RhoBTB2 (DBC2) functions as tumor suppressor via inhibiting proliferation, preventing colony formation and inducing apoptosis in breast cancer cells

RhoBTB2 was isolated recently as a tumor suppressor gene from sporadic breast cancer. Although RhoBTB2 was found to be frequently lost in breast cancer and a variety of cancers, its antitumor effect, however, remains unclear. In this study, we constructed a recombinant expression vector pEGFP-N1-RhoBTB2 and transfected it into RhoBTB2-negative breast tumor cell line T-47D. Stable transformanted cells were identified by fluorescence microscope, RT-PCR and Western blot. Cell viability was measured by MTT assay. Colony forming efficiency of breast tumor cells was detected by colony formation assay. Morphological change of apoptotic cells was observed by hematoxylin-eosin staining. Apoptotic ratio was determined by flow cytometry. Cell invasion and migration ability assay were performed using transwell system. Overexpression of RhoBTB2 in breast tumor cells significantly inhibited the proliferation and colony formation of tumor cells. In addition, RhoBTB2 also elevated the apoptotic ratio and caused typical changes of apoptotic morphology in breast tumor cells of RhoBTB2 overexpression. But RhoBTB2 did not influence the invasion and migration ability of breast tumor cells. Therefore, RhoBTB2 is an important tumor suppressor gene related with breast cancer and may play antitumor roles by inhibiting proliferation, preventing colony formation and promoting the apoptosis of tumor cells. However, the precise mechanism behind the antitumor effects of RhoBTB2 needs to be investigated further.     

Monoclonal Antibody to Single-Stranded DNA Is a Specific and Sensitive Cellular Marker of Apoptosis

The most widely used histochemical marker of apoptosis (in situend labeling, TUNEL) detects both apoptotic and necrotic cells and evaluates only late stages of apoptosis. Hence, a specific and sensitive cellular marker of apoptosis is needed to determine the role of apoptotic death in biology and pathology. The present study describes a novel immunohistochemical procedure for the staining of apoptotic cells using a monoclonal antibody (MAb) to single-stranded DNA. This MAb stained all cells with the morphology typical of apoptosis in etoposide-treated HL-60, MOLT-4, and R9 cell cultures, in which apoptosis was accompanied by high, moderate, and low levels of internucleosomal DNA fragmentation, respectively. TUNEL stained all apoptotic cells in HL-60 cultures, nearly 60% of apoptotic cells in MOLT-4 cultures, and only 14% of apoptotic cells in R9 cultures. Apoptotic R9 cells, which progressed into secondary necrosis, retained MAb staining and became TUNEL-positive. Necrotic cells in MOLT-4 cultures treated with sodium azide were stained by TUNEL, but were negative for MAb staining. All floating cells at a late stage of apoptosis in MDA-MB-468 cultures treated with cisplatin were stained by both MAb and TUNEL. However, among adherent cells in the early stages of apoptosis, MAb stained nearly 20 times more cells than TUNEL. In histological sections of human tumor xenografts, MAb detected clusters of apoptotic cells in viable tumor tissue, but did not stain cells in areas of central ischemic necrosis. In contrast, TUNEL stained nuclei in necrotic areas. Thus, MAb to single-stranded DNA is a specific and sensitive cellular marker of apoptosis, which differentiates between apoptosis and necrosis and detects cells in the early stages of apoptosis.     

Alterations in tumorigenicity of embryonal carcinoma cells by IGF-I triple-helix induced changes in immunogenicity and apoptosis

IGF-I antisense gene therapy has been applied successfully to animal models of glioma, hepatoma and teratocarcinoma. The antisense strategy has shown that tumor cells transfected with vectors encoding IGF-I antisense RNA lose tumorigenicity, become immunogenic and are associated with tumor specific immune response involving CD8+ lymphocytes. An IGF-I triple helix approach to gene therapy for glioma was recently described. The approach we have taken is to establish parameters of change using the IGF-I triple helix strategy. PCC-3 embryonal carcinoma cells derived from murine teratocarcinoma which express IGF-I were used as a model. The cells were transfected with vector which encodes an oligoribonucleotide that forms RNA-IGF-I DNA triple-helix structure. The triple-helix stops the production of IGF-I. Cells transfected in this manner underwent changes in phenotype and an increase in MHC-I and B-7 cell surface molecules. They also showed enhancement in the production of apoptotic cells (60-70%). The "triple helix" transfected cells lost the ability to induce tumor when injected subcutaneously in syngeneic 129 Sv mice. When co-transfected in vitro with expression vectors encoding both MHC-I and B-7 cDNA in antisense orientation, the "triple-helix" transfected cells were down-regulated in expression of MHC-I and B-7 and the number of apoptotic cells was significantly decreased. Injection of the doubly co-transfected cells into 129 Sv mice was associated with induction of teratocarcinoma. Comparison between antisense and triple-helix transfected cells strategies showed similar immunogenic and apoptotic changes. The findings suggest that triple-helix technology may offer a new clinical approach to treatement of tumors expressing IGF-I.     

