Investigation of the teratogenic potential of ochratoxin A and B using the FETAX system

BACKGROUND: Ochratoxin A (OTA) is a mycotoxin produced by certain Aspergillus and Penicillium species. It has been observed to be teratogenic in a number of animal models including rat, mouse, hamster, and chick, with reduced birth weight and craniofacial abnormalities being the most commonly observed malformations. Neither the potential of OTA to cause malformations in humans nor its teratogenic mode of action is known. The FETAX system is an embryotoxicity assay system, with a high correlation to animal models and epidemiological data. Analysis of OTA-mediated teratogenesis using this system could provide a useful tool for the generation of high numbers of samples for mechanistic studies. METHODS: Using the standard ASTM 96-hr exposure protocol, the effect of OTA and its structural analogue OTB on the development of Xenopus laevis embryos in vitro was assessed. The accumulation of both substances in Xenopus embryos was also examined using tritiated OTA and OTB. RESULTS: Both OTA and OTB caused craniofacial malformations, while OTA also caused reduced embryo growth. As expected, OTA was far more potent in inducing these effects than OTB. This could at least in part be due to greater levels of OTA being accumulated within the embryos. CONCLUSIONS: The ability of FETAX to differentiate between close structural analogues indicates the assay has great potential for the elucidation of the embryotoxic and teratogenic mechanisms of action. Hence, the model could provide a suitable system for the investigation of other known teratogens or for the pre-screening of new agents for teratogenic potential.     

A novel embryotoxic estimation method of VPA using ES cells differentiation system

Valproic acid (VPA), which has a wide range of therapeutic applications, is known as a potent teratogen that induces neural tube defects in vertebrates. Here, we have characterized the tissue-specific, embryotoxic effects of VPA on developmental processes using a novel system with differentiating mouse ES cells. Under our cultivating condition, ES cells differentiated into cardiomyocytes, although various cell types can be differentiated. VPA affected cell viability and differentiation from undifferentiated ES cells to cardiomyocytes in a dose-dependent manner. The analysis of tissue-specific markers also revealed that VPA potently inhibited mesodermal and endodermal development but promoted neuronal differentiation in a lineage-specific manner. Taking the in vivo teratogenicity of VPA into account, this assay system could be useful in predicting the degree of embryotoxicity of VPA. We, thus, propose that the in vivo embryotoxic effects of various medicines can be estimated fast and accurately using this in vitro cell differentiation system.     

Effects of teratogenic antibodies on cultured visceral yolk-sac endodermal cells: cytotoxicity and morphological studies

Primary cultures of visceral yolk-sac (VYS) endodermal cells were used to assess the effects of teratogenic and nonteratogenic antibodies. When assessed by cytotoxicity assay, teratogenic antibodies appeared to be lethal to the cultured cells at high concentrations (1.25-5 mg of antibodies per ml of culture medium). At a nonlethal dosage, the teratogenic antibodies induced morphological changes, including retraction and rounding up of living cells. The cytotoxic effect as well as the effect on cell morphology appeared to be dose-dependent and specific to VYS endodermal cells. The mechanisms of cell killing were not the same as those attributed to complement-mediated cell lysis. The nonteratogenic antibodies did not have any cytotoxic effect nor did they cause any cell morphological alterations. The results of this investigation, when interpreted by correlating the dose-dependent effects of the teratogenic antibodies on cultured endodermal cells with the in vivo teratogenic effect, suggest that teratogenic antibodies when given at a teratogenic dose cause congenital abnormalities without killing the VYS endodermal cells.     

Role of the community effect of cardiomyocyte in the entrainment and reestablishment of stable beating rhythms

To investigate the roles that the community effect and entrainment function of cultured cardiomyocyte play in decreasing beating fluctuation and reestablishing synchronized beating, we developed a single-cell-based two-dimensional network culture assay to measure and compare the dynamics of beating rhythm synchronization of individual cells before and after they form networks. Studying the formation of two-cell networks, we found that their synchronized beating tended to be determined by the cardiomyocyte whose beat rate fluctuated less than that of the other cardiomyocyte. We further found that the strength of this tendency increased with the number of cells in the network. These results indicate that (1) beating fluctuation is one of the important factors influencing the reestablishment of a stable synchronous beating rhythm, (2) the larger networks reduce fluctuation, and (3) the formation of a spatial network can itself stabilize cardiomyocyte beat rates.     

