beta 1 mRNA and was associated with an increase in cells in S-phase. No apoptotic effects were noted. Longer (> or = 12 h) TAM incubation induced TGF-beta 1 protein (about 3-fold) and mRNA expression (about 2-fold) in both cell lines, and was associated with G1/G0 blockade and induction of apoptosis. The accumulation of TAM-induced TGF-beta 1 mRNA was increased by cycloheximide, but was not affected by 17 beta-oestradiol. Long-term incubation with TAM had no significant effect on TGF-beta 1 gene copy number. TAM-induced internucleosomal DNA cleavage was inhibited in both cell lines by the addition of an anti-TGF-beta 1 antibody. TAM has dose- and time-dependent effects on TGF-beta 1 expression associated with changes in cell cycle kinetics. These effects are independent of ER status and may be the result of a direct regulatory effect of TAM on TGF-beta 1 transcription. It also appears that induction of TGF-beta 1 plays an important role in TAM-induced apoptosis in breast cancer cells.