Cutting edge: differential effect of apoptotic versus necrotic tumor cells on macrophage antitumor activities.

Macrophages (Mphi) play essential roles both in tumor defense and normal tissue homeostasis by removal of transformed as well as damaged and disintegrating cells. Whereas tissue necrosis is known to provoke inflammatory responses, removal of apoptotic cells has been assumed to be immunologically inert. We now show that while Mphi exposure to necrotized tumor cells causes pronounced stimulation of Mphi antitumor activity, exposure of Mphi to apoptotic tumor cells in contrast results in impairment of Mphi-mediated tumor defense and even support of tumor cell growth. Given the fact that apoptosis is a consequence of various cancer treatment modalities, this may lead to a suppression of local antitumor reactions and thus actually counteract endogenous immune-mediated tumor defense mechanisms.     

The expression of "tissue" transglutaminase in two human cancer cell lines is related with the programmed cell death (apoptosis).

The expression of "tissue" transglutaminase (tTG) in two human tumor cell lines (the cervix adenocarcinoma line HeLa-TV and the neuroblastoma cells SK-N-BE-2) was found to be in correlation with the rate of physiological cell death (apoptosis) in culture. We investigated the effect of retinoic acid (RA) and alpha-difluoromethylornithine (DFMO) in order to elucidate the relationship between tTG expression and apoptosis. RA led to a 6-fold increase of tTG activity in HeLa-TV cells and to a 12-fold increase in SK-N-BE(2) cells, which was paralleled in both cell lines by a proportional increase in the number of apoptotic bodies recovered from the cultures. On the contrary, DFMO determined a dramatic reduction of tTG expression and of the apoptotic index. Immunohistochemical analysis using an anti-tTG antibody showed that the enzyme was accumulated in both cell lines within typical apoptotic bodies. Immunocytochemistry and cell cloning of SK-N-BE(2) line demonstrated that tTG was absent in cells showing neurite outgrowth, indicating that the enzyme expression is not associated with neural differentiation, even though both phenomena are elicited by retinoic acid. On the whole, these data indicate that also in tumors tTG activation takes place in cells undergoing apoptosis. The enzyme is activated in apoptotic cells to form cross-linked protein envelopes which are insoluble in detergents and chaotropic agents. The number of insoluble protein envelopes as well as the N,N-bis(gamma-glutamyl)polyamine cross-links is related with both tTG expression and apoptotic index, strongly suggesting the participation of the enzyme in the apoptotic program.(ABSTRACT TRUNCATED AT 250 WORDS)     

Caspase-Mediated Apoptosis in AK-5 Tumor Cells: A Cell-Free Study Using Peptide Inhibitors and Antisense Strategy

Anin vitrosystem has been employed to study the apoptotic mechanisms in the AK-5 tumor which is a spontaneously regressing rat histiocytoma. Cytosolic extracts of tumor cells primed for apoptosis using dexamethasone and immune serum from tumor-regressing animals were able to induce apoptosis in intact nuclei and reproduce the classical morphological and biochemical features typical of apoptotic cells. The cleavage of lamin A and PARP to signature fragments by these extracts and the inhibition of the same using peptide inhibitors signify the pivotal role of ICE and ICE-related proteases in apoptosis. Lamin A cleavage was insensitive to YVAD but PARP cleavage was blocked by both YVAD and DEVD. Cell extracts derived from cells overexpressing theBcl-2gene andNedd-2antisense gene, respectively, failed to induce apoptosis in exogenously added nuclei, suggesting thatBcl-2gene product is downregulating a key event in apoptotic cascade. The study also demonstrates the coherent action of different ICE-related proteases in apoptosis and their functional redundancy. This system may prove useful for analyzing complex molecular mechanisms underlying apoptosis in tumor cells.     