Embryotoxicity of 1,2-dibromoethane in chick embryos in ovo: early and late effects.

Embryotoxic effects of 1,2-dibromoethane (DBE), a compound still widely used in industry, have been analyzed using chick embryos in ovo. Administration on embryonic days (ED) 3,4 or 5 induced dose-dependent embryotoxicity, manifested namely as the early embryonic death. A serious disturbance of the vascular system represented probably the main cause of strong embryolethality and growth retardation in the group of survivors. Amniotic bands in the parietal region and defects of brain and aorta prevailed in the malformation spectrum registered on ED 10. The local character of early induced changes suggests a direct effect of DBE itself in the embryotoxic action. This process is probably accomplished through interaction with lipids in cell membranes owing to the hydrophobic character of DBE molecules. The results, however, did not exclude an involvement of reactive metabolites in final embryotoxicity via the formation of DNA-adducts. In any case, a decreasing embryotoxicity of DBE with the age of treated embryos documented that the onset of liver function, assumed to occur on ED 5, did not increase the efficacy of DBE bioactivation. Our results confirmed the short-term embryotoxic properties of DBE reported in rat embryonic cultures. In addition, the in ovo system enabled us to reveal also long-term consequences represented namely by the formation of amniotic bands, not detectable in studies in vitro. The results obtained with the chick embryo in ovo confirmed the suitability of this system for embryotoxicity testing.     

Inhibition of histone deacetylase activity on specific embryonic tissues as a new mechanism for teratogenicity

BACKGROUND: the inhibition of histone deacetylase (HDAC) has been reported as an effective mechanism on therapy in neoplastic diseases. Among HDAC inhibitors, Trichostatin A (TSA) and Valproic Acid (VPA) prevent the tumorigenesis in rodent and human models. Malformations as neural tube and axial skeletal defects are well-known VPA side effects. Recent hypotheses suggest the HDAC inhibitor activity as the teratogenic mechanism of VPA. The teratogenic potency of TSA is, at the moment, unknown. The aim of the present work is to investigate the HDAC inhibition on embryos exposed in utero to TSA or VPA and to compare the teratogenic potential of these two molecules on the axial skeleton morphogenesis. METHODS: Pregnant CD mice were i.p. treated on day 8 post coitum (9.00 a.m.) with 400 mg/kg VPA or with 0, 2, 4, 8, 16 mg/kg TSA. Embryos explanted 1 hr after the treatment from some females exposed to 400 mg/kg VPA or to 16 mg/kg TSA were processed for Western blotting and immunohistochemical analysis, in order to evaluate the histone hyperacetylation in the total embryo homogenates and to visualize the hyperacetylated tissues. Foetuses at term were processed for skeletal examination. RESULTS: Both VPA and TSA were able to induce hyperacetylation on embryos, specifically at the level of the caudal neural tube and of somites. At term, TSA showed teratogenic effects at the axial skeleton, quite similar to those observed after VPA exposure. CONCLUSIONS: In conclusion, both VPA and TSA are teratogenic in mice. A direct correlation between somite hyperacetylation and axial abnormalities could suggest the HDAC inhibition as the mechanism of the teratogenic effects.     

Evaluation of Bupropion Hydrochloride Developmental Cardiotoxic Effects in Chick Cardiomyocyte Micromass Culture and stem cell derived Cardiomyocyte Systems

The use of antidepressant drug bupropion hydrochloride (BPN) during pregnancy results in increased cardiovascular anomalies. In this study, BPN developmental cardiotoxic effects in in vitro system were evaluated using chick cardiomyocyte micromass (MM) culture system and mouse embryonic stem cell derived cardiomyocyte (ESDC) system. In MM system, the cardiomyocyte contractile activity significantly decreased only at BPN 200 μM, while in ESDC system BPN concentration above 75 μM resulted in decreased contractile activity. The increase in drug concentration also affected the cardiomyocyte viability and total cellular protein content in both systems, but in ESDC system the cell viability failed to attain significant difference. The drug failed to induce reactive oxygen species production in both systems, but has affected the cardiac connexin43 expression especially in MM system. We observed that BPN showed developmental cardiotoxic effects irrespective of the stage of cardiac development in both in vitro systems     

Sensitivity of the primordia of various organs to chemical substances

In the experiments performed in chick embryos, using the chick embryo screening test (CEST)-1 and CEST-2 technique, 100 various classes of chemical substances have been tested for teratogenic activity. Estimation of three morphogenetic systems (caudal part of the body, facial skull and extremities) has demonstrated that some of the substances do not produce any damaging effect, other produce some lesions in selected germs, the third influence two or three germs simultaneously. Similar picture is also specific for the effect of the chemical substances to the rat intrauterine development. Owing to data obtained, we come to the conclusion that for screening of embryotoxicity of various xenobiotics under in vivo and in vitro experimental conditions one should not orient to one single germ.     