Actin depolymerization and polymerization are required during apoptosis in endothelial cells

In order to understand the role of actin microfilaments in the apoptotic process, we followed their evolution during tumor necrosis factor-alpha (TNF)-induced apoptosis in bovine aortic endothelial (BAE) cells. Using Western blotting analysis and immunofluorescence microscopy, we observed that the actin microfilaments network was disrupted in apoptotic cells. Depolymerization of F-actin was concomitant with internucleosomal DNA degradation and with the morphological changes associated with apoptotic cell death. However, using the actin microfilament disrupting agent, cytochalasin, we present evidence that the formation of blebs leading to apoptotic cell fragmentation requires neopolymerization of actin. Indeed, in the presence of cyochalasin, induction of apoptosis (internucleosomal DNA degradation) in BAE cells by TNF and cycloheximide was not associated with these classical morphological markers of apoptosis. Moreover, when added to BAE cells showing incipient apoptotic fragmentation, cytochalasin E reversed this process. We also observed an accumulation of actin at the basis of the apoptotic bodies in formation in these cells. Together, these results suggest that the actin network of flattened cells is disrupted concomitantly to the morphological modifications associated to the apoptotic cell death, and that the cytochalasin-sensitive reorganisation of actin is required to the formation of apoptotic blebs.     

CD8+ T Cells Specific to Apoptosis-Associated Antigens Predict the Response to Tumor Necrosis Factor Inhibitor Therapy in Rheumatoid Arthritis

CD8⁺ T cells specific to caspase-cleaved antigens derived from apoptotic T cells (apoptotic epitopes) represent a principal player in chronic immune activation, which is known to amplify immunopathology in various inflammatory diseases. The purpose of the present study was to investigate the relationship involving these autoreactive T cells, the rheumatoid arthritis immunopathology, and the response to tumor necrosis factor-α inhibitor therapy. The frequency of autoreactive CD8⁺ T cells specific to various apoptotic epitopes, as detected by both enzyme-linked immunospot assay and dextramers of major histocompatibility complex class I molecules complexed with relevant apoptotic epitopes, was longitudinally analyzed in the peripheral blood of rheumatoid arthritis patients who were submitted to etanercept treatment (or other tumor necrosis factor inhibitors as a control). The percentage of apoptotic epitope-specific CD8⁺ T cells was significantly higher in rheumatoid arthritis patients than in healthy donors, and correlated with the disease activity. More important, it was significantly more elevated in responders to tumor necrosis factor-α inhibitor therapy than in non-responders before the start of therapy; it significantly dropped only in the former following therapy. These data indicate that apoptotic epitope-specific CD8⁺ T cells may be involved in rheumatoid arthritis immunopathology through the production of inflammatory cytokines and that they may potentially represent a predictive biomarker of response to tumor necrosis factor-α inhibitor therapy to validate in a larger cohort of patients.     

Enhanced apoptotic cell clearance capacity and B cell survival factor production by IL-10-activated macrophages: implications for Burkitt's lymphoma

Burkitt's lymphoma (BL) is typified by frequent tumor cell apoptosis and significant macrophage infiltration. Since BL cells have an inherent tendency to undergo apoptosis at a high rate, we reasoned that macrophages in BL are functionally enhanced in at least two activities that have implications for tumor pathogenesis: 1) engulfment of apoptotic cells, an anti-inflammatory process known to suppress immune responses, and 2) production of BL cell survival factors that limit the extent of tumor cell apoptosis. In this study, we show that the microenvironment of BL is rich in the pleiotropic cytokine IL-10, which can be produced by both tumor cells and macrophages, and that IL-10-activated human macrophages have enhanced capacity to engulf apoptotic cells in vitro. This was found to be dependent on the macrophage tethering receptor of apoptotic cells, CD14. Furthermore, IL-10-activated macrophages were found to produce markedly higher levels of the B cell survival factor, B cell-activating factor of the TNF family/B lymphocyte stimulator (BAFF/BLyS) than macrophages matured in the absence of IL-10. Coculture of macrophages with BL cells further enhanced BAFF secretion. Significantly, we show that enhancement of BL cell survival by IL-10-activated macrophages is mediated by a BAFF-dependent component and that BAFF is produced at high levels by tumor-associated macrophages in situ. These results indicate that macrophages, regulated by IL-10, have the potential to promote BL pathogenesis, first, through suppression of antitumor immunity following enhanced engulfment of apoptotic tumor cells and, second, through increased production of tumor cell growth/survival factors.     