Embryotoxicity of T-2 toxin and secalonic acid in embryonic chicks varies with the site of administration.

A crucial role of the site of administration in the sensitivity of the alternative system using chick embryo for testing embryotoxicity was demonstrated by morphological evaluation of the effects of T-2 toxin and secalonic acid D, and by incorporation of [14C]sodium acetate radioactivity. Secalonic acid D, administered to 2-, 3-, and 4-day-old embryos in doses higher than 1 microgram produced mostly malformations of the face (bilateral cleft beak, microphthalmia) while the teratogenic effects of T-2 toxin were being limited to the embryonic trunk of 2-day-old embryos (rumplessness) after administering doses higher than 0.001 microgram. In case of subgerminal and intraamniotic injections, the doses of both mycotoxins needed for producing embryotoxic effects comparable to those obtained with the more commonly used yolk sac injections appeared to be lower by one and two orders of magnitude, respectively. The results stress the need of using the shortest transport channel of test substances from the site of application to the target tissues of the embryo, when the maximum sensitivity and reproducibility of the test system are to be expected.     

Identification of early-responsive genes correlated to valproic acid-induced neural tube defects in mice

BACKGROUND: Valproic acid (VPA) causes the failure of neural tube closure in newborn mice. However, the molecular mechanism of its teratogenesis is unknown. This study was conducted to investigate the genomewide effects of VPA disruption of normal neural tube development in mice. METHODS: Microarray analysis was performed on the head part of NMRI mouse embryos treated for 1 hr with VPA on gestational day (GD) 8. Subsequently, we attempted to isolate genes that changed in correlation with the teratogenic action of VPA by employing reduced teratogenic VPA analogs, valpromide (VPD) and valnoctamide (VCD), in a real-time PCR study. RESULTS: Microarray results demonstrated that during neurulation, many genes, some of whose functions are known and some unknown, were either increased or decreased after VPA injection. Some genes were affected by VPD or VCD in the same way as VPA, but others were not changed by the analogs. In this way, our system identified 11 increased and 20 decreased genes. Annotation analysis revealed that the increased genes included gadd45b, ier5, per1, phfl3, pou3f1, and sox4, and the decreased genes included ccne2, ccnl, gas5, egr2, sirt1, and zfp105. CONCLUSIONS: These findings demonstrate that expression changes in genes having roles in the cell cycle and apoptosis pathways of neural tube cells were strongly expected to relate to the teratogenic, but not antiepileptic, activity of VPA. Our approach has allowed the expansion of the catalog of molecules immediately affected by VPA in the developing neural tube.     

Membrane expression of DR4, DR5 and caspase-8 levels, but not Mcl-1, determine sensitivity of human myeloma cells to Apo2L/TRAIL

The improved recombinant form of the death ligand Apo2L/TRAIL (Apo2L/TRAIL.0) is not cytotoxic for normal human cells and is a good candidate for the therapy of multiple myeloma (MM), a B-cell neoplasia that remains incurable. We have analyzed the molecular determinants of myeloma sensitivity to Apo2L/TRAIL.0 in a number of MM cell lines, the mechanisms of resistance and a possible way of overcoming it. Expression of one death receptor for Apo2L/TRAIL (DR4 or DR5) is sufficient to transduce death signals, though DR5 was more efficient when both receptors were present. Membrane expression of decoy receptors (DcR1, DcR2) and intracellular levels of c-FLIP(L), XIAP and Mcl-1 were not predictive of resistance to Apo2L/TRAIL. Inhibition of Mcl-1 degradation did not prevent Apo2L/TRAIL-induced apoptosis. In IM-9 cells, resistance was associated to a reduced caspase-8 expression. U266 cells, though expressing significant levels of DR4 and caspase-8, were nevertheless resistant to Apo2L/TRAIL. This resistance could be overcome by co-treatment with valproic acid (VPA), a histone deacetylase inhibitor. VPA caused the redistribution of DR4 to plasma membrane lipid rafts and restored DR4 signaling. Overexpression of Mcl-1 in U266 cells did not prevent Apo2L/TRAIL cytotoxicity in VPA-sensitized cells. These results, taken together, support the possible use of Apo2L/TRAIL.0 in the treatment of MM.     