Moloney murine leukemia virus-induced tumors show altered levels of proapoptotic and antiapoptotic proteins

Moloney murine leukemia virus (M-MuLV) is a replication-competent, simple retrovirus that induces T-cell lymphomas when inoculated into neonatal mice. The tumor cells are typically derived from immature T cells. During preleukemic times, a marked decrease in thymic size is apparent in M-MuLV-inoculated mice. We previously demonstrated that this thymic regression is correlated with enhanced levels of thymocyte apoptosis (C. Bonzon and H. Fan, J. Virol. 73:2434-2441, 1999). In this study, we investigated the apoptotic state of M-MuLV-induced tumors. M-MuLV-induced tumors were screened for expression of the apoptotic proteins Fas and Bcl-2 by three-color flow cytometric analysis. Single-positive (SP; CD4(+) CD8(-) and CD4(-) CD8(+)) tumor cells generally displayed lower cell surface expression of Fas than SP thymocytes from uninoculated control mice. Double-positive (DP; CD4(+) CD8(+)) M-MuLV-induced tumor cells fell into two categories: those with normal high levels of Fas and those with low levels of Fas. Additionally, the vast majority of DP tumors showed elevated Bcl-2 levels. The DP tumor cells retaining normal/high Fas expression were capable of transducing an apoptotic signal upon anti-Fas engagement. In addition, DP and CD4(+) SP tumor populations displayed higher levels of Fas ligand than normal thymocytes with the same phenotypes. In contrast, CD8(+) SP and CD4(-) CD8(-) tumors did not show elevated Fas ligand expression. There was no significant correlation between Fas and Fas ligand expression in the DP tumors, suggesting that Fas Ligand expression was not the driving force behind Fas down-regulation. These results suggest that both the Fas death receptor and mitochondrial pathways of apoptotic death are active in M-MuLV-induced tumors and that they must be modulated to permit cell survival and tumor outgrowth.     

Defective apoptosis as potential mechanism in the tumorogenesis of myelolipoma.

Apoptosis is considered an important mechanism of selective deletion that occurs during hematopoiesis. Myelolipoma is a rare benign tumor composed of adipose tissue and hematopoietic cells. The pathogenesis of this benign tumor is still unclear. Analysing the structural levels and apoptosis of normal human bone marrow (NHBM) and human myelolipoma (HM), the apoptotic events resulted abundantly present in NHBM compared to HM, which showed a small number of apoptotic cells. By contrast, Fas expression was strongly present both in NHBM and HM. These findings suggest that an altered function of Fas in myelolipoma is not able to trigger the apoptotic machinery. In conclusion, we hypothesize that drastic reduction of apoptosis in myelolipoma can be considered one of the growth regulatory mechanisms.     

EphrinA5-EphA7 complex induces apoptotic cell death via TNFR1

A previous study showed that the EphA7 receptor regulates apoptotic cell death during early brain development. In this study, we provide evidence that the EphA7 receptor interacts with death receptors such as tumor necrosis factor receptor 1 (TNFR1) to decrease cell viability. We showed that ephrinA5 stimulates EphA7 to activate the TNFR1-mediated apoptotic signaling pathway. In addition, a pull-down assay using biotinylated ephrinA5-Fc revealed that ephrinA5-EphA7 complexes recruit TNFR1 to form a multi-protein complex. Immunocytochemical staining analysis showed that EphA7 was co-localized with TNFR1 on the cell surface when cells were incubated with ephrinA5 at low temperatures. Finally, both the internalization motif and death domain of TNFR1 was important for interacting with an intracytoplasmic region of EphA7; this interaction was essential for inducing the apoptotic signaling cascade. This result suggests that a distinct multi-protein complex comprising ephrinA5, EphA7, and TNFR1 may constitute a platform for inducing caspase-dependent apoptotic cell death.     