Autophagy is involved in ethanol-induced cardia bifida during chick cardiogenesis

Excess alcohol consumption during pregnancy has been acknowledged to increase the incidence of congenital disorders, especially the cardiovascular system. However, the mechanism involved in ethanol-induced cardiac malformation in prenatal fetus is still unknown. We demonstrated that ethanol exposure during gastrulation in the chick embryo increased the incidence of cardia bifida. Previously, we reported that autophagy was involved in heart tube formation. In this context, we demonstrated that ethanol exposure increased ATG7 and LC3 expression. mTOR was found to be inhibited by ethanol exposure. We activated autophagy using exogenous rapamycin (RAPA) and observed that it induced cardiac bifida and increased GATA5 expression. RAPA beads implantation experiments revealed that RAPA restricted ventricular myosin heavy chain (VMHC) expression. In vitro explant cultures of anterior primitive streak demonstrated that both ethanol and RAPA treatments could reduce cell differentiation and the spontaneous beating of cardiac precursor cells. In addition, the bead experiments showed that RAPA inhibited GATA5 expression during heart tube formation. Semiquantitative RT-PCR analysis indicated that BMP2 expression was increased while GATA4 expression was suppressed. In the embryos exposed to excess ethanol, BMP2, GATA4 and FGF8 expression was repressed. These genes are associated with cardiomyocyte differentiation, while heart tube fusion is associated with increased Wnt3a but reduced VEGF and Slit2 expression. Furthermore, the ethanol exposure also caused the production of excess ROS, which might damage the cardiac precursor cells of developing embryos. In sum, our results revealed that disrupting autophagy and excess ROS generation are responsible for inducing abnormal cardiogenesis in ethanol-treated chick embryos.     

A Developmental Toxicology Assay Platform for Screening Teratogenic Liability of Pharmaceutical Compounds

Increasing need for proactive safety optimization of pharmaceutical compounds has led to generation and/or refinement of in vitro developmental toxicology assays. Our laboratory has developed three in vitro developmental toxicology assays to assess teratogenic liability of pharmaceutical compounds. These assays included a mouse molecular embryonic stem cell assay (MESCA), a dechorionated zebrafish embryo culture (ZEC) assay, and a streamlined rat whole embryo culture (rWEC) assay. Individually, the assays presented good (73–82%) predictivity. However, it remains to be determined whether combining or tiering the assays could enhance performance. Seventy‐three compounds representing a broad spectrum of pharmaceutical targets and chemistry were evaluated across the assays to generate testing strategies that optimized performance. The MESCA and ZEC assays were found to have two limitations: compound solubility and frequent misclassification of compounds with H1 receptor or GABAnergic activity. The streamlined rWEC assay was found to be a cost‐effective stand‐alone assay for supporting poorly soluble compounds and/or ones with H1 or GABAnergic activity. For all other compounds, a tiering strategy using the MESCA and ZEC assays additionally optimized throughput, cost, and minimized animal use. The tiered strategy resulted in improved performance achieving 88% overall predictivity and was comparable with 89% overall predictivity achieved with frequency analysis (final teratogenic classification made from most frequent teratogenic classification from each individual assay). Furthermore there were 21 compounds in the test set characterized as definitive or suspect human teratogens and the multiassay approach achieved 95 and 91% correct classification using the tiered or frequency screening approach, respectively     

Polycomb homologs are involved in teratogenicity of valproic acid in mice

BACKGROUND: Valproic acid (VPA) is widely used to treat epilepsy and bipolar disorder and is also a potent teratogen, but its teratogenic mechanisms are unknown. We have attempted to describe a fundamental role of the Polycomb group (Pc-G) in VPA-induced transformations of the axial skeleton. METHODS: Pregnant NMRI mice were given a single subcutaneous injection of vehicle or VPA (800 mg/kg) on gestation day (GD) 8. The expression of genes encoding Polycomb and trithorax groups was measured by quantitative real-time RT-PCR using total RNA isolated from the embryos exposed to vehicle or VPA for 1, 3, and 6 hr. In addition, the use of two less teratogenic antiepileptic chemicals valpromide (VPD) and valnoctamide (VCD) provide reliable evidence to support the relationship between VPA teratogenicity and the Polycomb group. RESULTS: At a teratogenic level, VPA inhibits the expression of the Polycomb group genes, including Eed, Ezh2, Zfp144, Bmi1, Cbx2, Rnf2, and YY1 in the mouse embryos. In contrast, neither VPD nor VCD have significant effects on the expression of those genes affected by VPA. The trithorax group (trx-G) gene MLL, which is known to be required to maintain homeobox gene expression such as the Polycomb gene, is not affected by a teratogenic dose of VPA. CONCLUSIONS: We propose that, during embryonic development, VPA may affect the gene silencing pathway mediated by the Polycomb group complex. The epigenetic mechanism of VPA teratogenicity on anteroposterior patterning is suspected.     