Tumor suppression by a proapoptotic calcium-activated chloride channel in mammary epithelium

Little is known of the roles played by ion channels in cancer. Here we describe a pair of closely related calcium-activated chloride channels whose differential regulation in normal, apoptotic, and transformed mouse cells suggests that channel function is proapoptotic and antineoplastic. While mCLCA1 predominates over mCLCA2 under normal physiological conditions, this relationship is reversed by apoptotic stress both in developing mammary gland and in cultured HC11 mammary epithelial cells. Consistent with an apoptosis-promoting role, splicing of mCLCA2 is disrupted in apoptosis-resistant tumor cell lines and in HC11 cells selected for resistance to detachment-induced apoptosis (anoikis). Unexpectedly, mCLCA1 message is also down-regulated in these cells by at least 30-fold. These results suggest that both genes antagonize survival of mammary tumor cells by sensitizing them to anoikis. When MCF7 or HEK293 tumor cells were transfected with plasmids encoding either mCLCA1 or mCLCA2, colony formation was greatly reduced relative to a vector-transfected control, demonstrating that calcium-sensitive chloride channel (CLCA) expression is deleterious to tumor cell survival. Furthermore, mammary epithelial cells overexpressing mCLCA2 had twice the rate of apoptosis of normal cells when subjected to serum starvation and formed multinuclear giants at a high frequency in normal culture, suggesting that mCLCA2 can promote either apoptosis or senescence.     

p53-independent pRB degradation contributes to a drug-induced apoptosis in AGS cells

The retinoblastoma （RB） tumor suppressor protein, pRB, plays an important role in the regulation of mammalian cell cycle. Furthermore, several lines of evidence suggest that pRB also involves in the regulation of apoptosis. In the present study, the degradation of pRB was observed in apoptotic gastric tumor cells treated with a new potent anti-tumor component, tripchlorolide （TC）. The inhibition of pRB degradation by a general cysteine protease inhibitor IDAM resulted in the reduction of the apoptotic cells. Furthermore, the survival of the gastric tumor cells under the TC treatment was enhanced by an over-expression of exogenous pRB. These results suggest that the pRB degradation of the gastric tumor cells under the TC treatment involves in the apoptotic progression. In addition, the same extent of TCinduced pRB-degradation was detected in the gastric tumor cells containing a p53 dominant-negative construct, indicat- ing that this kind of pRB degradation is p53-independent.     

Single-cell analysis demonstrates how nutrient deprivation creates apoptotic and quiescent cell populations in tumor cylindroids

Understanding how quiescent and apoptotic populations form in tumors is necessary because these cell types can considerably diminish therapeutic efficacy. Most cancer therapeutics are ineffective against quiescent cells because they target rapidly proliferating cells. Distinguishing apoptosis is important because apoptotic cells are committed to death and do not require treatment. Regrowth of quiescent cell can lead to tumor re-occurrence and metastasis, which are the leading causes of cancer mortality. We hypothesized that cylindroid cultures and acridine orange staining could be used to determine how nutrient diffusion creates apoptotic and quiescent regions in tumors. To test this hypothesis we developed a microscopy technique to measure cellular DNA and RNA content in single cells using thin cylindroids and acridine orange staining. Cell classification was compared to flow cytometry of cells grown in defined monolayer cultures. The presence of apoptosis was confirmed by morphological nuclear analysis. The effect of diffusion was determined by varying incubation time, cylindroid size, and exposing cylindroids to nutrient-deficient media. Four overlapping regions were identified as a function of cylindroid radius: an outer viable/quiescent region; a second quiescent/apoptotic region; a third late-stage apoptotic region; and an inner dead region. In monolayer cultures the absence of glutamine and growth factors induced apoptosis and hypoxia induced quiescence. Treating with nutrient-deficient media suggested that cells became quiescent near the periphery because of glucose and oxygen limitations, and became apoptotic and died further from the edge because of glutamine and growth factor limitations. These results show that cellular microenvironments can be identified in cylindroids using simple acridine orange staining and that single cell fluorescence can be measured in three-dimensional culture. The developed techniques will be useful for developing cancer therapies and determining how cell death and apoptosis are induced in three-dimensional tumor tissue.     

Pigment epithelium-derived factor (PEDF)-induced apoptosis and inhibition of vascular endothelial growth factor (VEGF) expression in MG63 human osteosarcoma cells

Pigment epithelium-derived factor (PEDF) has been shown to be the most potent inhibitor of angiogenesis in the mammalian eye, thus suggesting that loss of PEDF is involved in angiogenic eye diseases such as proliferative diabetic retinopathy. Angiogenesis is required for tumor growth and progression as well. We, along with others, have recently found that PEDF could inhibit growth of melanoma and hepatocellular carcinoma in nude mice through its anti-angiogenic effects on tumor endothelial cells. However, the possibility of the direct effect of PEDF on tumor cells has remained. In this study, we investigated the effects of PEDF on growth and vascular endothelial growth factor (VEGF) expression in MG63 human cultured osteosarcoma cells. PEDF decreased viable cell number as well as DNA synthesis in MG63 cells in a dose-dependent manner. Furthermore, PEDF was found to increase caspase-3/7 activity and to subsequently induce apoptotic cell death in MG63 cells. PEDF also inhibited VEGF expression in MG63 cells at both mRNA and protein levels. Our present study provides novel beneficial aspects of PEDF on osteosarcoma cells; one is induction of apoptotic cell death of tumor cells, and the other is the suppression of VEGF expression, which would lead to inhibition of tumor angiogenesis. PEDF therefore might be a promising therapeutic agent for treatment of patients with osteosarcoma.     

Commentary: ATG5: A distinct role in the nucleus

Both apoptotic and autophagic pathways are activated in cells during anticancer treatment using DNA-damaging agents. Thus, the outcome is balanced between apoptotic cell death and enhanced autophagy, with the possibility of prolonged cell survival. It seems intuitively obvious that this survival mechanism might interfere with the desired tumor cell killing. We addressed this question by tipping the balance in favor of autophagy, using etoposide or cisplatin at low, sublethal doses. Over 4 days, only a little apoptosis was observed, but both drugs sharply increased autophagic flux. Surprisingly, cells underwent a cell cycle arrest at G₂/M, followed later by mitotic catastrophe with formation of multipolar spindles, missegregated chromosomes, or enlarged, irregular, sometimes multiple nuclei. Why? The answer is that even a low level of DNA damage not only upregulates autophagy, but also provokes the recruitment of an autophagy-related protein, ATG5, to the nucleus, where it binds BIRC5/survivin, thereby interfering with correct assembly of the chromosome passenger complex needed for cytokinesis.     

ATG5

Both apoptotic and autophagic pathways are activated in cells during anticancer treatment using DNA-damaging agents. Thus, the outcome is balanced between apoptotic cell death and enhanced autophagy, with the possibility of prolonged cell survival. It seems intuitively obvious that this survival mechanism might interfere with the desired tumor cell killing. We addressed this question by tipping the balance in favor of autophagy, using etoposide or cisplatin at low, sublethal doses. Over 4 days, only a little apoptosis was observed, but both drugs sharply increased autophagic flux. Surprisingly, cells underwent a cell cycle arrest at G₂/M, followed later by mitotic catastrophe with formation of multipolar spindles, missegregated chromosomes, or enlarged, irregular, sometimes multiple nuclei. Why? The answer is that even a low level of DNA damage not only upregulates autophagy, but also provokes the recruitment of an autophagy-related protein, ATG5, to the nucleus, where it binds BIRC5/survivin, thereby interfering with correct assembly of the chromosome passenger complex needed for cytokinesis.     

Comparative image and flow cytometric TUNEL analysis of fine needle samples of breast carcinoma.

The assessment of apoptosis in solid tumors is of interest because of its biological role in tumor evolution and response to therapy. A commonly used method for apoptosis measurement is the TUNEL 3' end-labeling technique, which has shown wide variations in results when applied to solid tumors. Thirty-one fine needle breast carcinoma samples were analyzed by fluorescent TUNEL assay and DNA content using image analysis and flow cytometry. TUNEL positivity, seen both in cells with apoptotic morphology and in a subset of morphologically normal cells, was categorized into five staining patterns and quantitated. Values for patterns of TUNEL-positive cells were compared with TUNEL positivity measured by flow cytometry. Flow cytometric quantitation showed a mean of 24.3% positive cells, which correlated (P < 0.02) with total positive cells (all patterns) measured by image (22.4%). Image analysis quantitation of morphologically apoptotic cells (4.2%) did not correlate with flow cytometric TUNEL positivity and the majority of TUNEL-stained cells were morphologically normal (17%). Image analysis allows discrimination of TUNEL-positive morphologically apoptotic and nonapoptotic cells, which are included in the total number of TUNEL-positive events measured by flow cytometry.