丙戊酸钠对抗鱼藤酮诱导的SH-SY5Y细胞损伤的线粒体机制

研究丙戊酸钠（sodiumvalproate,VPA）对抗鱼藤酮（Rotenone）诱导的SH-SY5Y细胞损伤的作用及线粒体机制。以l,10μmol／LVPA预处理SH－SY5Y细胞3h,再加入400nmol／LRotenone作用24h。MTT法检测与相差显微镜观察相结合,分析VPA对抗Rotenone损伤的作用;JC－1染色法与Mito－Tracker染色法分析线粒体膜电位及线粒体数量的变化;Clark氧电极法检测细胞呼吸功能;DCFH-DA探针法检测细胞中Ros的含量;并在离体线粒体上观察VPA对Ca^2＋诱导的线粒体肿胀的影响。结果发现,1,10p．mol／LVPA预处理SH．SY5Y细胞3h可对抗400nmol／LRotenoneI起的细胞损伤,并且可以提高损伤细胞中线粒体的膜电位,增加线粒体的数量,此外,还可以增强损伤细胞的呼吸功能,降低细胞中ROS的含量,但VPA并不能直接作用于离体的线粒体发挥神经保护作用。由此,VPA具有良好的神经保护作用,其机制与增强线粒体功能和数量、从而改善细胞功能有关,这为其应用于帕金森病的预防与治疗提供了实验依据。     

Induction and enhancement of cardiac cell differentiation from mouse and human induced pluripotent stem cells with cyclosporin-A

Induced pluripotent stem cells (iPSCs) are novel stem cells derived from adult mouse and human tissues by reprogramming. Elucidation of mechanisms and exploration of efficient methods for their differentiation to functional cardiomyocytes are essential for developing cardiac cell models and future regenerative therapies. We previously established a novel mouse embryonic stem cell (ESC) and iPSC differentiation system in which cardiovascular cells can be systematically induced from Flk1(+) common progenitor cells, and identified highly cardiogenic progenitors as Flk1(+)/CXCR4(+)/VE-cadherin(-) (FCV) cells. We have also reported that cyclosporin-A (CSA) drastically increases FCV progenitor and cardiomyocyte induction from mouse ESCs. Here, we combined these technologies and extended them to mouse and human iPSCs. Co-culture of purified mouse iPSC-derived Flk1(+) cells with OP9 stroma cells induced cardiomyocyte differentiation whilst addition of CSA to Flk1(+) cells dramatically increased both cardiomyocyte and FCV progenitor cell differentiation. Spontaneously beating colonies were obtained from human iPSCs by co-culture with END-2 visceral endoderm-like cells. Appearance of beating colonies from human iPSCs was increased approximately 4.3 times by addition of CSA at mesoderm stage. CSA-expanded human iPSC-derived cardiomyocytes showed various cardiac marker expressions, synchronized calcium transients, cardiomyocyte-like action potentials, pharmacological reactions, and ultra-structural features as cardiomyocytes. These results provide a technological basis to obtain functional cardiomyocytes from iPSCs.     

Retinoids regulate tight junctional resistance of cultured human cervical cells

The objective ofthe study was to determine the effect of retinoids on paracellularresistance across the cervical epithelium and the mechanisms involved.The experimental model was cultures of human CaSki cells on filters,which retain phenotypic characteristics of the endocervical epithelium.End points for paracellular resistance were measurements oftransepithelial electrical resistance and fluxes of pyranine (atrisulfonic acid that traverses the epithelium via the intercellularspace). Paracellular resistance was significantly increased in cellsgrown in retinoid-free medium; the effect could be blocked and reversedwith all-trans-retinoic acid (tRA) and with agonists of RAR and RXR receptors but only partially with retinol.The effect of tRA was dose dependent and saturable, with a 50%effective concentration of 0.8 nM. The increases in paracellular resistance induced by vitamin A deficiency required longer incubation in retinoid-free medium than decreases in resistance induced by retinoic acid. tRA had only a minimal effect on paracellular resistance in cells maintained in regular medium. Retinoid-free medium increased and tRA decreased the relative cation mobility across CaSki cultures. Also the effects of tRA were nonadditive to those of cytochalasin D(which decreases tight junctional resistance) and additive to those ofionomycin (which decreases the resistance of the lateral intercellularspace), suggesting that tRA modulates tight junctional resistance. Itis concluded that vitamin A determines the degree of paracellularresistance across cervical cells by a mechanism that involvesmodulation of tight junctional resistance.

     

Neurodevelopment and mood stabilizers

Mood disorders and schizophrenia share a number of common properties, including: genetic susceptibility; differences in brain structure and drug based therapy. Some genetic loci may even confer susceptibility for bipolar mood disorder and schizophrenia, and some atypical antipsychotic drugs are used as mood stabilizers. As schizophrenia is associated with aberrant neurodevelopment, could this also be true for mood disorders? Such changes could arise pre- or post-natal, however the recent interest in neurogenesis in the adult brain has suggested involvement of these later processes in the origins of mood disorders. Interestingly, the common mood stabilizing drugs, lithium, valproic acid (VPA) and carbamazepine, are teratogens, affecting a number of aspects of animal development. Recent work has shown that lithium and VPA interfere with normal cell development, and all three drugs affect neuronal morphology. The molecular basis for mood stabilizer action in the treatment of mood is unknown, however these studies have suggested both targets and potential mechanisms. Lithium directly inhibits two evolutionarily conserved signal transduction pathways: the protein kinase Glycogen Synthase Kinase-3 (GSK-3) and inositol signaling. VPA can up-regulate gene expression through inhibition of histone deacetylase (HDAC) and indirectly reduce GSK-3 activity. VPA effects are not conserved between cell types, and carbamazepine has no effect on the GSK-3 pathway. All three mood stabilizers suppress inositol signaling, results further supported by studies on the enzyme prolyl oligopeptidase (PO) and the sodium myo-inositol transporter (SMIT). Despite these intriguing observations, it remains unclear whether GSK-3, inositol signaling or both underlie the origins of bipolar disorder.     

Adaptation to the action of several teratogens as a consequence of preliminary administration of pesticides to females]

The effect of DDT and lindane pesticides on the intensity of the teratogenic action of sodium acetylxalicylate (SA) and of the cabomate benlate group of pesticides was studied on Wistar rats given the mentioned pesticides from the onset of pregnancy. The teratogens were administered on the 10th and the 12th days of gestation, respectively. Preliminary administration of these pesticides was found to weaken the teratogenic and the embryotoxic action of benlate given in a dose of 250 mg/kg, and of SA administered in a dose of 400 mg/kg. When SA was given in a dose of 600 mg/kg preliminary administration of the pesticides decreased the postimplantation mortality of the embryos, but the number of fetuses with developmental anomalies was the same as in the isolated action of the preparation given in this dose.     

Apoptotic cell death in neuronal differentiation of P19 EC cells: Cell death follows reentry into S phase

Apoptotic cell death was observed during aggregate culture of the mouse embryonal carcinoma cell line P19 exposed to all-trans retinoic acid (tRA). This finding was confirmed by genomic DNA agarose gel electrophoresis and transmission electron microscopy. Apoptosis was associated with P19 cell neuronal differentiation; alternative causes of cell death, i.e., cavitation-related, cytotoxicity of tRA, or spontaneous cell death were excluded. Analysis by flow cytometry revealed that the apoptosis was likely to occur in multiplying cells that underwent to reentering into S phase. We therefore examined 5-bromo-2′-deoxyuridine (BrdU) incorporation and proliferating cell nuclear antigen (PCNA) expression and localization in the aggregates by immunofluorescent staining. Although the P19 cells in the aggregates exposed to tRA incorporated BrdU at an equivalent level to those not exposed to tRA, the cells showed diminished PCNA expression and nuclear accumulation. We propose that P19 apoptosis during neuronal differentiation is a model system in which programmed cell death occurs simultaneously with cell division leading to differentiation. J. Cell. Physiol. 172:25–35, 1997. © 1997 Wiley-Liss, Inc.